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In open angle glaucoma, dysfunction of the trabecular meshwork (TM) results in impaired aqueous humour outflow leading to an elevated intraocular pressure (IOP) that underlies optic nerve damage and irreversible blindness. Currently, no curative treatment is available for the disease. Indeed, most pharmacological and surgical interventions usually provide only temporary relief from elevated IOP while little progress has been made in targeting the root cause of this disease: correcting the dysfunctional TM. In this context, we hypothesized that regeneration/refunctionalization of the TM may represent an effective therapeutic option to halt disease progression or even reverse the pathologic process. We previously demonstrated in a rat model of glaucoma that the injection of mesenchymal stromal cells (MSCs) cultured under hypoxic conditions or their conditioned media (MSC-CM) into laser-damaged TM area results in tissue regeneration. Injection of MSC or conditioned media in our glaucoma model led to activation and proliferation of ocular progenitor cells culminating in TM regeneration and a decrease in IOP. However, the mechanistic basis for this regenerative process remained elusive. Thus, the aim of this thesis is to elucidate the mechanistic basis of MSC secretome-mediated TM regeneration and the subsequent decrease in IOP. We now demonstrate that injection of hypoxic MSC-CM into laser-induced glaucomatous eyes resulted in massive immune cell recruitment. We also demonstrate that these hypoxic MSC-CM conditioned cells produced pro-regenerative factors in vitro and in vivo. Next, employing a proteomic approach, we identified and verified the pro-regenerative effect of several factors secreted by hypoxic MSC-CM recruited cells, which in turn induced the activation/proliferation of ocular progenitor cells leading to TM regeneration and decreased IOP. Upon individual injection of the purified factors into glaucomatous rat eyes, we observed a partial and delayed but significant decrease in IOP that correlated with an increase in the activation and proliferation of neuronal progenitor cells in the TM area. The co-injection of these factors resulted in a significant decrease in IOP compared with individual factor injection. Importantly, this drop in IOP was associated with restoration of retinal functionality, thus demonstrating the importance of these factors in the TM regeneration process and disease control. The findings presented in this thesis provide a novel acellular therapeutic approach for glaucoma treatment via in situ TM regeneration. Moreover, the knowledge gained here could have a lasting impact on how we induce tissue regeneration in other degenerative diseases and lead to novel therapeutic advances in regenerative medicine. / Dans le glaucome à angle ouvert, le dysfonctionnement du trabéculum (TM), un tissu nécessaire à la filtration de l'humeur aqueuse, entraîne une élévation de la pression intraoculaire (PIO). Ceci cause des lésions au niveau du nerf optique et une cécité irréversible. Présentement, aucun traitement curatif n'a été développé pour cette maladie. Nous émettons l'hypothèse que la régénération et re-fonctionnalisation du trabéculum peut représenter une option thérapeutique efficace pour arrêter ou inverser la progression de la maladie dans de nombreux cas de glaucome. Nous avons précédemment démontré les effets régénérateurs des cellules mésenchymateuses (MSCs) et de leurs milieux conditionnés par l'hypoxie (MSC-CM) dans la régénération du TM suite à un dommage par laser. Ce processus a conduit à l'activation et à la prolifération des cellules progénitrices oculaires résultant en une diminution de la PIO dans un modèle de glaucome induit par laser chez le rat. Cependant, la base mécanistique de ce processus de régénération reste encore inconnue. Ainsi, le but de cette thèse de recherche est d'élucider cette base mécanistique de la régénération du TM médiée par le sécrétome des MSC et la diminution subséquente de la PIO. À cette fin, l'injection de MSC-CM hypoxique dans les yeux glaucomateux induits par laser a entraîné un important recrutement de cellules immunitaires. Sous l’action du MSC-CM, ces cellules produisent des facteurs pro-régénératifs in vitro et in vivo. Ensuite, nous avons utilisé une approche protéomique et vérifié l'effet pro-régénératif des facteurs sécrétés par ces cellules exposées au MSC-CM hypoxique, sur l'activation et la prolifération des cellules progénitrices oculaires et la PIO. Lors de l'injection de ces facteurs chez le rat glaucomateux, nous avons observé une augmentation significative de l'activation et de la prolifération des cellules progénitrices neuronales présentes dans la zone du TM, résultant en une diminution de la PIO. De plus, l’injection combinée de ces facteurs résulte en une diminution synergique importante de la PIO. Cette baisse de la PIO était associée à une restauration de la fonction rétinienne, démontrant ainsi l'importance de ces facteurs dans le processus de régénération du TM et de contrôle de la maladie. Les résultats présentés dans cette thèse pourraient amener à une nouvelle approche thérapeutique acellulaire pour le traitement du glaucome via la régénération du TM. De plus, les connaissances acquises au cours de cette thèse pourraient avoir un impact durable sur la manière d’aborder la régénération tissulaire dans d'autres maladies dégénératives et amener des avancées thérapeutiques nouvelles en médecine régénératrice

Le vieillissement est l'un premier facteur de risque pour plusieurs maladies telles que l’athérosclérose, la fibrose, l’Alzheimer, le diabète de type 2 et le cancer. L'accumulation de cellules sénescentes avec l'âge contribue au développement de maladies liées à l'âge en induisant une inflammation chronique causée par le phénotype sécrétoire associé à la sénescence (SASP). Il y a également une augmentation de la sécrétion de vésicules extracellulaires (EV) lors de la sénescence. Les EV sont des structures à bicouche lipidique permettant le transport de molécules actives vers des cellules réceptrices. De plus, les EV participent aux effets pathologiques des cellules sénescentes. Ainsi, nous proposons que les macrophages sénescents participent au développement de maladies liées à l’âge en induisant l'inflammation par l’action combinée des facteurs solubles du SASP et des EV. Tout d'abord, nous avons établi un modèle de macrophages sénescents induit par l’oncogène Raf-1. Une analyse transcriptionnelle de notre modèle a démontré un profil inflammatoire régulé par Nf-κB. La sécrétion d'EV est également augmentée par les macrophages sénescents. En outre, les EV dérivées de macrophages sénescents sont enrichies en miARN, tels que miR-21, miR-155 et miR-132, ainsi qu'en protéines ribosomiques, qu'en protéine Alix et qu'en protéine Mvp. Les sécrétions des macrophages sénescents induisent un échappement de la sénescence chez les cellules MEF, probablement par l'action combinée des molécules solubles du SASP et des EV. Nous concluons que les macrophages sénescents sécrètent des signaux prolifératifs et inflammatoires dans les cellules réceptrices, ce qui suggère leur rôle potentiel dans le développement de cancer. Le traitement avec le navitoclax élimine les macrophages sénescents et pourrait prévenir leurs effets pathologiques. / Aging is the first risk factor for several diseases such as atherosclerosis, fibrosis, Alzheimer’s, type 2 diabetes and cancer. The accumulation of senescent cells with age contributes to development of age-related diseases by inducing chronic inflammation. This inflammation is induced by the senescence-associated secretory phenotype (SASP). During senescence, there is also an increase of extracellular vesicles (EV) secretion. EVs are lipid bilayer structures that allow the transport of active molecules to recipient cells. In addition, EVs participate in pathologic effects of senescent cells. Thus, we propose that senescent macrophages participate in development of age-related diseases by inducing inflammation through the combined effect of SASP soluble factors and EV. First, we established a model of senescent macrophages induced by the oncogene Raf-1. Transcriptional analysis of our model demonstrated an Nf-κB-regulated inflammatory profile. EV secretion is also increased by senescent macrophages. Moreover, EVs derived from senescent macrophages are enriched in miRNA, such as miR-21, miR-155 and miR-132, as well as ribosomal proteins, Alix protein an Mvp protein. Secretion of senescent macrophages induce senescence escape in MEF cell, probably through the combined action of SASP soluble factor and EV. We conclude that senescent macrophages secrete proliferative and inflammatory signals in recipient cell, suggesting their potential role in cancer development. Treatment with navitoclax eliminates senescent macrophages and may prevent their pathological effects.

Bovine milk-derived extracellular vesicles (EVs) hold promises as oral drug delivery systems. Since EV bioavailability studies are difficult to compare, key factors regarding EV uptake and intestinal permeability remain little understood. This work aims to critically study uptake and transport properties of milk-derived EVs across the intestinal barrier in vitro by standardization approaches. Therefore, uptake properties were directly compared to liposomes in intestinal Caco-2 cells. Reliable staining results were obtained by the choice of three distinct EV labeling sites, while non-specific dye transfer and excess dye removal were carefully controlled. A novel fluorescence correction factor was implemented to account for different labelling efficiencies. Both EV and liposome uptake occurred mainly energy dependent with the neonatal Fc receptor (FcRn) providing an exclusive active pathway for EVs. Confocal microscopy revealed higher internalization of EVs whereas liposomes rather remained attached to the cell surface. Internalization could be improved when changing the liposomal formulation to resemble the EV lipid composition. In a Caco-2/HT29-MTX co-culture liposomes and EVs showed partial mucus penetration. For transport studies across Caco-2 monolayers we further established a standardized protocol considering the distinct requirements for EVs. Especially insert pore sizes were systematically compared with 3 µm inserts found obligatory. Obtained apparent permeability coefficients (Papp) reflecting the transport rate will allow for better comparison of future bioavailability testing.

[ES] A pesar de los nuevos avances en el tratamiento del cáncer de pulmón, su tasa de incidencia y mortalidad siguen en cabeza en todo mundo. Concretamente, el cáncer de pulmón no microcítico (CPNM) representa casi el 85% de todos los cánceres de pulmón, siendo su supervivencia a 5 años muy reducida. En base a dicho escenario, el objetivo principal de este trabajo es el de caracterizar de manera exhaustiva los exosomas secretados por las células del CPNM. Se sabe que estas microvesículas están involucradas en números procesos celulares, por lo que pueden contener gran cantidad de información acerca de las características moleculares del tumor. Para ello se han empleado cultivos primarios y líneas comerciales crecidas en diferentes condiciones, así como muestras de sangre periférica obtenida de los pacientes con CPNM. Un primer screening llevado a cabo en los exosomas secretados in vitro, ha permitido obtener un gran número de mRNAs y miRNAs relacionados con diferentes procesos biológicos y vías de señalización. Además, algunos genes como FDFT1 y SNAI1 han destacado por su sobreexpresión en exosomas procedentes de las células crecidas en formación de tumoresferas (modelos 3D), las cuales están enriquecidas en población de células madre tumorales. A su vez, otros marcadores presentes en el interior de estas microvesículas, se han mostrado relacionados con dos de los subtipos histológicos más frecuentes: adenocarcinoma (LUAD) y carcinoma escamoso (LUSC). Posteriormente, para validar los hallazgos obtenidos en exosomas, los marcadores más significativos fueron analizados in silico en una cohorte de muestras de tejido, compuesta por 661 pacientes con CPNM (TCGA database). Estos resultados han revelado una asociación entre la expresión del gen SNAI1 y la supervivencia de estos pacientes (OS y RFS p<0.05). Además, los genes XAGE1B, SEPP1 y TTF-1 (previamente determinados en exosomas), mantienen una relación significativa con el grupo de pacientes LUAD; mientras que CABYR, RIOK3 y CAPRIN1 se mantienen sobrexpresados en LUSC (Mann-Whitney test p<0.05). Estos marcadores también se han analizado en una cohorte de 186 pacientes con CPNM procedentes del Hospital General Universitario de Valencia, donde se corroboró la asociación de SNAI1 con la supervivencia de los pacientes en estadios tempranos (RFS en pacientes LUAD, p<0.05), así como la sobreexpresión de CABYR y RIOK3 en pacientes LUSC, y de XAGE1B y TTF-1 en LUAD. Por otra parte, el aislamiento de los exosomas presentes en la sangre periférica de pacientes en estadios avanzados, ha permitido identificar otros marcadores asociados a caracterísiticas clínico-patológicas relevantes. A su vez, el contenido de estas microvesículas ha sido empleado para la detección de mutaciones génicas ligadas al manejo clínico del CPNM. En resumen, los resultados obtenidos en este trabajo ponen de manifiesto el potencial de los exosomas como fuente de biomarcadores para el estudio de las diferentes etapas de desarrollo del CPNM. Estas microvesículas ofrecen una visión completa y en tiempo real, de las características de la enfermedad, pudiendo ser aisladas de forma repetida y mediante técnicas mínimamente invasivas. / [CA] A pesar dels avanços recents en el tractament del càncer de pulmó, les seues taxes d'incidència i mortalitat continuen sent altes a nivell mundial. Concretament, el càncer de pulmó de cèl·lules no petites (CPNM) representa gairebé el 85% de tots els càncers de pulmó, amb una taxa de supervivència a 5 anys molt limitada. Donat aquest escenari, l'objectiu principal d'aquest estudi és caracteritzar de manera exhaustiva els exosomes secretats per les cèl·lules de CPNM. Aquestes microvesícules estan involucrades en nombrosos processos tumorals i poden contenir una gran quantitat d'informació sobre les característiques moleculars de la malaltia. Per aconseguir-ho, es van utilitzar cultius primaris i línies cel·lulars (cultiu en diferents condicions), juntament amb mostres de sang perifèrica obtingudes de pacients amb CPNM. Un cribratge inicial en exosomes secrets in vitro va permetre identificar una quantitat significativa de mARNs i miARNs relacionats amb diversos processos biològics i vies de senyalització. A més, alguns gens com FDFT1 i SNAI1 van destacar per la seua sobreexpressió en exosomes derivats de cèl·lules crescuts en formació de tumorsferes (models 3D), que estan enriquides en poblacions de cèl·lules mare tumorals. A més, s'han trobat marcadors en aquestes microvesícules associats amb dos dels subtipus histològics més comuns: adenocarcinoma (LUAD) i carcinoma escamós (LUSC). Posteriorment, per validar els resultats obtinguts en exosomes, es van analitzar in silico els marcadors més significatius en una cohort de teixit de CPNM de la base de dades TCGA. Aquests resultats van revelar una associació entre l'expressió del gen SNAI1 i la supervivència dels pacients (OS i RFS, p <0,05). A més, l'expressió dels gens XAGE1B, SEPP1 i TTF-1 (prèviament identificats en exosomes) va mantenir una relació significativa amb el grup LUAD, mentre que CABYR, RIOK3 i CAPRIN1 van continuar sobreexpressats en els pacients de LUSC (prova de Mann-Whitney, p <0,05). Aquests marcadors també es van analitzar en una cohort de 186 pacients amb CPNM de l'Hospital General Universitari de València, on es va confirmar l'associació de l'expressió de SNAI1 i la supervivència dels pacients en estadi precoç (RFS en pacients de LUAD, p <0,05), així com la sobreexpressió de CABYR i RIOK3 en pacients de LUSC, i de XAGE1B i TTF-1 en LUAD. D'altra banda, els exosomes presents en mostres de sang de la cohort d'estadis avançats van permetre la identificació d'altres biomarcadors associats a característiques clíniques rellevants dels pacients. A més, la càrrega exosomàtica també es va utilitzar per detectar mutacions genètiques relacionades amb el tractament clínic del CPNM. En resum, els resultats obtinguts en aquesta tesi destaquen el potencial dels exosomes com a font de biomarcadors per a l'estudi de les diferents etapes del desenvolupament del CPNM. Aquestes microvesícules ofereixen una visió completa i en temps real de les característiques moleculars de la malaltia i poden ser obtingudes de manera repetida i amb una mínima invasió. / [EN] Despite recent advancements in lung cancer treatment, its incidence and mortality rates remain high worldwide. Specifically, non-small cell lung cancer (NSCLC) accounts for nearly 85% of all lung cancers, with a 5-year survival rate of 20%. Given this scenario, the primary objective of this study is to comprehensively characterize the exosomes secreted by NSCLC cells. These microvesicles are known to be involved in numerous tumoral processes, potentially containing a wealth of information about the molecular characteristics of the disease. To achieve this, primary cultures and cell lines, along with peripheral blood samples obtained from NSCLC patients were used. An initial screening in exosomes secreted in vitro allowed the identification of a significant number of mRNAs and miRNAs, related to various biological processes and signaling pathways. Moreover, some genes such as FDFT1 and SNAI1 stood out due to their overexpression in exosomes derived from cells grown in tumorspheres formation (3D models), which are enriched in cancer stem cell population. Additionally, markers found within these microvesicles were associated with two of the most common histological subtypes: adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC). Subsequently, to validate the findings seen in exosomes, the most significant markers were analyzed in silico in an NSCLC tissue cohort from the TCGA database. These results revealed an association between the expression of SNAI1 and patient survival (OS and RFS, p<0.05). Furthermore, XAGE1B, SEPP1, and TTF-1 expression (previously identified in exosomes) maintained a significant relationship with the LUAD group, while CABYR, RIOK3, and CAPRIN1 remained overexpressed in LUSC patients (Mann-Whitney test, p<0.05). These markers were also analyzed in a cohort of 186 NSCLC patients from the University General Hospital of Valencia. The association of SNAI1 expression and the survival of early-stage patients (RFS in LUAD patients, p<0.05) was confirmed, as well as the overexpression of CABYR and RIOK3 in LUSC patients, and of XAGE1B and TTF-1 in LUAD. Furthermore, exosomes present in blood samples of the advanced-stage cohort, allowed the identification of other biomarkers associated with clinically relevant characteristics of the patients. Moreover, exosomal cargo was also used to detect gene mutations related to the clinical management of NSCLC. In summary, the results obtained in this thesis highlight the potential of exosomes as a source of biomarkers for the study of the different stages of NSCLC development. These microvesicles offer a comprehensive and real-time view of the disease's molecular features and can be obtained repeatedly and in a minimally invasive way. / Duréndez Sáez, ME. (2024). Liquid Biopsy in non-small cell lung cancer: exosomes as a tool for the study of biomarkers [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/203438

[ES] La cardiopatía isquémica, caracterizada por la falta de suministro adecuado de oxígeno al tejido cardíaco, es una afección grave que puede desencadenar un infarto agudo de miocardio y contribuir al desarrollo de la insuficiencia cardíaca (IC). A pesar de las terapias actuales, la IC sigue siendo una enfermedad con alta morbilidad y mortalidad, lo que destaca la necesidad de estrategias terapéuticas más efectivas. En este contexto, las células madre mesenquimales (MSCs) y, en especial sus vesículas extracelulares (EVs), han surgido como opciones prometedoras por sus propiedades regenerativas, pro-angiogénicas e inmunomoduladoras. Sin embargo, el reto actual se centra en mejorar la eficacia terapéutica de las EVs, ya sea mejorando su biodisponibilidad en el tejido cardíaco o potenciando sus capacidades intrínsecas, con el fin de hacer viable una terapia basada en las mismas. En este trabajo, nos hemos centrado en la mejora de las EVs mediante la modificación genética de las MSCs. Hemos seguido dos enfoques: la carga de oncostatina M (OSM) en la superficie de las EVs y la sobreexpresión inducible del dominio intracelular de Notch1 (N1ICD) junto con el factor inducible por hipoxia 1-alfa (HIF1A) en MSCs para enriquecer las EVs con factores terapéuticos, con la expectativa de mejorar su eficacia en el contexto de la isquemia cardíaca. Los resultados obtenidos muestran que las EVs cargadas con OSM poseen propiedades antifibroticas superiores a las EVs nativas, además de reducir el daño cardíaco provocado por la infusión de isoproterenol in vivo. Por otro lado, la sobreexpresión de N1ICD y HIF1A actúa a modo de precondicionamiento genético favoreciendo la carga de diferentes moléculas terapéuticas en las EVs. Estas EVs han demostrado ejercer efectos beneficiosos in vitro como la reducción de la fibrosis, la protección de los cardiomiocitos y reducción de la hipertrofia, la disminución de especies reactivas de oxígeno, y el aumento de la angiogénesis. En el estudio in vivo estas EVs redujeron el daño provocado por la infusión de isoproterenol. En conclusión, hemos generado dos tipos de EVs con un potencial terapéutico superior a las EVs nativas en el contexto de la patología cardíaca. Este trabajo abre la puerta al diseño de nuevas estrategias terapéuticas basadas en EVs, abordando de manera integral los diversos aspectos de la enfermedad cardíaca. / [CA] La cardiopatia isquèmica, caracteritzada per la falta de subministrament adequat d'oxigen al teixit cardíac, és una afecció greu que pot desencadenar un infart agut de miocardi i contribuir al desenvolupament de la insuficiència cardíaca (IC). Malgrat les teràpies actuals, la IC continua sent una malaltia amb alta morbiditat i mortalitat, la qual cosa destaca la necessitat d'estratègies terapèutiques més efectives. En este context, les cèl·lules mare mesenquimals (MSCs) i, especialment les seues vesícules extracelul·lars (EVs), han sorgit com a opcions prometedores per les seues propietats regeneratives, pro-angiogèniques i inmunomoduladores. No obstant això, el repte actual se centra en millorar l'eficàcia terapèutica de les EVs, ja siga millorant la seua biodisponibilitat en el teixit cardíac o potenciant les seues capacitats intrínseques, amb la finalitat de fer viable una teràpia basada en estes. En este treball, ens hem centrat en la millora de les EVs mitjançant la modificació genètica de les MSCs. Hem seguit dos enfocaments: la càrrega d'oncostatina M (OSM) en la superfície de les EVs i la sobreexpressió induïble del domini intracel·lular de Notch1 (N1ICD) juntament amb el factor induïble per hipòxia 1-alfa (HIF1A) en MSCs per a enriquir les EVs amb factors terapèutics, amb l'expectativa de millorar la seua eficàcia en el context de la isquèmia cardíaca. Els resultats obtinguts mostren que les EVs carregades amb OSM posseeixen propietats antifibròtiques superiors a les EVs natives, a més de reduir el dany cardíac provocat per la infusió d'isoproterenol in vivo. D'altra banda, la sobreexpressió de N1ICD i HIF1A actua a mode de precondicionament genètic afavorint la càrrega de diferents molècules terapèutiques en les EVs. Estes EVs han demostrat exercir efectes beneficiosos in vitro com la reducció de la fibrosi, la protecció dels cardiomiòcits i reducció de la hipertròfia, la disminució d'espècies reactives d'oxigen, i l'augment de l'angiogènesis. En l'estudi in vivo estes EVs van reduir el dany provocat per la infusió d'isoproterenol. En conclusió, hem generat dos tipus de EVs amb un potencial terapèutic superior a les EVs nadiues en el context de la patologia cardíaca. Este treball obri la porta al disseny de noves estratègies terapèutiques basades en EVs, abordant de manera integral els diversos aspectes de la malaltia cardíaca. / [EN] Ischemic heart disease, characterized by a lack of adequate oxygen delivery to the heart tissue, is a serious condition that can trigger acute myocardial infarction and contribute to the development of heart failure (HF). Despite current therapies, HF remains a disease with high morbidity and mortality, highlighting the need for more effective therapeutic strategies. In this context, mesenchymal stem cells (MSCs) and, especially their extracellular vesicles (EVs), have emerged as promising options due to their regenerative, pro-angiogenic and immunomodulatory properties. However, the current challenge focuses on improving the therapeutic efficacy of EVs, either by improving their bioavailability in cardiac tissue or by enhancing their intrinsic capabilities, in order to make a therapy based on them viable. In this work, we have focused on the improvement of EVs through genetic modification of MSCs. We have followed two approaches: loading of oncostatin M (OSM) on the surface of EVs and inducible overexpression of Notch1 intracellular domain (N1ICD) together with hypoxia-inducible factor 1-alpha (HIF1A) in MSCs to enrich EVs with therapeutic factors, with the expectation of improving its effectiveness in the context of cardiac ischemia. The results obtained show that OSM-loaded EVs have superior antifibrotic properties than native EVs, in addition to reducing cardiac damage caused by isoproterenol infusion in vivo. On the other hand, the overexpression of N1ICD and HIF1A acts as genetic preconditioning, favouring the loading of different therapeutic molecules in EVs. These EVs have been shown to exert beneficial effects in vitro such as reducing fibrosis, protecting cardiomyocytes and reducing hypertrophy, decreasing reactive oxygen species, and increasing angiogenesis. In the in vivo study, these EVs reduced the isoproterenol-induced myocardial damage. In conclusion, we have generated two types of EVs with a therapeutic potential superior to native EVs in the context of cardiac pathology. This work opens the door to the design of new therapeutic strategies based on EVs, comprehensively addressing the various aspects of heart disease. / Buigues Caravaca, M. (2024). Modificación genética de células estromales mesenquimales para potenciar la eficacia de las vesículas extracelulares en el ámbito de la terapia cardíaca [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/204408

MicroRNAs are a class of small non-coding RNAs involved in post-transcriptional regulation. Their role in almost all processes of the cell, make microRNAs ubiquitary players of cell development, growth, differentiation, cell to cell communication and cell death. Thus, cells’ physiological or pathological conditions are reflected by variations in the levels of expression of microRNAs, enabling them to be used as biomarkers of such states. In the past decade, there has been an exponential increase of studies using microRNAs as potential biomarkers for cancer, neurodegenerative diseases, inflammation and cardiac diseases, from tissues and liquid biopsies. However, none of them has reached the clinics yet, due to inconsistency of results through the literature and lack of assay standardization and reproducibility. Technological limitations of microRNAs detection have been, to date, the biggest challenge for using these molecules in clinical settings. In fact, although microarrays, RT-qPCR and RNA-seq are well-established technologies, they all require complex procedures and trained personnel, for performing RNA extraction, labelling of the target and PCR amplification. All these steps introduce variability and, in addition, since no universally standardized protocol – from sample extraction to analyte detection - has been produced yet, methodological procedures are difficult to reproduce. For this reason, we developed a new platform for the rapid detection of microRNAs in biofluids composed of an innovative silicon-photomultiplier (SiPM) based detector and a new chemistry for nucleic acid testing (Chem-NAT). Chem-NAT exploits a dynamic labelling chemistry which allows the sensitive detection of nucleic acids till single base level. On the other hand, SiPM-based device, compared to normal vacuum photomultipliers, grants miniaturization and higher capacity of fitting in a bench-top solution for clinical settings, among other advantages. The new platform – ODG – has been validated for the direct detection – neither RNA extraction nor PCR amplification needed - of microRNA-21 in plasma of lung cancer patients. In this work, we also explored the use of microRNAs as biomarkers in metastatic castration resistant prostate cancer (mCRPC). We collected plasma samples from mCRPC patients before and after abiraterone acetate treatment – androgen deprivation type of drug – and performed a miRnome analysis for discovering microRNAs predicting the efficacy of the drug. We chose miR-103a-3p and miR-378a-5p and we validated them via TaqMan RT-qPCR. We discovered that the ratio between the two microRNAs is able to predict the efficacy of abiraterone acetate and follow the responsiveness in time. In liquid biopsies, extracellular vesicles are getting increasing importance for diagnostic and prognostic purposes. Therefore, in this work we also explored the expression of some microRNAs in extracellular vesicles from plasma, isolated via nickel-based method. We discovered that microRNA-21 and microRNA-223 are not enriched in vesicles from healthy individuals.

La leishmaniose est une maladie zoonotique affectant à la fois les chiens et les humains, transmise par la piqûre d’un phlébotome. Elle est causée par un parasite protozoaire du genre Leishmania et se manifeste sous trois formes principales, dont la plus grave est souvent mortelle chez l’humain. Or, la pharmacopée utilisée pour combattre cette maladie est très limitée, avec des médicaments qui sont non-spécifiques au parasite en plus d’être les mêmes chez les chiens et les humains. Cela contribue à l'aggravation de la résistance aux antiparasitaires, en particulier à l'antimoine, l'un des médicaments de première ligne contre cette maladie. Une découverte récente de notre laboratoire a mis en cause les vésicules extracellulaires (EVs) dans le développement de résistance chez Leishmania, en raison de leur capacité à transmettre horizontalement des gènes au sein d’une population de parasites. Dans ce projet de maîtrise, nous avons cherché à exploiter cette découverte et à créer des EVs qui faciliteraient le transfert de gènes permettant la resensibilisation des parasites résistants. Pour ce faire, nous avons créé des parasites surexprimant le gène AQP1, qui code pour une protéine de transport de l’antimoine. Nous avons ensuite produit des EVs à partir de ce parasite modifié, et nous en avons caractérisé la taille, la morphologie et le contenu protéique. Enfin, nous avons tenté la resensibilisation des parasites résistants, en utilisant ces EVs surexprimant AQP1. L’impact sur les parasites résistants a ensuite été évalué en termes de leur sensibilité à l’antimoine. Nous avons pu déterminer que certaines des méthodes utilisées avaient un effet significatif de resensibilisation à l’antimoine. Ces découvertes mettent de l’avant une approche innovante pour lutter contre la résistance aux antimicrobiens, et explorent une application potentielle des EVs dans la médecine moderne. / Leishmaniasis is a zoonotic disease affecting both dogs and humans, transmitted by the bite of a sandfly. It is caused by a protozoan parasite of the genus Leishmania and manifests in three main forms, the most severe of which is often fatal in humans. However, the pharmacopeia used to combat this disease is very limited, with drugs that are non-specific to the parasite and are the same for both dogs and humans. This contributes to the worsening of resistance to antiparasitic drugs, particularly to antimony, one of the first-line drugs against this disease. A recent discovery from our team implicated extracellular vesicles (EVs) in the development of resistance in Leishmania, due to their ability to horizontally transmit genes within a parasite population. In this master's project, we sought to exploit this discovery and create EVs that would facilitate the transfer of genes enabling the resensitization of resistant parasites. To do this, we created parasites overexpressing the AQP1 gene, which encodes an antimony transport protein. We then produced EVs from this new strain and characterized their size, morphology, and protein content. Finally, we attempted to resensitize resistant parasites using these AQP1-overexpressing EVs. The impact on resistant parasites was then assessed in terms of their sensitivity to antimony. We determined that some of the methods we used had a significant resensitization effect to antimony. These discoveries highlight an innovative approach to combat drug resistance and explore a potential application of EVs in modern medicine.

Le développement récent de l’endoscopie interventionnelle digestive nous a conduit à prendre en charge deux types de pathologies préoccupantes. Il s’agit d’une part des fistules digestives souvent responsables d’une morbi-mortalité élevée et d’autre part des sténoses œsophagiennes après résection tumorale endoscopique étendue. Dans ces deux situations, des phénomènes inflammatoires chroniques conduisent soit à l’absence de fermeture de la fistule soit à une fibrose importante responsable de sténose de l’œsophage. La thérapie cellulaire a déjà été utilisée pour diminuer ces phénomènes inflammatoires et entrainer une cicatrisation. La thérapie tissulaire par cellules souches organisées en construction 3D représente un avantage important en permettant de cibler le site d’action par dépôt direct du feuillet cellularisé. Notre objectif était d’évaluer l’effet thérapeutique de ces nouveaux outils pour fermer les fistules digestives et pour prévenir la survenue des sténoses œsophagiennes. La première étape a consisté a évaluer l’efficacité du traitement par des cellules souches mésenchymateuses provenant de moelle osseuse humaine, marquées puis organisées en doubles feuillets, dans un modèle de fistule entéro-cutanée post-chirurgicale chez la souris nude. L’évaluation clinique et en imagerie (IRM et microscopie confocale) a montré une meilleure cicatrisation avec une augmentation de la microvascularisation et une accélération de la fermeture de la fistule chez les souris greffées. Les effets observés semblent liés à une augmentation précoce de la synthèse des facteurs de réparation (EGF et le VEGF) et des cytokines anti-inflammatoires (TGF-ß2 et IL-10). Après avoir développé un modèle inédit de fistule oeso-cutanée chez le porc grâce à la mise en place par voie endoscopique et chirurgicale de prothèses plastiques entre la lumière œsophagienne et la peau, nous avons évalué l’efficacité thérapeutique d’un gel contenant des vésicules extracellulaires issues de cellules souches isolées du tissu adipeux de porc. Ce gel injecté dans la fistule par voie endoscopique a permis la fermeture des fistules. Enfin, la troisième partie de notre travail a consisté à évaluer l’efficacité de la greffe allogénique de doubles feuillets de cellules souches mésenchymateuses pour prévenir la survenue des sténoses œsophagiennes dans un modèle porcin après dissection sous muqueuse étendue. Il existait une réduction significative du taux de sténose œsophagienne cicatricielle dans le groupe greffé avec une fibrose moins importante. En conclusion, l’effet paracrine antifibrosant des cellules souches mésenchymateuses organisées en feuillets est efficace à la fois pour fermer les fistules entéro-cutanées chez la souris et pour prévenir les sténoses œsophagiennes chez le porc. Un gel avec des vésicules extracellulaires issues des cellules souches a de la même façon un effet cicatrisant anti-inflammatoire permettant la fermeture des fistules œsophagiennes chez le porc. Ces résultats sont très encourageants et posent la question d’une évaluation future chez l’homme. / Recent developments in digestive interventional endoscopy lead us to manage two types of digestive disease. First, it is digestive fistulas associated in many cases with high morbi-mortality; and second is oesophageal stenosis after extended superficial endoscopic resection. In both situations, chronic inflammatory process resulted in delayed or no fistula healing for the first case or oesophageal stenosis due to fibrosis. Cellular therapy has proved to be successful in reducing the inflammatory process and to promote tissue healing. Tissue therapy with 3D construct stem cells represents a major advantage by allowing a direct adaptation on the right place. Our objective was to evaluate the therapeutic effect of new strategy to close the digestive fistula and to prevent oesophageal stenosis. First step was to evaluate the effect of labelled human bone marrow derived mesenchymal stem cells engraftment in the form of double cellsheet in a post-surgical fistula model in nude mice. Clinical and radiological (MRI and probe based confocal microscopy) evaluation showed a better fistula healing with higher microvascularization and a faster fistula closing in grafted mice. These effects appear to be related to an increase production of factors involved in tissue repair (EGF et le VEGF) and anti-inflammatory cytokines (TGF-ß2 et IL-10). We developed an unpublished eso-cutaneous fistula in a porcine model after plastic catheters placement by surgical and endoscopic way between the oesophageal lumen and the skin. We evaluated the therapeutic effect of a hydrogel with extracellular vesicles extracted from porcine adipose derived stem cells. The hydrogel with vesicles was injected into the fistula by endoscopy. Radiological and histological evaluation 15 days after injection showed a fistula tract closure in treated group.The third part of this work was to evaluate the effect of allograft of adipose derived stem cells 3D construct to prevent the stenosis after extended endoscopic submucosal dissection in a porcine model. There was a significant reduction of number and degree of stenosis with decrease fibrosis infiltration in the grafted group.In summary, thanks to their paracrine and antifibrotic effect, the mesenchymal stem cells organised as 3D construct allowed fistula closure in an entero-cutaneous model in mice and prevention of stenosis after extended oesophageal submucosal dissection in a porcine model. Moreover, endoscopic hydrogel and extracellular vesicles injection allowed oesophageal fistula healing in a porcine model. These promising results pose the challenge of future clinical studies.

A artrite séptica (AS), também chamada de artrite infecciosa, é uma doença inflamatória das articulações iniciada por um agente infeccioso. O agente causal mais comum da SA é Staphylococcus aureus (S. aureus). A patogênese da SA inclui uma resposta inflamatória complexa envolvendo sistema imune inato e adaptativo. As citocinas liberadas a partir de macrófagos, tais como TNF-?, IL-1? e IL-6, foram classicamente apontadas como os principais mediadores da inflamação grave que precede a destruição da cartilagem e osso e a disfunção articular permanente mediante a AS. A evidência radiológica está frequentemente presente, mas não diferencia o afrouxamento mecânico do septo das articulações. Portanto, se houver algum indício de suspeita de infecção, deve ser aspirado para avaliação microbiológica. Recentemente, as tecnologias de imagem como a micro tomografia computadorizada (?CT) foram amplamente utilizadas para modelos pré-clínicos de distúrbios articulares auto-imunes. No entanto, as características radiológicas da AS em camundongos ainda são amplamente desconhecidas. No estudo atual, os camundongos NMRI foram inoculados intravenosamente ou intra-articularmente com a cepas de S. aureus Newman ou LS-1. Os sinais radiológicos e clínicos da artrite séptica foram acompanhados durante 10 dias usando ?CT. Avaliamos as correlações entre alterações radiológicas conjuntas e sinais clínicos, alterações histológicas e níveis séricos de citocinas. Nos dias 5-7 após a infecção intravenosa, a destruição óssea verificada por ?CT tornou-se evidente na maioria das articulações infectadas. Os sinais radiológicos de destruição óssea eram dependentes da dose bacteriana. O local mais comumente afetado pela artrite séptica foi o fêmur distal nos joelhos. A destruição óssea detectada pelo ?CT foi correlacionada positivamente com alterações histológicas na artrite séptica local e hematogênica. Os níveis séricos de IL-6 foram significativamente correlacionados com a gravidade da destruição das articulações. Coletivamente, nossos dados mostram que o ?CT é um método sensível para monitorar a progressão da doença e determinar a gravidade da destruição óssea em um modelo de artrite séptica do mouse; enquanto que a IL-6 é um potencial biomarcador de destruição óssea na artrite séptica. / Extracellular vesicles (EVs) are heterogeneous population of nano- and micro-sized vesicles secreted from almost every cell type. The process of EV secretion seems to be evolutionary conserved across the species kingdoms, ranging from simple prokaryotes to higher eukaryotes including bacteria, viruses, and parasitic protozoa such as leishmania and malarial parasites, fungi, plants and animals. Recent data suggests that Staphylococcus aureus (S. aureus) bacteria secretes EVs that could mediate host-pathogen interactions. EVs have been investigated in various bacterial species which modulate the secretion of immunoregulatory molecules such as cytokines and may have key role in infection. However, their role in S. aureus septic arthritis has not been explored yet. In current study, we postulate novel perspectives for the implementation of S. aureus-derived EVs in vitro as well as in vivo model of septic arthritis. EVs derived from S. aureus were applied to stimulate mice splenocytes in vitro as well as intra-articularly and the cytokine levels were measured. Our results showed that S. aureus derived EVs potentially provoke the production of proinflammatory cytokines. TNF-?, and IL-6 were significantly elevated in splenocytes in vitro after EV-based stimulation. Moreover, NMRI mice were injected with variable doses of EVs intraarticularly and mice were observed for 10 days to examine inflammation and development of septic arthritis. Bone and cartilage destruction was assessed by histochemistry analysis to score the joint erosion. Altogether, our results demonstrate the putative role of S. aureus-derived EVs in provoking inflammation and immunological responses suggesting that these vesicles could induce and disseminate systemic immune response during the development of septic arthritis.