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

URI permanent per a aquesta col·leccióhttps://hdl.handle.net/2445/104131

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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.
Fisiología digestiva y nutrición durante las primeras etapas de vida del pez gato amazónico Pseudoplaystoma punctifer (Castelnau, 1899) en cultivo
(Universitat de Barcelona, 2022-05-16) Castro Ruíz, Diana; Darias Cáceres, María José; Gisbert Casas, Enric; Universitat de Barcelona. Departament de Biologia Cel·lular, Fisiologia i Immunologia
[spa] El pescado es una de las principales fuentes de proteínas para la población de la cuenca amazónica, la cual posee la mayor biodiversidad de peces del mundo. La acuicultura se ha desarrollado de manera constante en la región durante las últimas décadas para contribuir a las necesidades alimentarias de una población en rápido crecimiento, y varias especies de peces nativos han sido identificadas como de interés desde hace tiempo. Entre ellas, el pez gato Pseudoplatystoma punctifer (Castelnau, 1855) es muy apreciado por la calidad de su carne y la ausencia de espinas intramusculares, aunque su cultivo comercial aún no se ha desarrollado en el Perú debido a las altas tasas de mortalidad durante las primeras etapas de vida, principalmente causadas por una alta incidencia de canibalismo y la dificultad para adaptarse a los alimentos formulados. Para superar estos obstáculos, el objetivo de esta tesis fue estudiar la ontogenia del sistema digestivo de P. punctifer y sus necesidades nutricionales durante las primeras etapas de vida. Los resultados revelaron que el sistema digestivo estaba completamente desarrollado y funcionalmente maduro entre 10 y 13 días post fertilización (dpf, ca. 11-14 mm TL), lo que indicó la transición del modo de digestión larvario al adulto y el momento apropiado para el destete. Los estudios nutricionales revelaron que una dieta con 45% de proteína cruda (harina de pescado) y 15% de lípidos (lecitina marina y de soja) permitió adelantar el destete, promovió un mejor y más rápido desarrollo de la función digestiva y un mejor crecimiento (6 veces mayor) y redujo la incidencia de canibalismo (a la mitad) al final del primer mes de vida en comparación con protocolos anteriores. Además, los resultados revelaron que la nutrición durante la etapa larvaria afectó la nutrición y el comportamiento de los juveniles tempranos. Específicamente, el DHA dietético (4% TFA) proporcionado durante la fase larvaria influyó en la expresión génica de las enzimas digestivas y contribuyó a reducir el comportamiento caníbal en la etapa juvenil temprana. En conclusión, esta tesis aporta valiosos conocimientos sobre la nutrición temprana de esta especie, sentando así las bases para la mejora de los protocolos de alimentación, adaptados a sus capacidades digestivas y necesidades nutricionales durante las primeras etapas de vida, y representa un paso importante hacia su cultivo a nivel comercial.
Activity-dependent mechanisms of axonal growth
(Universitat de Barcelona, 2022-04-01) Mesquida Veny, Francina; Hervera Abad, Arnau; Río Fernández, José Antonio del; Universitat de Barcelona. Departament de Biologia Cel·lular, Fisiologia i Immunologia
[eng] Spinal cord injuries (SCI) are a major cause of paralysis in young adults. In this type of injuries recovery is impaired as adult central nervous system (CNS) axons fail to regenerate. This results from both a loss of intrinsic growing capacities in developmental axons when they mature, together with the presence of extrinsic factors hampering this regeneration, including a glial scar together with the production of growth-inhibitory molecules, as well as a lack of injury resolution leading to a chronic inflammation. Unfortunately, despite research efforts, current therapies for this type of injuries only lead to mild improvements and among them, activity-based therapies seem to raise above the others. Activity- based therapies try to induce recovery by increasing neuronal activity, however, a proper physiological and molecular characterization of the rationale behind their success is still missing. Neuronal activity has been described to regulate transcriptional and epigenetic mechanisms; moreover, it also alters neuronal secretion with an impact on cellular dialogues. These characteristics indicate neuronal activity may be modulating both of the CNS barriers for regeneration. During this doctoral thesis we aimed to explore the influence of neuronal activity on SCIs, hypothesizing specific neuronal activations were the principal responsible for success in activity-based therapies. Particularly, we studied the role of precise manipulations of neuronal activity, using optogenetic and chemogenetic tools, in axonal growth of stimulated neurons as well as the impact these activations could have on neuronal extrinsic signalling. Our results show that optogenetic and chemogenetic stimulations of neuronal activity enhanced growth in both regenerating and refractory to regenerate neurons. However, this growth was hampered by the inhibitory molecules present in the injured CNS and did not result in functional recovery in rodent models of SCI. Our data indicated that the growth induction in specifically stimulated neurons resulted from local adjustments rather than inducing a pro-regenerative transcriptional state, as seen by our gene expression analysis of regeneration-associated genes (RAGs). Altogether, our results suggest recovery in activity-based therapies derives from the summation of various forms of plasticity, induced by their simultaneous recruitment of several circuits. In parallel, we observed that these precise modulations of neuronal activity, while unable to alter the predominant environment after SCI, could initiate previously undescribed intricate cellular dialogues. Specifically, we found an increase in the chemokine CCL21 upon nociceptor activation which triggered the response of several cell types in the injury. In proprioceptors, this CCL21 was responsible for a growth induction after CCR7 activation, which required the MEK-ERK pathway as well as the modulation of the actin cytoskeleton. Meanwhile, the CCL21 interaction with CXCR3 in other cells effectively aborted this regeneration. All in all, our work reveals the existence of a complex plethora of synergic mechanisms, far from understood, contributing to the outcome of activity-based therapies and reinforces the need for further mechanistic studies which would allow the optimization of their success.
New insights about the role of WT1 in blood vessel formation during development and repair
(Universitat de Barcelona, 2022-03-31) Ramiro Pareta, Marina; Martínez Estrada, Ofelia María; Universitat de Barcelona. Departament de Biologia Cel·lular, Fisiologia i Immunologia
[eng] The pleiotropic WT1 transcription factor (WT1) was first discovered in renal tumours. It contains four zinc-finger motifs at the C-terminus which are important for DNA binding to activate gene expression and is also involved in post-transcriptional processes. The expression of WT1 is essential during development of multiple organs, among them the cardiovascular structures. In the developing heart, WT1 is strongly expressed in the epicardium, and in cardiac endothelial cells. After cardiac injury, WT1 is temporally upregulated in both lineages in the infarcted area and border zone, which points to a potential role for WT1 in cardiac repair and regeneration. In this thesis, we focused on the role of Wt1 in endothelial cells, particularly in cardiac endothelial cells during coronary vessel formation, and in the adult heart after myocardial infarction. To support some of these findings, the mouse retinal vasculature was used as a model for two-dimensional angiogenic studies. We have demonstrated that Wt1 is present in CECs during coronary development, and that Wt1 deletion abrogates for endothelial proliferation, migration, and maturation during both retinal and coronary vasculature formation in vivo. Also, RNAseq analysis of embryonic Wt1-KOΔEC CECs suggests that Wt1 controls several downstream pathways. However, the importance of Wt1 functions in neovascularization in the adult heart after injury, specifically after 14 days of MI, has proven to be limited. The so-called recapitulation of embryonic programs in the neovascularization processes arising after injury was not sufficient for Wt1 to exert a differential effect on the adult heart after myocardial infarction. We observed no significant functional changes in cardiac output, assessed by ECG imaging, nor in tissue remodelling, visualized by analysis of the fibrotic scar in the infarcted region. However, we did observe changes in Wt1-KOΔEC CECs at the molecular and transcriptomic level, suggesting that WT1 may activate some genetic programmes in these cells, even if its effects are not able to alter evident cardiac functions.
Gestational exercise and intergenerational inheritance. Metabolic and functional outcomes in rat liver and skeletal muscle
(Universitat de Barcelona, 2022-04-05) Beleza, Jorge; Magalhäes, José; Torrella Guio, Joan Ramon; Universitat de Barcelona. Departament de Biologia Cel·lular, Fisiologia i Immunologia
[eng] Maternal environmental exposure during gestational period may affect offspring risk for chronic diseases. Accordingly, maternal metabolic disturbances induced by high-fat high-sucrose (HFHS) diets can have a negative impact on offspring liver and skeletal muscle (SM) tissue phenotypes. In contrast, gestational exercise (GE) seems to favorably modulate deleterious stimuli induced by HFHS- diet consumption, not only in mothers but also in their offspring. However, the mechanisms associated to intergenerational inheritance of favorable metabolic outcomes in the offspring are far to be understood, as well as the role of epigenetic regulation in the offspring program. In order to contribute to increase the scientific knowledge in this field, the present PhD thesis comprises two literature review publications and three animal-based experimental papers that were developed using a gestational diabetes mellitus (GDM) model. Therefore, the goal was to analyze the potential role of an exercised model that combines voluntary free-running wheel (FRW) with forced endurance exercise, against liver and SM offspring metabolic and functional impairments. Our data revealed that GE counteracted the negative impact of HFHS maternal diet and sedentarism in offspring liver and SM morphology. Moreover, offspring from HFHS exercised mothers exhibited an improvement in liver and SM parameters of mitochondrial biogenesis, oxidative phosphorylation subunits protein content and respiratory capacity, associated to improvements in maximal exercise performance. Additionally, improvements in biomarkers of mitochondrial performance seems to be regulated, at least in part, by the role of miRNAs through epigenetic modifications inhered by the offspring generation. Ultimately, these findings shed light on the importance of maternal lifestyle, specifically the relevance of GE, on the phenotypic programming of the offspring against GDM-associated metabolic impairments and ultimately to an enhanced functional performance. Hopefully, evidence from the present PhD thesis can contribute to encourage mothers to engage in an active lifestyle before and during pregnancy aiming positive outcomes in their offspring.
Análisis de los efectos globales que ocasiona la hipoxia hipobárica crónica sobre el estado físico-emocional de los pobladores que residen en altitudes mayores a los 2,500 msnm
(Universitat de Barcelona, 2022-03-11) Ortiz-Prado, Esteban; Calvopiña, Manuel; Viscor Carrasco, Ginés; Universitat de Barcelona. Departament de Biologia Cel·lular, Fisiologia i Immunologia
[spa] ANTECEDENTES: A nivel planetario, al menos 200 millones de personas a nivel mundial residen en poblaciones ubicadas a grandes alturas. Vivir o visitar regiones montañosas puede provocar cambios fisiológicos significativos entre quienes visitan estos lugares durante cortos periodos de tiempo o provocar modificaciones adaptativas más crónicas entre quienes residen allí durante varias generaciones. Los seres humanos dependen de la presencia, disponibilidad y utilización de oxígeno para funcionar correctamente y mantener la homeostasis fisiológica alrededor de la respiración. En condiciones de hipoxia, los mecanismos adaptativos a largo plazo o compensatorios a corto plazo generan alteraciones genéticas, anatómicas, fisiológicas y, en algunos casos, patológicas entre los seres humanos. OBJETIVOS: El objetivo de nuestro estudio ha sido doble: desde un punto de vista más general, el de estudiar el impacto epidemiológico que representa el vivir por encima de los 2.500 m sobre el nivel del mar, y, más detalladamente, estudiar las diferencias fisiológicas más evidentes entre dos poblaciones indígenas, genotípicamente idénticas que residen a baja y gran altura. RESULTADOS: a) Diferencias antropométricas Nuestra tesis ha demostrado que las mujeres que residen a bajas alturas tienden a presentar tallas más cortas y mayor peso que sus pares de las grandes alturas, aunque estas diferencias no fueron estadísticamente significativas (p = 0,333). A su vez, los hombres que residen a grandes alturas son más bajos de talla que sus homólogos que viven a bajas alturas, siendo estos resultados estadísticamente significativos (p = 0,019). En relación con la composición corporal, las mujeres de las alturas tienen un porcentaje menor de músculo corporal (-24,8%), mientras que los hombres de la misma región tienen un porcentaje de masa corporal muscular significativamente mayor (+ 13,5%) que sus homólogos de bajas alturas. El porcentaje de grasa corporal fue menor entre las mujeres de las bajas alturas (-15,5%), mientras que entre los hombres no logramos encontrar diferencias estadísticamente significativas. b) Diferencias hematológicas, de perfil lipídico y de riesgo cardiovascular Al comparar estos parámetros, nuestros resultados nos demuestran que la proporción de individuos con sobrepeso y obesidad fue mayor entre los habitantes de las bajas alturas (p <0,05). A la vez, el número de glóbulos rojos (RBC), la concentración de hemoglobina y la fueron significativamente más altos entre los habitantes de las grandes alturas, mientras que la saturación de oxigeno arterial (SpO2%) y el tamaño de los glóbulos rojos resultaron ser menores entre pobladores de las grandes alturas. Por otro lado, el grupo viviendo cerca del nivel del mar, mostró niveles más bajos de colesterol plasmático, lipoproteínas de baja densidad (LDL) y lipoproteínas de alta densidad (HDL). c) Optimismo y autopercepción Los habitantes de las zonas altas presentaron puntuaciones más bajas en todas las dimensiones estudiadas dentro de la encuesta de salud SF-36. Las diferencias estadísticamente significativas se encontraron dentro de la esfera de vitalidad (p = 0,005), Salud Mental (p = 0,002) y funcionamiento social (p = 0,005). En todos los casos, los participantes que vivían a bajas alturas puntuaban más que los que vivían a grandes alturas. Por otro lado, las mujeres de la amazonia, que residen a baja altura, resultaron ser más optimistas que sus homólogas de las grandes alturas. d) Función pulmonar y parámetros ventilatorios Los habitantes de Oyacachi (3,800 m) mostraron tener valores predictivos mayores que los habitantes de Limoncocha (230 m). Por ejemplo, la capacidad vital forzada (CVF) y el volumen espiratorio forzado en un segundo (VEF1) fueron significativamente mayores entre los habitantes de las tierras altas que la de sus pares amazónicos (valor p < 0,001). El índice de Tiffeneau (VEF1/CVF) fue significativamente mayor entre los habitantes de las bajas alturas que entre los que residen a mayor elevación, siendo esto evidente tanto en hombres como en mujeres. CONCLUSIONES: Vivir a grandes alturas se asocia a cambios fisiológicos adaptativos bien conocidos y descritos como son el recuento de eritrocitos, la concentración de hemoglobina o el nivel de hematocrito. Nuestros resultados también demuestran que existen diferencias clínicas en el perfil lipídico plasmático, teniendo niveles más altos de colesterol, HDL y LDL entre los habitantes de las grandes alturas. Las diferencias antropométricas varían según el sexo y la edad. Nuestros hallazgos sugieren que la población que reside por sobre los 3,800 m es en general más ligera de peso y más baja en talla que la de sus pares de bajas alturas. Los hombres que residen a grandes alturas tienen cuerpos con mayores porcentajes de masa muscular que sus homólogos de las tierras bajas, pero en términos de edad corporal, los pobladores de las alturas resultaron ser mayores que los de las bajas alturas. Los habitantes de las grandes alturas tuvieron mayor capacidad pulmonar que la de sus pares de las bajas alturas. Al analizar los patrones espirométricos obtenidos en estas poblaciones, resultó evidente que las mayores capacidades pulmonares están probablemente relacionadas con pulmones más grandes, lo que mejora la oxigenación sistémica a pesar de la baja disponibilidad de oxígeno.
Efecto de la hipoxia hipobárica intermitente y el frío en la recuperación de lesiones del músculo esquelético
(Universitat de Barcelona, 2021-12-17) Santocildes Martinez, Garoa; Torrella Guio, Joan Ramon; Viscor Carrasco, Ginés; Universitat de Barcelona. Departament de Biologia Cel·lular, Fisiologia i Immunologia
[spa] Los trastornos músculo-esqueléticos son una de las principales causas de necesidad de rehabilitación en el mundo. En el ámbito deportivo, las lesiones del sistema musculo- esquelético representan entre el 10-55% del total de las lesiones. Debido a su alta incidencia e impacto sobre la salud, el proceso de regeneración muscular ha sido ampliamente estudiado y caracterizado, pero todavía se continúa en la búsqueda de estrategias terapéuticas que puedan acelerar el proceso de regeneración muscular y mejorar el tejido muscular post-lesión. Gracias a su alta plasticidad el músculo esquelético se adapta y responde ante diferentes estímulos con la finalidad de mantener la homeostasis y mejorar su funcionalidad. La hipoxia hipobárica intermitente (HHI) ha demostrado que puede mejorar el transporte, liberación y uso del oxígeno a nivel muscular, pero su coexistencia con el frío en la biosfera abre las puertas a considerar el uso combinado del frío y la hipoxia como herramienta para lograr beneficiosas adaptaciones fisiológicas y musculares. Por todo ello, en la presente tesis se estudian los efectos de la exposición a la HHI y al frío, de forma independiente y combinada, en la fisiología de los animales y sobre los procesos de regeneración muscular. Se utilizaron machos-adultos de ratas Sprague-Dawley como modelo experimental. Los animales fueron quirúrgicamente lesionados en su músculo gastrocnemio y aleatoriamente divididos en diferentes grupos: 1) Grupo CTRL: animales con recuperación pasiva; 2) Grupo COLD: animales expuestos a frío intermitente (4°C); 3) Grupo HYPO: animales expuestos a HHI (altitud simulada 4500 m); 4) Grupo COHY: animales expuestos a frío e hipoxia intermitente simultáneamente (4500 m + 4°C). Las ratas fueron sometidas a las diferentes intervenciones durante 4 horas por día a lo largo de 9 o 21 días. Además, se incluyeron 2 grupos adicionales, uno con la finalidad de corroborar la simetría morfo-funcional entre el gastrocnemio derecho e izquierdo y el otro, para validar el método de lesión. Los resultados mostraron la existencia de una simetría tanto morfológica (área transversal de las fibras y proporción de fibras) como funcional (fuerza y resistencia a la fatiga) entre el gastrocnemio derecho e izquierdo de las ratas. El método de lesión utilizado demostró ser útil, produciendo alteraciones tanto en la arquitectura del tejido como en su capacidad funcional. El uso de exposiciones intermitentes garantizó un crecimiento normal y el incremento de la masa muscular de los animales induciendo, además, respuestas de aclimatación como incrementos del tejido adiposo marrón en los grupos expuestos al frío (COLD y COHY) e incrementos en el transporte de oxígeno en sangre en los animales sometidos a HHI (HYPO y COHY). A nivel muscular, el grupo COLD exhibió mejoras en la capilaridad muscular mediante incrementos de VEGF y del número de capilares, mientras que los grupos HYPO y COHY mejoraron la irrigación de las fibras gracias a la reducción del tamaño de las fibras, pero sin detectarse un proceso angiogénico. Los animales de los grupos COHY y COLD presentaron una acelerada regeneración muscular, observada a nivel histológico a los 9 días (reducción del porcentaje de fibras positivas en dMHC y con núcleos centrales) y a nivel funcional a los 21 días. El grupo HYPO mostró una completa recuperación de la lesión muscular (a nivel histológico y funcional) en 9 días. Además la HHI demostró ser un interesante anti- fibrótico reduciendo la deposición de colágeno I en el lugar de la lesión. El incremento en el cociente pSer473Akt/Akt total observado tras 9 días de tratamiento en los grupos COLD, HYPO y COHY, junto con el incremento en el cociente pThr172AMPKα/ AMPKα total observado en el músculo gastrocnemio del grupo HYPO, proporcionan indicios de los posibles mecanismos responsables de la mejora de la regeneración muscular observada.
Factores físicos y moleculares implicados en la migración celular y en el desarrollo de la corteza cerebral
(Universitat de Barcelona, 2022-01-12) López Mengual, Ana; Río Fernández, José Antonio del; Universitat de Barcelona. Departament de Biologia Cel·lular, Fisiologia i Immunologia
[spa] La migración celular adquiere especial relevancia durante el desarrollo embrionario y la regeneración tisular. Durante el desarrollo del individuo adulto, las células se multiplican, se diferencian y maduran, debiéndose de desplazar a sus regiones de destino mediante la migración. Una vez se ha formado el individuo adulto, estos tejidos de los que forman parte pueden sufrir daños, que conlleven a un estado alterado del mismo. En este proceso de regeneración del tejido en un intento de reestablecer la homeostasis tisular, la migración celular es fundamental. Por ellos, en esta tesis se analizan los factores mecánicos durante la migración celular en desarrollo cerebral y en regeneración neural, como una herramienta fundamental para entender estos procesos. En primer lugar, se analiza la idoneidad del uso de nanofibras de PLA 80/20 funcionalizadas con la quimiocina CXCL12 como sistema andamio para la migración de las células de la glía envolvente olfatoria (OECs). Las OECs migran mayores distancias sobre las nanofibras funcionalizadas, respondiendo así al gradiente quimiotáctico. Además, al sembrar las OECs y disponer las nanofibras funcionalizadas paralelas y suspendidas, nos aporta un buen sistema para trasplantar las células de forma direccionada en procesos de daño neural como terapia. En segundo lugar, se analiza el papel de los factores físicos en la migración y disposición de las células de Cajal-Retzius (CRcs) y de su origen migratorio. Se realizan mediciones mediante BIO-AFM del cerebro embrionario de ratón, obteniendo diferencias entre el palio y el subpalio. Estas diferencias determinan las tasas de migración diferenciales al trasplantar explantes de diversos orígenes fuera de su sitio habitual y analizar la migración de las CRcs. Además, las CRcs responden mediante entrada de calcio a la inhibición de canales catiónicos mecanosensibles, cambiando su tasa de migración.

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