Tesis Doctorals - Departament - Biologia Cel·lular, Fisiologia i Immunologia

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Regulation of muscle growth and metabolism in European sea bass and gilthead sea bream: Integrating myogenic regulatory factors, dietary composition, and physical activity
(Universitat de Barcelona, 2025-12-16) García Pérez, Isabel; Gutiérrez Fruitós, Joaquín; Blasco Mínguez, Josefina; Universitat de Barcelona. Departament de Biologia Cel·lular, Fisiologia i Immunologia
[eng] Aquaculture is one of the fastest-growing food production sectors, and its success and sustainability depend on improving growth efficiency and product quality in farmed species. Muscle is the main edible tissue in fish, underscoring the importance of understanding the cellular and molecular mechanisms that govern its development and growth, and how these mechanisms respond to nutritional inputs and rearing conditions such as sustained exercise. The present thesis examines these processes in European sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata), two species of major importance in Mediterranean aquaculture. Through the integration of in vitro, in vivo, and in silico approaches, this work provides new insights into how cellular pathways, myogenic regulators, non-coding RNAs (ncRNAs), metabolic markers, and systemic signals interact to control muscle growth and metabolic plasticity in these two species. A primary culture of white muscle cells was established and characterized for the ﬁrst time in European sea bass. Using immunoﬂuorescence and transcriptional analyses, this study presents a detailed overview of myogenesis regulation. The ﬁndings showed the coordination of multiple molecular pathways throughout myogenesis, including several molecules previously unknown or poorly described in ﬁsh, and revealed both conserved and species-speciﬁc regulatory dynamics. The panel of stage-speciﬁc markers presented oﬀers a valuable tool for evaluating myogenic development, with relevance for both basic research in muscle biology and applied aquaculture studies. The role of ncRNAs in muscle was also explored in both species. In European sea bass, miR-1/206 and miR-133 family members were enriched in skeletal and cardiac muscle, mainly driving diﬀerentiation programs. The miR-221/222 family was most abundant in brain, while in muscle it showed low basal expression that became induced under proliferative and remodeling conditions. Both members of this family were predicted to target the cell cycle regulator cdkn1bb. In gilthead sea bream, novel lncRNAs were identiﬁed, and their expression varied with developmental stage, muscle injury, fasting-refeeding, and during in vitro diﬀerentiation. Moreover, integrated transcriptomic and correlation analyses suggested that lncRNAs act mainly as miRNA sponges in muscle, ﬁne-tuning miRNA availability and modulating mRNA targets in response to pro-growth signals such as amino acids and Igf-1. In gilthead sea bream juveniles, both diet composition and sustained swimming modulated growth performance, muscle composition, and energy metabolism. A high-protein diet promoted a more anabolic endocrine state, with higher hepatic expression of Gh/Igfs axis components, as well as markers of mitochondrial biogenesis and dynamics, oxidative phosphorylation, and antioxidant defenses. A high-carbohydrate diet shifted energy metabolism toward greater glucose use, particularly in red muscle, but also in white muscle, reﬂecting the plasticity of both muscle types in adapting to carbohydrate-rich conditions. Exercise further enhanced growth, especially under the high-protein feeding, and reduced fat deposition in mesentery, liver, and muscle. It also decreased plasma triglycerides in ﬁsh fed the high-carbohydrate diet and increased markers of mitochondrial eﬃciency and glucose handling in red muscle, pointing to improved substrate allocation during sustained activity. These ﬁndings demonstrate that gilthead sea bream juveniles exhibit strong metabolic ﬂexibility, allowing them to successfully adapt to a diet containing 25% carbohydrates, and highlight the potential of combining exercise with protein-reduced diets to enhance nutrient utilization and support more sustainable farming practices for this species. Overall, the present thesis provides a comprehensive analysis of the molecular, cellular, and physiological regulation of muscle growth and metabolism in two major aquaculture species. By bridging cellular and molecular mechanisms with whole-organism performance, this work advances fundamental knowledge of ﬁsh muscle biology and oﬀers practical insights for optimizing aquaculture production through nutritional strategies and sustained exercise conditions.
Polyethylene glycol 35 as a therapeutic tool in pancreatic ischemia reperfusion injury: preservation of endothelial integrity and function
(Universitat de Barcelona, 2025-11-11) Llaves López, Andrea; Folch i Puy, Emma; Universitat de Barcelona. Departament de Biologia Cel·lular, Fisiologia i Immunologia
[eng] Pancreas transplantation is the only therapeutic option for patients with type 1 diabetes mellitus. Ischemia reperfusion injury (IRI) remains a major challenge in solid organ transplantation, especially in the case of the pancreas. This phenomenon has a direct impact on posttransplant outcomes and can compromise graft survival. The vascular endothelium is critically involved in many steps of tissue damage originated during IRI. In fact, IRI is widely accepted as the primary driver of early graft pancreatitis, primarily through its disturbances of microvascular perfusion, which cause subsequent endothelial dysfunction. One of the earliest structures to be affected is the endothelial glycocalyx, a carbohydrate-rich layer that plays a key role in the maintenance of vascular integrity. Damage to the endothelial glycocalyx has been shown to disrupt vascular homeostasis and exacerbate microcirculatory dysfunction during transplantation. Thus, strategies aimed at mitigating endothelial damage may improve posttransplant outcomes. Polyethylene glycols (PEGs) are non-immunogenic, non-toxic and water-soluble polymers composed of repeating units of ethylene glycol that have been reported to exert beneficial effects in experimental models of tissue injury. In this thesis, we present both in vitro and in vivo evidence that PEG35, a 35 kDa polyethylene glycol, protects the vascular endothelium against IRI. Moreover, we comprehensively describe the alterations in endothelial integrity and damage markers throughout the course of human pancreas transplantation. Our results suggest that PEG35 might be useful as a new therapeutic tool for the management of secondary complications associated with pancreas transplantation, such as graft pancreatitis.
Pdk1+ cells as drivers of stem cell activity and tumorigenesis in the intestine
(Universitat de Barcelona, 2025-11-21) Fortuny Martí, Lidia; Sebastián Muñoz, Carlos; Sebastián Muñoz, David; Universitat de Barcelona. Departament de Biologia Cel·lular, Fisiologia i Immunologia
[eng] Adult stem cells have been proposed as the cells of origin for several types of cancer, including colorectal cancer. Their intrinsic capacity for long-term self-renewal and their ability to survive and regenerate tissue following injury place them as key players in both tissue homeostasis and malignant transformation. While recent studies have shed light on the signalling pathways that regulate stem cell behaviour, the metabolic determinants of stem cell fate — including their quiescence, activation, and lineage commitment — remain poorly understood. Similarly, despite extensive body of work has demonstrated that metabolic reprogramming is a hallmark feature of most cancer cells, it remains unclear whether this metabolic rewiring represents a mere adaptation to the increased energetic and biosynthetic demands of uncontrolled proliferation, or whether it plays an active, instructive role in promoting tumour initiation and expansion. In this context, very little is known about how metabolism contributes to the earliest steps of malignant transformation, particularly in the subset of cells that act as tumour-initiating cells. Exploring these aspects could offer new insights into the biological basis of tumour heterogeneity and reveal novel vulnerabilities for therapeutic intervention. Using the intestinal epithelium as a model system, our laboratory has demonstrated metabolic heterogeneity among both normal epithelial cells and tumour cells in intestinal adenomas. In this context, we identified a rare population of highly glycolytic cells expressing elevated levels of Pyruvate Dehydrogenase Kinase 1 (Pdk1). In this thesis, we have tested the hypothesis that Pdk1+ cells drive stem cell activity and tumorigenesis in the intestine. To do so, we have employed 3D-organoid cultures, mouse models and novel genetically-encoded metabolic reporters in combination with lineage-tracing experiments, advanced imaging techniques and metabolic experiments. Our results indicate that Pdk1⁺ cells are spatially distributed throughout both the crypt and villus compartments, independent of proximity to blood vessels, suggesting that their metabolic phenotype is not dictated by the microenvironment but rather reflects a stable, cell-intrinsic state. Further characterisation revealed that Pdk1⁺ cells belong to the enteroendocrine (EE) lineage and display features of quiescent cells. Remarkably, we demonstrated that these cells possess intrinsic stem cell potential, they contribute to intestinal regeneration in vivo and contribute to organoids growth in vitro. Importantly, this stem cell activity is dependent on Pdk1 function, indicating that glycolytic metabolism plays a decisive role in maintaining their identity and regenerative capacity. Having established their stem cell-like behaviour, we next explored their potential involvement in tumorigenesis. Using inducible in vivo tumour models, we found that Pdk1⁺ cells can initiate intestinal tumours and contribute to tumour growth and maintenance, highlighting their functional relevance beyond initiation. Altogether, our findings suggest that metabolic state is not only a marker of cell identity, but also a functional regulator of both stem cell behaviour and tumour initiation. Glycolysis, mediated by Pdk1, emerges as a key determinant of regenerative potential in the intestinal epithelium and appears to confer tumour-initiating capacity on certain cell populations. From a clinical perspective, this work has important implications. Identifying metabolically distinct cell populations with intrinsic tumour-initiating properties offers a new angle for early detection and stratification of tumour risk. Moreover, targeting metabolic enzymes such as Pdk1 could represent a therapeutic strategy to eliminate cells with stem cell-like and tumour-initiating features before they contribute to tumour progression. Understanding how metabolism governs cell fate at the interface of regeneration and transformation may pave the way for novel approaches to prevent or intercept cancer at its earliest stages.
The role of IL-9 and IL-22 in the effector function of memory CLA+ T lymphocytes in atopic dermatitis
(Universitat de Barcelona, 2025-11-28) García Jiménez, Irene; Santamaria Babí, Luis F.; Universitat de Barcelona. Departament de Biologia Cel·lular, Fisiologia i Immunologia
[eng] Atopic dermatitis is a common chronic, heterogeneous, inflammatory skin condition affecting around 10% of the adult population worldwide, with a huge quality of life impact, particularly in patients with moderate-to-severe disease. The development of atopic dermatitis is complex and comprises a strong genetic predisposition, epidermal barrier dysfunction, cutaneous dysbiosis with particular overgrowth of Staphylococcus aureus (S. aureus), an abnormal immune activation, and intense pruritus, all leading to the appearance of cutaneous lesions. Atopic dermatitis lesions are highly infiltrated by Cutaneous Lymphocyte-associated Antigen (CLA)+ T cells, a subpopulation of effector memory T cells with cutaneous tropism, accounting for over 90% of skin infiltrating T cells. The CLA+ T cells recirculate between skin and blood and reflect the immune abnormalities present in atopic dermatitis lesions. In addition, they can be easily isolated from peripheral blood, thus being a valuable tool to translationally studying the immunological mechanisms taking place in the skin during atopic dermatitis. Our group has previously developed an ex vivo model of moderate-to-severe atopic dermatitis—based on the co-culture of circulating CLA+ memory T cells and autologous epidermal cells, activated with relevant disease-associated triggers such as S. aureus microorganism and house dust mite (HDM) allergen—that has generated valuable translational insights by allowing the functional stratification of patients and linking CLA+ T-cell cytokine production in vitro with the clinical features of patients. Over the past decade, safe and efficacious specific Th2-targeting agents have been approved. However, these therapeutic options lead to clear skin only in up to 30% of patients. Thus, targeting Th2 cytokines does not control disease activity in all patients, implying the involvement of other cytokines beyond IL-4, IL-13 and IL-31 in a considerable number of patients. These precedents let us to investigate the effector function of CLA+ memory T cells, focusing of the production of IL-9 and IL-22 in response to HDM allergen and staphylococcal enterotoxin B (SEB). First, the activation with HDM allergen and staphylococcal enterotoxin B (SEB) induced the secretion of IL-9 in both skin-tropic CLA+ and extracutaneous CLA− memory T cells. However, HDM induced a stronger IL-9 response than SEB. Additionally, total and specific IgE levels positively correlated with IL-9 in vitro response to HDM, but not SEB. Of note, the production of IL-9 was highly variable among patients, allowing patient stratification into IL-9 producers and non-producers. IL-9 responses triggered by HDM were associated with broader Th2, Th17 and Th22 cytokine inflammation compared to SEB, and in both cases were especially higher in initial stages of the disease. Next, the study of IL-22 response to HDM allergen by CLA+ memory T cells defined two groups of patients: those producing IL-22 (IL22P) and those with no IL-22 response (IL22NP). Cutaneous lesions from IL22P patients showed an increased degree of epidermal thickening and higher IL22 mRNA expression in comparison with lesional skin from IL22NP and healthy skin from controls. In addition, the IL22P group showed elevated IgE levels against HDM and SEB in plasma. In summary, this translational doctoral thesis demonstrates that adult patients with moderate-to-severe atopic dermatitis present a heterogeneous production of IL-9 and IL-22 by circulating CLA+ memory T cells in response to disease-associated environmental triggers, which enables the functional stratification of patients into producers and non-producers. This novel translational insight expands the understanding of disease-associated endotypes and may contribute to the selection of more personalized treatment approaches.
Estrategias nutricionales y de actividad física para la mejora productiva de dorada y lubina
(Universitat de Barcelona, 2025-10-14) Sánchez Moya, Albert; Gutiérrez Fruitós, Joaquín; Fernández Borrás, J. (Jaume); Universitat de Barcelona. Departament de Biologia Cel·lular, Fisiologia i Immunologia
[spa] La creciente demanda global de animales acuáticos y la necesidad de reducir la dependencia de las harinas y aceites de pescado (FO) han impulsado la búsqueda de estrategias que optimicen tanto la formulación de piensos como las condiciones de cultivo en acuicultura. En este contexto, la presente tesis doctoral propone diferentes estrategias para el estudio y optimización del cultivo de dos de las especies de mayor interés en la acuicultura mediterránea, la dorada (Sparus aurata) y la lubina (Dicentrarchus labrax). El primer capítulo explora, en dietas para la dorada, la posibilidad de sustituir hasta un 75 % del FO por aceites vegetales tales como palma, colza, soja y lino, administrados tanto de forma aislada como en combinaciones entre ellos. Tras 18 semanas de ensayo, los resultados mostraron que las combinaciones de dos o más aceites vegetales obtienen resultados similares en cuanto a crecimiento. Sin embargo, cuando las dietas son monosustituidas con colza o soja, el crecimiento se ve reducido. En general, las dietas con mayor proporción de ácidos grasos saturados presentaron un mayor crecimiento somático y un aumento de la deposición visceral de lípidos, sugiriendo cierta especiﬁcidad tisular en la acumulación de lípidos según el aceite utilizado. Los parámetros relacionados con los metabolitos plasmáticos, la textura, el color, la actividad digestiva y la expresión de génica de las distintas familias de genes, presentaron algunas diferencias signiﬁcativas entre dietas, aunque sin un patrón claro. En el segundo capítulo se estudia el efecto combinado de la composición de la dieta, —variando la ratio energía:proteína—, y el efecto de la natación moderada sostenida sobre el balance óxido-reductor en la dorada. Para ello, se estudiaron marcadores bioquímicos, actividad enzimática y expresión génica de genes antioxidantes, chaperonas y un proapoptótico. Tras 6 semanas, los resultados mostraron un aumento del ambiente pro-oxidativo en el grupo alimentado con la dieta alta en lípidos y con una actividad natatoria voluntaria. Contrariamente, cuando estos animales eran sometidos a una natación forzada, la mayoría de estos marcadores bioquímicos y de defensa antioxidante mejoraban. Además, se observó cierta especiﬁcidad en la respuesta en función del tejido (hígado, músculo blanco y músculo rojo). La aplicación de un ejercicio sostenido puede ser una práctica útil en alevines de dorada, y particularmente en aquellos alimentados con dietas ricas en energía. En el tercer capítulo, se estudia el efecto de la inclusión de la cisteamina (CSH) como aditivo en piensos de dorada y su efecto sobre el crecimiento y el eje somatotropo. La inclusión de CSH (1,65 g/Kg) mejoró el crecimiento un 16 % tras 9 semanas, y un 27 % (1,65 g/Kg) y un 32 % (3,3 g/Kg) tras 18 semanas. Además, la CSH potenció el eje somatoropo en distintos tejidos, aumentó la ratio anabólica IGF:GH y la sensibilización a estas hormonas mediante la sobreexpresión de sus receptores. Por otro lado, la aplicación de la CSH (200 µM) in vitro en miocitos primarios de dorada, provocó una respuesta a nivel de expresión génica similar a lo observado en el modelo in vivo. La CSH se revela como un prometedor aditivo para la mejora del crecimiento en piensos para alevines de dorada. En el cuarto capítulo, se estudió el uso de la prueba de esfuerzo extenuante para la caracterización de las capacidades natatorias aeróbicas y anaeróbicas en la lubina. En el primer experimento, se observaron diferencias en las capacidades aeróbicas (MMR, AMS) y anaeróbicas (Ucrit) entre distintas líneas comerciales de lubina, sugiriendo un posible compromiso entre rendimiento metabólico y desarrollo somático. En el experimento 2 y 3 se reveló que Ucrit, MMR y AMS presentan repetibilidad intra-día; además, Ucrit también fue estable tras un mes, validando la prueba como herramienta ﬁable para el fenotipado de la lubina. Se determinó que escalones de cinco minutos resultan idóneos para estimar la respuesta anaeróbica, mientras que los de quince minutos capturan mejor la capacidad aeróbica. También se concluyó que los protocolos a velocidad constante, aunque precisos, son poco prácticos debido a su duración excesiva. Por último, la aplicación de una fase de calentamiento mejora la respuesta natatoria.
The role of the Wnt pathway in the regulation of the central nervous system growth
(Universitat de Barcelona, 2025-10-28) Lozano Fernández, Mª Pilar; Marti Gorostiza, Elisa; Universitat de Barcelona. Departament de Biologia Cel·lular, Fisiologia i Immunologia
[eng] The central nervous system (CNS) arises from the neural tube (NT), an embryonic structure formed through highly coordinated morphogenetic processes. Proper growth and differentiation of the NT is essential to generate the diversity of cell types thar compose the mature organ. The spatiotemporal distribution of morphogens such as BMP and Wnt, secreted in the dorsal region of the NT, and Shh, secreted in the ventral region of the NT, play a critical role in establishing dorsoventral (D-V) patterning by enabling neural progenitor cells (NPCs) to acquire specific identities based of their position within NT. These signals regulate the expression of key transcriptional determinants that drive neural cell fate specification via genetic regulatory networks (GRNs). Importantly, these signals also contribute to controlling the proliferative behavior of NPCs, thereby orchestrating tissue growth. Thus, morphogen gradients integrate positional information with proliferative cues to ensure the proper expansion and organization of the embryonic neural tissue. In this thesis, we investigate the role of the Wnt signaling pathway in regulating embryonic NT growth, using the chick embryo as a model system. We characterize the intracellular distribution of β-catenin, the key effector of Wnt signaling, and identify its accumulation at the centrosome during symmetric proliferative progenitor-progenitor divisions in early neurodevelopmental stages. Notably, we show that the centrosome pool of β-catenin corresponds to its stabilized form (S33/S37/Thr41-dephospho-β-catenin), implicating centrosome β-catenin as a downstream target of Wnt pathway activation. Functional assays reveal that increases β-catenin levels promote symmetric PP divisions over neurogenic progenitor-neuron (PN) divisions, acting through both transcriptional and non-transcriptional mechanisms. Furthermore, we identify Nek2 kinase as a centrosome interactor of β-catenin. This interaction appears spatially restricted and enhances the promotion of symmetric PP divisions through synergistic mechanism that requires the C-terminal region of β-catenin. These findings suggest that β-catenin may exert instructive functions at the centrosome, influencing NPC division modes and contributing to the growth of the embryonic NT.
Fisiologia femenina i rendiment esportiu: biomecànica muscular, susceptibilitat a les lesions i capacitat aeròbica
(Universitat de Barcelona, 2025-07-18) Rotllan Serra, Cristina; Viscor Carrasco, Ginés; Esquius de la Zarza, Laura; Universitat de Barcelona. Departament de Biologia Cel·lular, Fisiologia i Immunologia
[cat] En les últimes dècades, la participació de les dones en la pràctica esportiva ha augmentat considerablement, tant a nivell amateur com professional. Aquest increment ha anat acompanyat d’un interès creixent per comprendre les implicacions de la ﬁsiologia femenina en les respostes i adaptacions a l’exercici físic. Històricament, la recerca en ciències de l’esport ha patit un biaix androcèntric, ja que la majoria d’estudis s’han realitzat en població masculina, i els resultats s’han extrapolat a les dones sense tenir en compte les seves particularitats ﬁsiològiques ni les ﬂuctuacions hormonals que es produeixen al llarg de la vida d’una dona. Com a conseqüència, encara no existeixen guies especíﬁques sobre la pràctica d’exercici físic, la prevenció ni la recuperació de lesions adaptades al sexe femení. Fins que les implicacions de les ﬂuctuacions hormonals femenines en el rendiment esportiu no siguin millor compreses i integrades en la recerca i l’entrenament, les dones no podran assolir tot el seu potencial esportiu. La heterogeneïtat dels estudis recents i les diferències en els resultats han posat de manifest les limitacions metodològiques de la recerca sobre els efectes del cicle menstrual en l’exercici físic i el rendiment esportiu. Entre els principals punts febles s’inclouen la variabilitat en les metodologies emprades, els diferents dissenys experimentals i la manca de consens en la deﬁnició de les fases del cicle menstrual, fet que diﬁculta l’obtenció de conclusions sòlides i generalitzables. Amb l’objectiu d’ampliar el coneixement sobre la ﬁsiologia femenina en el rendiment esportiu, inicialment es va dur a terme un estudi observacional en què es va analitzar la resposta muscular al llarg d’una setmana de competició en una cursa ciclista femenina, amb participants que presentaven cicles menstruals naturals. Amb la ﬁnalitat d’analitzar de manera més precisa els efectes de les ﬂuctuacions hormonals al llarg de les diferents fases del cicle menstrual sobre el rendiment esportiu, es va millorar la metodologia per a l’establiment del perﬁl hormonal, incorporant tests d’orina per detectar l’augment de l’hormona luteïnitzant i determinar així el moment de l’ovulació. En aquest context, es va dissenyar un estudi experimental controlat amb dones eumenorreiques, en el qual es van estudiar les respostes cardiorespiratòries a un test submàxim i el control postural en condicions d’hipòxia hipobàrica. Finalment, amb la intenció d’explorar els efectes d’un altre perﬁl hormonal, es va analitzar el rendiment aeròbic i la potència anaeròbica en un grup d’esportistes d’elit que utilitzaven anticonceptius hormonals. Els resultats van mostrar canvis signiﬁcatius de les propietats musculars al llarg d’una setmana de competició ciclista professional, es va observar una disminució del to i la rigidesa muscular, possiblement atribuïble a una major fatiga neuromuscular post-competició i a l’estrès pre-competitiu. En particular, es va detectar un increment en la rigidesa del múscul rectus femoris després de la cursa, la qual cosa podria augmentar la susceptibilitat a lesions per sobrecàrrega en aquest múscul. D’altra banda, l’anàlisi del rendiment aeròbic en condicions d’hipòxia hipobàrica no va mostrar diferències signiﬁcatives entre les fases del cicle menstrual. No obstant això, es van observar canvis estadísticament signiﬁcatius en el volum corrent i en la saturació d’oxigen arterial entre fases del cicle menstrual, probablement deguts a l’efecte estimulant de la progesterona sobre la ventilació i un patró respiratori femení. Finalment, en el grup d’esportistes que utilitzaven anticonceptius hormonals, no es van observar alteracions signiﬁcatives ni en el rendiment aeròbic ni en la potència anaeròbica, la qual cosa suggereix que aquest tipus de perﬁl hormonal no afecta de manera rellevant el rendiment en aquestes variables.
Cognitive decline in Mfn2 KO mice: role of Mfn2 in neuronal plasticity
(Universitat de Barcelona, 2025-04-11) Riqué Pujol, Guillem; Soriano Zaragoza, Francesc X. (Francesc Xavier); Martinez Estrada, Ofelia María; Universitat de Barcelona. Departament de Biologia Cel·lular, Fisiologia i Immunologia
[eng] Mitofusin 2 (Mfn2) is a protein initially characterized as a mitochondrial fusion protein with a bioenergetic function, like Mitofusin 1. Beyond its role in bioenergetics, Mfn2 also acts as an ER-mitochondria tether, which plays a critical role in the regulation of intracellular Ca2+ homeostasis. Both efficient bioenergetics and tightly regulated Ca²+ homeostasis are fundamental for neuronal physiology, as they support synaptic activity, plasticity, and overall neuronal health. Mfn2 is highly expressed in brain, with reduced expression observed during aging and various neurodegenerative diseases, suggesting its involvement in the pathophysiology of different neurodegenerative disorders and cognitive decline. The main goal of this doctoral thesis has been studying the role of Mfn2 in mitochondrial associated membranes (MAMs) and their functions in the brain. To do so, we analyzed the impact of Mfn2 deletion in neurons in a Mfn2 knockout (KO) mouse model and the mechanism through which loss of Mfn2 alters neuronal function. The results of this thesis show that Mfn2 KO mice present a cognitive impairment before neuronal death. This cognitive decline is caused by a loss of synapses due to an overall downregulation of synaptic genes. Through bioinformatic analyses we found the expression of the repressor Rest to correlate with altered synaptic gene expression in Mfn2 KO neurons. The upregulation of this transcription factor in Mfn2 KO neurons is induced by the unfolded protein response (UPR), which is triggered by loss of ER- mitochondria coupling. Therefore, we propose a model in which MAMs regulate synaptic gene expression through REST upon UPR activation, impairing cognition in our Mfn2 KO mice. On the other side, exploring the metabolic adaptations driven by synaptic activity, in this study we found that synaptic activity regulates ER-mitochondria interactions and enhances mitochondrial bioenergetics. Activity-mediated mitochondrial bioenergetics boost required HIF1A stabilization, which induced the COXIV complex component HIGD1A. By elucidating the mechanisms through which Mfn2 influences synaptic function and energy metabolism, this research provides new insights into potential therapeutic targets for neurodegenerative diseases characterized by impaired mitochondrial dynamics and synaptic dysfunction.
Development of 3D spheroids from fish liver cells as in-vitro models to assess the effects of plastics in aquatic systems
(Universitat de Barcelona, 2024-10-18) Wang, Tiantian; Porte Visa, Cinta; Universitat de Barcelona. Departament de Biologia Cel·lular, Fisiologia i Immunologia
[eng] Plastic pollution poses a significant environmental threat, exacerbated by the accumulation of single-use items, especially in aquatic ecosystems. These plastics can degrade into mesoplastics and smaller micro- and nanoplastics, causing harm to aquatic organisms physically and chemically. Despite the new design of biodegradable and compostable plastics (BPs), these materials still require additives that could lead to toxic effects before complete degradation. This thesis investigated the toxicity of BPs, conventional plastics, and mesoplastics collected from Spanish beaches, and explored how plastic photodegradation and composting influence toxic responses in PLHC-1 cells. Additionally, it assessed the cellular and molecular responses of polystyrene micro- and nanoplastics in ZFL cells, focusing on lipid metabolism and antioxidant related pathways. Considering recent trends in environmental toxicity assessment using 3D cell culture techniques to simulate toxicity gradients and emulate cell functions under in vivo conditions, this thesis developed fish liver spheroids (PLHC-1, ZFL) as test models. They were characterized in terms of growth pattern, lipidomic signatures and gene expressions. PLHC-1 spheroids (7-day post seeding) were used to evaluate the toxicity of a mixture of plastic additives and their responses, while ZFL spheroids were characterized in terms of cyp1a gene expression in response to β-naphthoflavone. In addition, the effect of serum supplement (FBS) in the exposure medium was investigated in terms of changes of the lipidome of PLHC-1 spheroids and modulation of the toxicity of plastic additives. Regarding the toxicity of BPs, results showed that BP extracts significantly reduced the cell viability of PLHC-1 cells compared to conventional plastics and this toxicity increased after photodegradation or composting, while extracts from recycled plastics induced EROD activity and micronucleus formation. These results highlight the need to identify and further investigate the compounds causing toxicity in these plastics and to investigate the environmental consequences of uncontrolled dumping before the widespread use of BPs. Meanwhile, beach mesoplastic extracts contained chemicals that induced cytotoxicity and genotoxicity in PLHC-1 cells, with further photodegradation increasing the overall toxicity. These results emphasize the utility of cell-based assays in assessing plastic pollution risks. Regarding the toxicity of MNPs, lipidomic analysis allowed the identification of an upregulation of ceramides (C16, C22, C24:1) induced by NP exposure (but not MPs), which was associated to the activation of ceramide-mediated apoptotic pathways. The study highlighted the potential of lipidomics for elucidating the mechanisms underlying nanoplastic toxicity. Furthermore, the analysis of fish liver spheroids showed a more mature liver cell phenotype than cell monolayers. Liver spheroids were characterized by a decrease in total membrane lipids (particularly PEs and PCs) with a concomitant increase in highly unsaturated phospholipids, indicating significant changes in cell membranes and inter- and extra-cellular interactions, additionally reflected by the higher PC/PE ratio observed in spheroids, which is closer to that of healthy liver. Concerning the responses to toxicants, fish liver cell spheroids exhibited increased cell viability and reduced generation of reactive oxygen species (ROS) production after exposure to a mixture of plastic additives, yet demonstrated increased sensitivity in lipidomic responses and detoxification gene expression compared to cells cultured in monolayers. Additionally, the presence of FBS in the exposure medium has been shown to significantly affect lipidomic responses induced by plastic additive mixture. These results further support the use of fish cell spheroids as more realistic in vitro approaches for aquatic toxicity studies, but they also emphasize the importance of optimizing and standardizing culture conditions before its wide application in toxicological studies. Overall, the current thesis describes the toxicity and molecular and subcellular mechanisms of action of plastic-derived toxicants using in vitro cell systems, emphasizes the importance of comprehensive environmental risk assessments associated with new emerging plastics, and highlights the key role of fish liver cell spheroids and lipidomics in advancing this field.
Regulación del metabolismo neuronal. Papel de la actividad sináptica e implicación en la fisiopatología del síndrome de Leigh
(Universitat de Barcelona, 2024-07-19) Tena Morraja, Paula; Soriano Zaragoza, Francesc X. (Francesc Xavier); Reina del Pozo, Manuel; Universitat de Barcelona. Departament de Biologia Cel·lular, Fisiologia i Immunologia
[spa] La mayor parte del gasto energético en el cerebro se utiliza en procesos relacionados con la transmisión sináptica. La actividad sináptica provoca adaptaciones estructurales y funcionales en las que se potencian las sinapsis, dando lugar a un aumento de la fuerza de estas sinapsis y, en consecuencia, también a un aumento del gasto energético. Por lo tanto, las neuronas con actividad sináptica persistente deben experimentar una remodelación bioenergética de larga duración para soportar una mayor demanda energética relacionada con la actividad. Los resultados de esta tesis muestran que la actividad sináptica aumenta la bioenergética mitocondrial más allá de la duración de la actividad sináptica mediante la regulación del metabolismo neuronal del hierro a nivel transcripcional con la consiguiente mejora de la absorción celular y mitocondrial de hierro. La importación a los mitocondríos es necesaria para la síntesis de hemo y clúster hierro-azufre (ISC), que son cruciales para la generación de energía en los mitocondríos, ya que son necesarios para el funcionamiento de los complejos de cadenas respiratorias. Mecánicamente, se encontró que CREB induce la expresión del transportador de hierro mitocondrial Mfrn1, que es necesario para el aumento bioenergético mediada por la actividad. Por el contrario, se encontró que el metabolismo desregulado del hierro podría participar en la fisiopatología del síndrome de Leigh. El síndrome de Leigh (LS) es una enfermedad mitocondrial grave y progresiva caracterizada por lesiones cerebrales simétricas, déficits motores y respiratorios y muerte a una edad temprana. El análisis de muestras de tronco cerebral de ratones Ndufs4 KO presintomáticos y sintomáticos, que recapitula la patología humana del síndrome de Leigh, reveló alteraciones en el metabolismo del hierro y la expresión génica de la antiferroptosis que resulta en un aumento de la per oxidación lipídica, un sello distintivo de la ferroptosis. Para identificar nuevas vías celulares subyacentes a la patogénesis del LS, se llevó a cabo un análisis de los desencadenantes de señalización celular modificados por el cannabidiol (CBD), cuya administración alarga la vida útil y mejora los signos clínicos en ratones Ndufs4 KO. El tratamiento con CBD disminuye la liberación de lactato de manera dependiente del AKT reduciendo la absorción de glucosa y normalizando la relación de expresión LDHA/LDHB, que se altera en ratones Ndufs4 KO. Estos hallazgos avanzan en nuestra comprensión de las adaptaciones metabólicas tanto en condiciones fisiológicas como patológicas, ofreciendo información para posibles intervenciones terapéuticas.
Nutrition and gut microbiota modulation as tools for regulating fat accumulation in aquaculture fish
(Universitat de Barcelona, 2024-07-12) Ruiz Hernández, Alberto; Gisbert Casas, Enric; Adree, Karl; Universitat de Barcelona. Departament de Biologia Cel·lular, Fisiologia i Immunologia
[eng] The sustainability concerns, increasing cost, and insufficient availability of fish oil associated to aquaculture growth, have led to the search of sustainable alternatives in aquafeeds. However, fish oil replacement increases the levels of body fat deposition. An excessive fat accumulation in digestive tissues may cause deregulations in nutrient digestion and absorption, reduced feed efficiency and growth, as well as negatively affect the organoleptic qualities of the fillet. Hence, it is imperative to explore complementary strategies to balance fat accumulation when diminishing fish oil content in aquafeeds. This thesis proposes two strategies to reduce the levels of fat accumulation in fish: the supplementation of diets with additives with digestive and hypolipidemic properties, and the modulation of the gut microbial communities through an intestinal microbiota transplant (IMT). To test these strategies, gilthead seabream (Sparus aurata), the most important farmed species in the Mediterranean, was used as a biological model. Under this context, the effect on fat accumulation, and fish health and condition, of three different additives was evaluated: i) a blend of bile salts containing sodium cholate, sodium deoxycholate, and sodium taurocholate at dietary inclusion levels of 0.06% and 0.12%; ii) a combination of capsicum, black pepper, and ginger oleoresins, and cinnamaldehyde (SPICY additive) at 0.1% and 0.15% in the diet; and iii) a combination of turmeric, capsicum, black pepper, and ginger oleoresins (SO additive) at 0.2% in the diet. Results revealed that the tested blend of bile salts at 0.06% in the diet and the SPICY additive at 0.1% and 0.15% promoted somatic growth. Additionally, the SPICY additive reduced the values of feed conversion ratio at both inclusion levels. Furthermore, the blend of bile salts at 0.12% increased lipid apparent digestibility, which was attributed to the higher activity of the bile salt-activate lipase and increased secretion of bile salts into the intestine. A higher bile salt-activate lipase activity was also observed for the SPICY and SO additives at all the inclusion levels tested. Dietary supplementation with the tested additives demonstrated a promising reduction in the levels of fat accumulation in the visceral cavity and digestive organs (liver and intestine) without compromising the proximate composition of the fillet. Moreover, the three additives modulated the local immune response in the intestine, as well as the gut bacterial composition in gilthead seabream without affecting the diversity and structure. Regarding the IMT, two marine carnivorous fish species that thrive in different environmental conditions were selected, Atlantic salmon (Salmo salar) as microbiota's donor, and gilthead seabream as recipient. This approach was designed to develop an IMT protocol and to study the dynamics of the gut bacterial communities after the IMT and under the influence of different dietary treatments. The purpose was further applying this protocol in the gilthead seabreams submitted to the above-mentioned nutritional assays to test the reduction in fat accumulation levels. However, this assay was not conducted since the microbial modulation induced by the tested additives was not robust enough for expecting it to have a determinant role in fat accumulation. Nonetheless, the results of the inter-specific IMT provided insight to the paramount role of the diet in shaping the gut microbial communities after an IMT, modulating richness, diversity, structure, and composition over time. Unfortunately, many experimental factors, such as the high number of individuals typically managed in aquaculture, suggest that implementation of IMTs as a reliable strategy in the sector are yet remote. Results from the current thesis indicated that feed additives are a safe strategy to improve the health and condition in farmed fish as well as modulate body fat accumulation without affecting the nutritional quality of the fillet.
Respuestas cardiorrespiratorias, metabólicas, mecánicas y de estrés oxidativo, agudas y crónicas, en la danza aeróbica realizada sobre una plataforma de disipación de aire en personas adultas jóvenes y mayores
(Universitat de Barcelona, 2023-12-15) Moreira Reis, Alessandra; Garnacho Castaño, Manuel Vicente; Alva Bocanegra, Norma V. (Norma Violeta); Universitat de Barcelona. Departament de Biologia Cel·lular, Fisiologia i Immunologia
[spa] INTRODUCCIÓN: La danza aeróbica (AD) es una modalidad de ejercicio de las más practicadas en el mundo que ha demostrado múltiples beneficios en la salud de aquellas personas que la practican, especialmente en la mejora del fitness cardiorrespiratorio y muscular. La AD ha evolucionado a diversas modalidades de ejercicio utilizando múltiples superficies y materiales. Una de estas modalidades de AD es realizada sobre una plataforma de disipación de aire (ADP). Esta tesis doctoral examinó las respuestas agudas y crónicas, cardiorrespiratorias, metabólicas, mecánicas, musculares y el estrés oxidativo realizando una AD sobre una ADP ya que, hasta la fecha, este conocimiento era inexistente. Para ello, se establecieron 3 objetivos principales de acuerdo con los tres estudios planteados: 1º Comparar las respuestas agudas cardiorrespiratorias y metabólicas, la fatiga muscular y la percepción subjetiva del esfuerzo (RPE) entre una ADP y una superficie dura durante una sesión de AD grabada en video (primer estudio) 2º Comparar el componente lento del consumo de oxígeno (V̇ O2, V̇ O2sc), la eficiencia ventilatoria, el lactato sanguíneo y la RPE entre una sesión de AD en una ADP y una prueba de tapiz rodante a una intensidad de primer umbral ventilatorio (VT1) (segundo estudio). 3º Valorar los efectos de un programa de entrenamiento supervisado de 12 semanas de AD sobre una ADP en la composición corporal, el fitness cardiorrespiratorio y muscular y el estrés oxidativo en personas mayores obesas y con sobrepeso (tercer estudio). METODOLOGÍA: La presente tesis doctoral constó de 3 estudios experimentales. En el estudio 1 (primer objetivo), 25 mujeres adultas sanas (edad 23,3 ± 2,5 años) fueron reclutadas y realizaron aleatoriamente dos sesiones idénticas de AD: ADP vs. superficie rígida. Las variables ventilatorias, la frecuencia cardíaca, las concentraciones de lactato en sangre (fatiga metabólica), la altura de vuelo y la potencia en el salto con contramovimiento (fatiga muscular) fueron comparadas entre ambas sesiones de AD sobre distintas superficies. En el estudio 2 (segundo objetivo), 17 mujeres adultas (edad 23,5 ± 2,2 años) fueron seleccionadas y efectuaron 3 sesiones de evaluación. En la primera sesión, se completó una prueba incremental hasta el agotamiento en tapiz rodante para determinar las respuestas cardiorrespiratorias pico y a VT1. En la segunda sesión, que tuvo lugar una semana después, las participantes fueron asignadas aleatoriamente a la prueba de AD en un ADP o al test en tapiz rodante a una velocidad a carga constante correspondiente a la intensidad de VT1. En la sesión 3, realizada una semana después, las participantes realizaron el protocolo pendiente que no se realizó en la sesión 2, en las mismas condiciones. El V̇ O2sc, la eficiencia ventilatoria (pendiente de la VE·VCO −1), las concentraciones de lactato en sangre y la RPE fueron comparadas entre ambas sesiones. En el tercer estudio, 32 personas mayores (67,1 ± 3,6 años) fueron asignadas a 3 grupos en función de su índice de masa corporal (IMC): Grupos de normopeso, sobrepeso y obesidad. Se comparó los efectos de un programa de entrenamiento de 12 semanas de AD sobre una ADP entre los tres grupos experimentales. Las variables principales comparadas en este estudio fueron el peso, la grasa corporal (%), la masa libre de grasa, la masa magra, el V̇ O2 pico (fitness cardiorrespiratorio), la peroxidación lipídica (estrés oxidativo), la altura de vuelo y la potencia de las extremidades inferiores en un salto con contramovimiento, la fuerza muscular de los brazos, la agilidad y el equilibrio (fitness muscular). RESULTADOS: En el primer estudio, se observó un incremento significativo de la frecuencia cardíaca (p < 0,05) y en los niveles de lactato (p < 0,01) en la ADP en comparación con la superficie rígida. Sin embargo, la fatiga muscular y la RPE fueron similares entre ambas superficies (p < 0,05). En el segundo estudio, no se encontraron diferencias significativas en el V̇ O2sc, la eficiencia ventilatoria y la RPE (p > 0,05) entre la prueba de tapiz rodante y la sesión de AD en una ADP. Sin embargo, se detectaron concentraciones más altas de lactato en sangre en la sesión de AD en una ADP a los 10 min (p = 0,003) y 20 min (p < 0,001) en comparación con la prueba en tapiz rodante. En el tercer estudio, se observó una reducción del peso en las personas mayores con sobrepeso (p = 0,016) y obesas (p < 0,001); también se redujeron las concentraciones de malondialdehído en plasma en todos los grupos experimentales (p < 0,05). Los grupos experimentales con sobrepeso y obesidad mejoraron significativamente su VO2 pico (p < 0,01). El grupo normopeso y el de obe- sidad mejoraron la producción de potencia de las extremidades inferiores (p < 0,05). CONCLUSIÓN: En el primer estudio, una sesión de AD sobre un ADP indujo una mayor frecuencia cardiaca (FC), ventilación pulmonar (VE), tasa de intercambio respiratorio (RER), equivalente respiratorio de oxígeno (VE·VO −1), equivalente de dióxido de carbono (VE·VCO −1) y concentraciones de lactato en sangre, produciendo una mayor respuesta cardiorrespiratoria y metabólica en comparación con una superficie más dura, sin inducir mayor fatiga muscular y RPE. En el segundo estudio, la sesión de AD en una ADP produjo valores similares de V̇ O2sc, pendiente de la VE·VO −1 y de RPE en comparación con la prueba en tapiz rodante a una intensidad a carga constante de VT1 en mujeres adultas jóvenes sanas, a pesar de que la sesión de AD en una ADP indujo mayores concentraciones de lactato en sangre que la prueba en tapiz rodante. En el tercer estudio, un programa de entrenamiento de 12 semanas de AD en una ADP puede ser una estrategia efectiva para regular adaptaciones cardiorrespiratorias y musculares y mejorar los efectos del estrés oxidativo en personas mayores obesas y con sobrepeso.
Development of 3D in vitro platforms for the study of muscle function and axonal growth and regeneration
(Universitat de Barcelona, 2023-10-27) Wells Cembrano, Karen Isabel; Río Fernández, José Antonio del; Universitat de Barcelona. Departament de Biologia Cel·lular, Fisiologia i Immunologia
[eng] The use of in vitro models in biomedical research offers an invaluable tool for exploring biological processes of health and disease. While animal models remain highly informative and necessary to ensure clinical research safety, laboratory models are essential for reducing animal experimentation and enabling fine manipulation and high- throughput analysis. However, there is a concern that traditional in vitro models, based on two-dimensional cultures of mammalian cells and relying on primary culture and non- human cell lines, could lead to inaccurate or misleading results, ultimately jeopardizing the success of novel therapies in clinical trials. Thankfully, exponential advancements in biomedical and biotechnological research have facilitated the creation of increasingly complex and biomimetic models, leading to a paradigm shift in this field. The first pillar of this shift is the use of three-dimensional culture to recapitulate the extracellular matrix environment, through the use of scaffold-based models, lab-on chip devices, and organotypic slice cultures. The second pillar involves transitioning to human cell-derived models to achieve clinically relevant outcomes. The combination of these models with optical techniques such as optogenetics and calcium imaging enables precise manipulation and analysis of cell activity, opening new possibilities for relevant biomedical research. In this work, our general objective was to develop and utilize 3D in vitro models of excitable tissues for two main purposes. On the one hand, our objective was to develop a 3D platform for studying muscle physiology, utilizing human immortalized myoblasts as a clinically relevant cell source. Our next focus was to use this system to model the autoimmune neuromuscular disease myasthenia gravis (MG). On the other hand, we were interested in studying the effect of neuronal activity on axonal regeneration in central nervous system (CNS) neurons. For this, we sought to develop a 3D axotomy platform which could complement previous results obtained in 2D culture. In parallel, we wished to investigate the effect of neuronal activity in a previously established 3D model, namely entorhino-hippocampal organotypic slice cultures (OSCs). In both models, we were interested in applying optogenetic stimulation to Channelrhodopsin-2 (ChR2)-modified neurons. In the first chapter, we show the validation of in vitro culture systems for human myoblast differentiation. First, we used 2D cultures with hydrogel overlays as an approach for comparing differentiation and functionality of human myoblast cell lines, as well as for evaluating MG serum antibody binding to in vitro endplates. Later, we developed an anchored 3D in vitro platform for the culture of aligned, differentiated, and contractile human muscle, which responded to electrical, chemical, and optogenetic stimulation. Muscle function was analyzed using motion analysis algorithms or by calcium imaging. Treatment with MG patient serum recapitulated endplate destruction observed in vivo, and some functional effects were observed, although further experiments are needed. In the second chapter, we developed a 3D axotomy platform using embryonic cortical explants cultured in collagen gels. This model provided a reliable source of axonal projections for axotomy, in contrast to stem-cell derived cultures, which were also evaluated as potential neuronal sources. Using this axotomy platform, we observed that optogenetic stimulation was detrimental to axonal regeneration. In the third chapter, we used entorhino-hippocampal OSCs for a similar purpose, benefitting from our research group's experience in this area. This model recapitulates key neurobiological processes and forms near-in vivo glial scars after lesion; making it a biologically relevant model of CNS development and lesion. We applied optogenetic stimulation on developing connections and axotomized cultures, to study the effect of neuronal activity on axonal pathfinding and regeneration after lesion. We observed that increased neuronal activity resulted in loss of target specificity in developing axons, which could affect regenerative potential in lesioned neurons. In line with the second chapter, optical stimulation was also found to impair axonal regeneration in this model.
The role of CLA+T lymphocytes in the development of Atopic Dermatitis
(Universitat de Barcelona, 2023-10-26) Sans de San Nicolàs, Lídia; Santamaria Babí, Luis F.; Universitat de Barcelona. Departament de Biologia Cel·lular, Fisiologia i Immunologia
[eng] Atopic dermatitis is the most common chronic immune-mediated inflammatory skin disease affecting up to 20% of children and 10% of adults. The complex pathophysiology of the disease comprises genetic susceptibility, epidermal barrier dysfunction, cutaneous dysbiosis with abundance of S. aureus, an abnormal cutaneous immune system activation with a core Th2 response and pruritus. Cutaneous Lymphocyte-associated Antigen (CLA)+ T cells represent the subset of memory T cells that belong to the cutaneous immune system. CLA+ T cells recirculate between blood and skin through the thoracic duct, specifically respond to skin-related antigens, and represent over 90% of T cells infiltrating the skin. Therefore, CLA+ T cells constitute peripheral cellular biomarkers and, because they can be found in general circulation, they are a source of translational information on the immunological mechanisms taking place in the skin during disease. We have studied atopic dermatitis in the context of the cutaneous immune response through the effector function of CLA+ T cells. For this, we have stablished a novel ex vivo model of adult non- treated moderate-to-severe atopic dermatitis based on circulating CLA+ T cells cocultured with a suspension of autologous epidermal cells obtained from lesional biopsies in the same patients. Then, we have studied the T-cell effector function in response to relevant disease triggers such as S. aureus microorganism and house dust mite (HDM) allergen, as well as the association between cytokine response to the stimuli and patient’s clinical data. First, the study of IL-13 response to S. aureus enterotoxin B (SEB) by CLA+ T cells defined two groups of patients, Th2 high and Th2 low, within a clinically homogeneous population. In the Th2 high group, in contrast to the Th2 low group, the IL-13 response positively correlated with severity, in terms of EASI score, and levels of CCL17, sIL-2R and S. aureus-specific IgE in plasma. Additionally, in this group the IL-13 response directly correlated with CCL26 and indirectly correlated with LCN2 mRNA expression in cutaneous lesions. Conversely, in the Th2 low group, the CLA+ T-cell response to SEB skewed towards Th17, Th22 and Th1. Next, the role of the neuroimmune cytokine IL-31 was examined in our model by studying the CLA+ T-cell response to HDM, and a bimodal (present or absent) IL-31 response in relation with the HDM-specific IgE levels in plasma was observed. Patients producing IL-31 by HDM-activated CLA+ T cells showed increased HDM-specific and total IgE levels and reported an increased inflammatory profile compared to patients with no IL-31 response. Interestingly, the IL-31 response directly correlated with patient’s pruritus intensity and plasma levels of CCL27 and periostin. Of note, patients with no IL-31 response reported raised presence of HDM- specific and total IgE levels compared to control subjects, suggesting that the degree of IgE sensitization to HDM in this group was not enough for inducing IL-31 response. In summary, this novel ex vivo model of adult non-treated moderate-to-severe atopic dermatitis has allowed to functionally identify Th2 high and Th2 low responders from a clinically homogeneous population based on the SEB-CLA+-IL-13 axis, as well as stratifying patients into IL-31 producers and non-producers in relation with the degree of IgE sensitization to HDM by analyzing the CLA+ T-cell response to HDM and its association with clinical features. Altogether, this translational work expands the understanding of the heterogeneous inflammatory response of the disease and may contribute to improving the effectiveness of targeted therapies.
Anàlisi de les funcions de la proteïna NCAM2 en desenvolupament i plasticitat neuronal
(Universitat de Barcelona, 2023-07-07) Ortega Gascó, Alba; Soriano García, Eduardo; Pujadas Puigdomènech, Lluís; Universitat de Barcelona. Departament de Biologia Cel·lular, Fisiologia i Immunologia
[eng] The Central Nervous System (NS) is a fascinating and complex structure formed by millions of interconnected neurons that facilitate cognitive tasks such as memory and learning. The development and maintenance of this system rely on genetic programs that regulate the spatial and temporal expression of numerous genes. Cell adhesion molecules (CAMs) are key proteins in the development and maintenance of the adult CNS participating in the architecture of the system and in signal transduction. These proteins exhibit a wide range of structural and functional diversity, allowing them to regulate different processes, including the renewal and proliferation of neural stem cells (NSCs), neuronal migration and differentiation, as well as the integration of neurons into functional circuits. The present thesis investigates the role of neuronal cell adhesion molecule 2 (NCAM2) in neuronal development and the regulation of adult neuronal plasticity. NCAM2, also known as OCAM and RNCAM, is a glycoprotein located in the cell membrane from the NCAM family. The NCAM family comprises two members, NCAM1 and NCAM2, which are generated by genomic duplication. Alternative splicing of Ncam2 produces two isoforms of the protein: NCAM2.1 and NCAM2.2. Both isoforms share an extracellular domain composed of five immunoglobulin-like modules (IgI-IgV) and two fibronectin type III modules (FnIII1-2). Through the extracellular domain, they establish trans homophilic interactions that are responsible for a large part of their functions. The intracellular domain varies depending on the isoform, while NCAM2.1 has a transmembrane domain and a cytoplasmic tail, NCAM2.2 is attached to the membrane via a glycosylphosphatidylinositol (GPI) anchor. The different structure of the isoforms diversifies their cellular localization, interactions, and biological functions. The implications of NCAM2 have been extensively studied in the olfactory bulb, where it participates in neurite growth, axo-dendritic compartmentalization, and selective axon fasciculation. However, its role outside of this region largely unexplored to date. The main objective of this thesis has been to determine the functions of the NCAM2 protein in the cortex and hippocampus. It has been observed that NCAM2 controls the process of neuronal polarization and morphogenesis. Silencing the protein during neuronal polarization events leads to aberrant dendritic phenotypes and the formation of shorter and more branched axons. The mechanisms through which NCAM2 mediates its functions in neuronal morphogenesis include interaction with cytoskeletal proteins (actin, tubulin, and neurofilaments), cytoskeleton-associated proteins (MAP1B, MAP2, or CAPZ), and other intercellular effectors (CAMKII or 14-3-3). Moreover, the effect of the NCAM2 protein on the regulation of adult neural stem cells (NSCs) and synaptic plasticity in the hippocampus has been analyzed. The results in the context of neurogenesis revealed that regulated levels of Ncam2 are necessary for the proper activation of quiescent NSCs, their division, and neuronal differentiation. Increased levels of NCAM2 lead to cell arrest at a neural progenitor stage and hinder the formation of new neurons. In synaptic plasticity, our data show that the protein is necessary for synaptic formation and its proper maintenance during adulthood. Silencing NCAM2 leads to a constriction and a reduction in density of spines. Conversely, upregulated levels may promote contact stabilization by increasing the size of dendritic spines. In summary, the results obtained in this thesis highlight the relevance of NCAM2 in neuronal development and plasticity, thus reinforcing the crucial role of CAMs in the proper functioning of the CNS. The NCAM2 gene has been associated with neurodevelopmental disorders or neurodegenerative diseases such as autism, Down syndrome, or Alzheimer's disease. The evidence provided in the thesis, could enhance our understanding of the neurogenic and synaptic deficits that occur in these pathologies and opening new promising avenues for future research.
Evaluation of tau seeding, spreading, and cytotoxicity using in vitro and in vivo models of tau pathology
(Universitat de Barcelona, 2023-02-01) Sala Jarque, Julia; Río Fernández, José Antonio del; Universitat de Barcelona. Departament de Biologia Cel·lular, Fisiologia i Immunologia
[eng] Abnormal folding, hyperphosphorylation, aggregation, and subsequent deposition of the microtubule-associated protein tau, is the hallmark of a group of devastating neurodegenerative diseases known as tauopathies, including Alzheimer’s disease. One striking aspect of Alzheimer’s disease is that the presence of tau-related lesions in the brain occurs in a systematic, sequential manner, maintaining a predictable distribution pattern between synaptically connected neurons that varies very little among individuals. Increasing evidence suggests that the progression of tau pathology in the diseased brain behaves like a prion. The “prion-like” hypothesis suggests that “pathological” tau engages in self-seeded fibrillization and propagates through cell-to-cell spreading. However, despite intensive research, the cellular and molecular mechanisms involved and the pathological processes linking neuronal death and tau dysfunction are not fully understood. Although Alzheimer’s disease was first described in 1906 and has an increasing prevalence in the aging population, there is currently no treatment to prevent or cure this or any other tauopathy. Progress limitations are partially explained by the lack of appropriate models to study human tauopathies. Indeed, tau-targeting therapies that had demonstrated an improvement in the pathology in several models (i.e., in vitro and in vivo) were unable to produce positive results in clinical trials. These incongruences could be related to the fact that most experimental models rely on the over-expression of mutated tau species and the use of recombinant tau fibrils, which do not reproduce the sporadic nature of most human tauopathies. Through this doctoral thesis, we examined various aspects of tau pathology, including tau seeding, spreading, and cytotoxicity, by implementing experimental approaches that better mimicked sporadic tauopathies. Nevertheless, at the beginning of this work, the reliability of the only commercially available cell line designed to be used as a cell-based assay to detect and report proteopathic seeding in biological samples was questioned in one publication. Given that this cell-based assay was central to the validation of the samples employed in this thesis, we conducted a thorough characterization of this cell line, known as the Tau biosensor cell line, and its ability to produce fluorescent tau aggregates. Our results show that the Tau biosensor cell line is a reliable cell-based assay that forms amyloid-like inclusions upon the addition of extracellular seed- competent tau species. Next, we investigated the impact of extracellular seed-competent tau on the neuronal activity of primary cortical cultures derived from wild-type mice. We established an experimental setup that included microfluidic devices and calcium imaging, which allowed us to specifically treat the axons with tau, as well as monitor changes in spontaneous neural activity in a time-course manner. Although we demonstrate that cortical neurons in our microfluidic platforms display typical patterns of neuronal network activity, we do not detect changes after treating them with seed-competent tau. We then investigated how the presence of various extracellular seed-competent tau may affect neural metabolism (i.e., as an indicator of cellular viability) also in primary cortical cultures. Nevertheless, similar to what we observed in the analysis of neuronal activity, within the course of 10 days, no differences between tau-treated and untreated cells are found. Finally, recent evidence suggests that the cellular prion protein is involved in the pathology of other prion-like proteins, such as amyloid-β and α-synuclein; however, much less is known about its role in tauopathies. We inoculated human Alzheimer’s disease-derived samples into the hippocampus of transgenic mouse models with different expressions of the cellular prion protein. We found that all mice, regardless of their genotype, have similar profiles of tau-related lesions in their brains. Hence, our findings indicate that the cellular prion protein does not have a paramount role in the onset, seeding, or spreading of tau pathology. Taken together, our work underscores the need for more pathologically relevant models to study certain aspects of sporadic human tauopathies, which could lead to the development of effective therapeutic strategies.
Implicación del Sistema de Melanocortinas en la Reproducción del Pez Cebra
(Universitat de Barcelona, 2023-01-20) Navarro Rojas, Sandra Ximena; Cerdà-Reverter, José Miguel; Rocha, Ana Maria dos Santos; Universitat de Barcelona. Departament de Biologia Cel·lular, Fisiologia i Immunologia
[spa] El sistema de melanocortinas es probablemente uno de los sistemas hormonales más complejos involucrados en la regulación de la ingesta de alimentos y la homeostasis energética. Las melanocortinas son péptidos derivados de un complejo precursor peptídico denominado proopiomelanocortina (POMC). Su procesado genera diversos péptidos de melanocortina entre los que se encuentran las hormonas estimulantes de melanocitos (MSHs) y la hormona adrenocorticotrópica (ACTH). Las funciones de las melanocortinas se median a través de 5 receptores diferentes (MC1R-MC5R). Tanto ACTH como MSH activan los MCRs con diferente afinidad, pero MC2R solo es activado por ACTH. La señalización de melanocortinas también puede ser modulada por dos antagonistas endógenos, la proteína de señalización de agutí (ASIP) y la proteína relacionada con agutí (AGRP) que compiten con los péptidos de melanocortina al unirse a MCR. Entre los MCR, el MC4R está involucrado en el control de la ingesta y la reproducción. Las mutaciones negativas dominantes de Mc4r se han relacionado con un tamaño corporal más grande y un inicio tardío de la pubertad en Xiphophorus. Investigaciones en el pez cebra demuestran que la supresión de la actividad central del Mc4r es esencial para el crecimiento larvario. De forma similar, la supresión del Mc4r resulta en una disminución de la expresión de fshb y lhb. Esto sugiere que, en los peces, el Mc4r participa no solo en el control de la ingesta de alimentos, sino que también en la reproducción. Experimentos desarrollados en nuestro laboratorio, han demostrado que Asip1 funciona como un antagonista endógeno tanto de Mc1R como de Mc4R. Además, la generación de una cepa transgénica de pez cebra que sobreexpresa asip1 de pez dorado (asip1-Tg), demostró la participación del sistema de melanocortinas en la regulación del patrón pigmentario dorsoventral y estimulación del crecimiento. En consecuencia, este modelo transgénico supone una excelente oportunidad para estudiar la relación entre el sistema de melanocortinas, el crecimiento y la reproducción. Los resultados presentados en esta tesis demuestran que la disminución de la actividad del sistema de melanocortina inducida por la sobreexpresión de asip1 retrasa significativamente el crecimiento temprano de los animales transgénicos, exhibiendo un crecimiento reducido hasta completar el desarrollo gonadal. Además, no tuvo efecto fenotípico sobre la pubertad y mermó el potencial reproductivo en comparación con los peces WT, lo que se refleja en una menor calidad del huevo y diámetro del vitelo, un retraso en el tiempo de eclosión y el crecimiento larvario. La caracterización de los niveles de expresión relativa de los receptores de melanocortinas, proteínas accesorias, agonistas y antagonistas en gónadas de pez cebra así como la expresión de mc1r y mc4r en oocitos previtelogénicos y vitelogénicos y en células germinales masculinas, respalda el papel del sistema de melanocortinas en la fisiología gonadal. Además, nuestros experimentos in vitro sugieren que los pétidos de melanocortinas pueden modular la síntesis de esteroides gonadales. Finalmente, la expresión de mc4r en gonadotropos y la modulación de la síntesis de gonadotropinas por los péptidos melanocortina sugiere un posible rol paracrino en la síntesis y liberación de hormonas reproductivas desde la adenohipófisis.
Secretory and synaptic proteins in brain and cerebrospinal fluid in normal and Alzheimer’s disease animal models
(Universitat de Barcelona, 2022-11-25) Sánchez Domínguez, Irene; Aguado Tomàs, Fernando; Universitat de Barcelona. Departament de Biologia Cel·lular, Fisiologia i Immunologia
[eng] The central nervous system represents a huge network architecture where intercellular communication governs and coordinates the proper function of the brain. Regulated secretion of neuropeptides and growth factors from dense-core vesicles (DCV) exert key roles in neural circuit development, function, and plasticity. However, these organelles have received significantly less attention than synaptic vesicles. Here, we investigated different aspects of DCV in the healthy and pathological brain by analyzing their molecular components. First, we studied the developmental acquisition of peptidergic regulated secretion in cortical neurons in situ and in vitro. We found that the competence for regulated secretion is determined sequentially for different DCV subsets. Importantly, the association between acquisition of regulated competence and levels and location of synaptotagmin-IV pointed out this vesicular component as a key mechanism in the developmental maturation of DCV secretion. Second, due to the significance of astrocytes in the neural circuitry physiology, we developed new adeno-associated vectors to express neuropeptides tagged with fluorescent proteins specifically in neurons, but also in astrocytes to evaluate glial peptidergic secretion into neuronal networks. Neuronal stimulation triggered a robust astrocyte DCV secretion in a calcium-dependent manner, presumably by NMDA glutamate receptors, although in a lesser extent than classical neuronal secretion. Last, alterations in synaptic function underlie early pathogenic events in Alzheimer’s disease (AD) which precedes symptoms onset but initiate a remarkably impairment of cognitive function. In line with recent research directed toward the identification of early biomarkers of synaptic degeneration, we studied age- and sex- dependent protein changes in the brain and cerebrospinal fluid (CSF) of TgF344-AD rats, evidencing alterations in DCV and other synaptic components even prior to Aβ deposition, markedly in males. The CSF proteome revealed that this transgenic model undergoes alterations in biological pathways involved in glia-neuron communication, neuronal assembly, memory or neuroinflammation. Hence, we consider TgF344-AD rats as a suitable model for the search of early fluid biomarkers that sustain the development of new therapeutic strategies for pre-clinical AD trials. Altogether, this dissertation shows new evidence of neuronal and glial communication in the physiology and neuropathology of the cerebral cortex.
Effects of the pharmacological activation of Liver X Receptors in the tumor microenvironment
(2022-07-22) Font Díaz, Joan
[eng] Nuclear receptors are a superfamily of ligand-dependent transcription factors that are involved in numerous biological processes in homeostasis and disease. Liver X receptors (LXRs) are members of the nuclear receptors family that are regulated by oxidized forms of cholesterol (oxysterols) and other byproducts of cholesterol metabolism and biosynthesis. In addition, there are synthetic agonists, such as T0901317 (T1317), which have higher affinity and stability. LXRs are key factors in the regulation of lipid homeostasis and in the modulation of inflammation. There are two LXRs isoforms, LXRα (NR1H3) and LXRβ (NR1H2), encoded by two different genes and with differential tissue distribution. In order to bind to the DNA and regulate the expression of target genes, LXRs form heterodimers with the retinoid X receptors (RXRs), another member of the nuclear receptors family. LXRs can also repress the expression of genes, for example interacting with co-repressor complexes through transrepression. In the last decade, there has been a growing interest in LXRs as therapeutic targets against cancer. Activation of LXRs with high doses of synthetic agonists exerts direct antiproliferative, cytostatic and pro-apoptotic effects in vitro in many cancer cell lines. In addition, pharmacological activation of LXRs can inhibit tumor progression in pre- clinical models in mice. Interestingly, in our own previous studies using a syngeneic model of Lewis lung carcinoma, activation of the LXR pathway with T1317 suppressed tumor growth in wild type but not in LXR-deficient mice, underlining the importance of functional expression of LXRs in the host for the antitumoral effects of the agonist. In addition, the expression of the chemokines CCL17 and CCL22, key attractants for regulatory T (Treg) cells, and the transcription factor IRF4 was inhibited in T1317-treated TAMs in vivo and ex vivo. Based on these observations, we further explored the actions of the LXR agonist in the tumor microenvironment. Tumor-associated macrophages (TAMs) are the most abundant immune cells in the tumor microenvironment, and they strongly contribute to the establishment of an immunosuppressive environment. We studied two TAM subpopulations with distinct phenotypic characteristics and intra-tumoral localization, named MHCIIhigh TAMs and MHCIIlow TAMs after their differential expression of MHCII. Collectively, treatment with T1317 impacted the transcriptional profile of TAMs at multiple levels, suppressing several mechanisms used by these cells for the maintenance of the immunosuppressed environment. Among these effects, activation of LXRs caused a decrease in the abundance of Tregs in the tumor, without affecting their immunosuppressive or proliferative capabilities, nor their peripheral abundance. Concomitantly with the inhibition of CCL17 expression in TAMs, the results suggested that LXR activation reduced Treg intratumoral abundance through blocking their recruitment. In this sense, a functional systemic expression of IRF4 was found necessary for the T1317-mediated inhibition of tumor growth. In addition, the inhibitory effect of LXR activation on CCL17, CCL22 and IRF4 expression was also observed in human macrophages derived from peripheral mononuclear cells from healthy donors, suggesting that the crosstalk between LXRs and the IRF4- CCL17/CCL22 axis is evolutionary conserved and may be also relevant in humans. Overall, these results shed new light on the mechanisms of LXR agonists as antitumoral drugs targeting the tumor microenvironment.
Characterization of a new animal model of Alzheimer's disease: relevance of Tau phosphorylation in hippocampal interneurons
(Universitat de Barcelona, 2022-06-03) Dávila Bouziguet, Eva; Pascual Sánchez, Marta; Soriano García, Eduardo; Universitat de Barcelona. Departament de Biologia Cel·lular, Fisiologia i Immunologia
[eng] The neuropathological hallmarks of Alzheimer’s disease are senile plaques, extracellular deposits of amyloid-β peptide (Aβ), and neurofibrillary tangles, intracellular aggregates of hyperphosphorylated Tau protein (P-Tau). At the functional level, Alzheimer’s disease involves synaptic dysfunction, aberrant network activity, and cognitive impairment, eventually resulting in dementia. Remarkably, individuals presenting Aβ and P-Tau without cognitive impairment have been reported. This cognitive resilience to Alzheimer’s disease is believed to involve synaptic preservation. In Alzheimer’s disease, Aβ and P-Tau exert toxicity either independently or synergistically. However, Tau is pivotal in mediating the synaptotoxicity induced by Aβ, neurodegeneration, and cognitive decline. Tau is a canonical microtubule-binding protein. Nevertheless, its physiological roles are currently recognized to extend far beyond microtubule stabilization, including participation in synaptic plasticity and DNA protection. Although the regulation of Tau function depends on phosphorylation, the association of P-Tau with Alzheimer’s disease and other tauopathies has established the notion that it is invariably pathological. Aberrant network activity, hyperexcitability, and altered oscillatory activity, which indicate an imbalance between excitation and inhibition in neural circuits, have been described in Alzheimer’s disease. Hence, dysfunction of the GABAergic system has been suggested to underlie network abnormalities. Studies from our group have revealed synaptic alterations in the GABAergic septohippocampal pathway and its implication in the emergence of abnormal hippocampal oscillatory activity in J20 and VLW animals, Alzheimer’s disease mouse models that accumulate Aβ and P-Tau, respectively. GABAergic neurons in the medial septum control GABAergic hippocampal interneurons, which govern principal cell activity, hence modulating hippocampal oscillations. Given the myriad of physiological roles of Tau and the impact of phosphorylation on its activity, we have studied its phosphorylation pattern in physiological and pathological conditions in mice and human subjects. Our data show that control mice and human subjects present Tau phosphorylated at Thr205 and Ser262 in the soma of hippocampal interneurons, pointing to a physiological role of these P-Tau species in this neuron population. Moreover, we have observed these P-Tau species in the soma of hippocampal interneurons in Alzheimer’s disease patients and in J20 and VLW mice. The presence of Tau phosphorylated at Thr205 correlates with the Aβ plaque burden in J20 animals, suggesting an inductive effect of Aβ on Tau phosphorylation at this site. Conversely, the accumulation of Tau phosphorylated at Ser262 is unchanged in pathological conditions, implying a physiological role of this P-Tau. In addition, our data indicate that 3 the presence of mutant human Tau in pyramidal neurons in VLW mice induces the phosphorylation of endogenous murine Tau at Thr231 in hippocampal interneurons. Furthermore, to recapitulate the complete spectrum of Alzheimer’s disease pathology, we have crossed J20 and VLW mice. In the resulting J20/VLW animals, which simultaneously present Aβ and P-Tau, our findings reveal no differences in Aβ burden or P-Tau levels compared to single transgenic mice. However, J20/VLW animals present a distinct Tau phosphorylation pattern in hippocampal interneurons. Our data show that GABAergic septohippocampal innervation is dramatically altered in J20 and VLW animals, whereas it is preserved in J20/VLW mice. Moreover, we have found that hippocampal oscillations are partially conserved in J20/VLW animals, in contrast to single transgenic mice. Finally, J20/VLW mice display preserved cognitive function, contrary to J20 and VLW animals. Taken together, our findings point to a physiological role of Tau phosphorylation in the somatodendritic compartment of hippocampal interneurons. Moreover, they suggest that a particular Tau phosphorylation signature in this neuron population protects against the loss of GABAergic septohippocampal innervation, thereby avoiding alterations in hippocampal oscillatory activity and, thus, preventing cognitive impairment in J20/VLW mice. Lastly, these data highlight J20/VLW mice as a suitable animal model to explore the cognitive resilience to Alzheimer’s disease.

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Tesis Doctorals - Departament - Biologia Cel·lular, Fisiologia i Immunologia

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Amb motiu del tancament d'estiu, la validació de documents es reprendrà a partir del 28 d'agost de 2026. Disculpeu les molèsties.

Con motivo del cierre de verano, la validación de documentos se reanudará a partir del 28 de agosto de 2026. Disculpad las molestias

Due to the summer closure, document validation will resume starting August 28, 2026. We apologize for any inconvenience.

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