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We, the participants of the First Organoid Intelligence Workshop - "Forming an OI Community" (22-24 February 2022), call on the international scientific community to explore the potential of human brain-based organoid cell cultures to advance our understanding of the brain and unleash new forms of biocomputing while recognizing and addressing the associated ethical implications. The term "organoid intelligence" (OI) has been coined to describe this research and development approach (1) in a manner consistent with the term "artificial intelligence" (AI) - used to describe the enablement of computers to perform tasks normally requiring human intelligence. OI has the potential for diverse and far-reaching applications that could benefit humankind and our planet, and which urge the strategic development of OI as a collaborative scientific discipline. OI holds promise to elucidate the physiology of human cognitive functions such as memory and learning. It presents game-changing opportunities in biological and hybrid computing that could overcome significant limitations in silicon-based computing. It offers the prospect of unparalleled advances in interfaces between brains and machines. Finally, OI could allow breakthroughs in modeling and treating dementias and other neurogenerative disorders that cause an immense and growing disease burden globally. Realizing the world-changing potential of OI will require scientific breakthroughs. We need advances in human stem cell technology and bioengineering to recreate brain architectures and to model their potential for pseudo-cognitive capabilities. We need interface breakthroughs to allow us to deliver input signals to organoids, measure output signals, and employ feedback mechanisms to model learning processes. We also need novel machine learning, big data, and AI technologies to allow us to understand brain organoids.

Biochemistry; Molecular medicine Bioquímica; Medicina molecular Bioquímica; Medicina molecular Imbalanced mitochondrial dNTP pools are known players in the pathogenesis of multiple human diseases. Here we show that, even under physiological conditions, dGTP is largely overrepresented among other dNTPs in mitochondria of mouse tissues and human cultured cells. In addition, a vast majority of mitochondrial dGTP is tightly bound to NDUFA10, an accessory subunit of complex I of the mitochondrial respiratory chain. NDUFA10 shares a deoxyribonucleoside kinase (dNK) domain with deoxyribonucleoside kinases in the nucleotide salvage pathway, though no specific function beyond stabilizing the complex I holoenzyme has been described for this subunit. We mutated the dNK domain of NDUFA10 in human HEK-293T cells while preserving complex I assembly and activity. The NDUFA10E160A/R161A shows reduced dGTP binding capacity in vitro and leads to a 50% reduction in mitochondrial dGTP content, proving that most dGTP is directly bound to the dNK domain of NDUFA10. This interaction may represent a hitherto unknown mechanism regulating mitochondrial dNTP availability and linking oxidative metabolism to DNA maintenance. We thank Dr, Luke Formosa (Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Monash University, Melbourne, Australia) for his valuable advice and assistance on NDUFA10 molecular studies and Dr. Francesc Canals and his team (Proteomics Laboratory, Vall d’Hebron Institute of Oncology [VHIO], Universitat Autònoma de Barcelona, Barcelona, Spain) for their assistance with LC-MS/MS analyses. This work was supported by the Spanish Ministry of Industry, Economy and Competitiveness [grants BFU2014-52618-R, SAF2017-87506, and PID2020-112929RB-I00 to Y.C.], by the Spanish Instituto de Salud Carlos III [grants PI21/00554 and PMP15/00025 to R.M.], co-financed by the European Regional Development Fund (ERDF), and by an NHMRC Project grant to M.R. (GNT1164459).

MicroRNAs (miRNA) are major regulators of intercellular communication and key players in the pathophysiology of cardiovascular disease. This study aimed to determine the miRNA fingerprint in a cohort of 53 patients with acute myocardial infarction (AMI) with non-ST-segment elevation (NSTEMI) relative to miRNA expression in healthy controls (n = 51). miRNA expression was initially profiled by miRNA array in the serum of patients undergoing cardiac catheterization during NSTEMI (n = 8) and 1 year past the event (follow-up, n = 8) and validated in the entire cohort. In total, 58 miRNAs were differentially expressed during AMI (p < 0.05), while 36 were modified at follow-up (Fisher’s exact test: p = 0.0138). Enrichment analyses revealed differential regulation of biological processes by miRNA at each specific time point (AMI vs. follow-up). During AMI, the miRNA profile was associated mainly with processes involved in vascular development. However, 1 year after AMI, changes in miRNA expression were partially related to the regulation of cardiac tissue morphogenesis. Linear correlation analysis of miRNA with serum levels of cytokines and chemokines revealed that let-7g-5p, let-7e-5p, and miR-26a-5p expression was inversely associated with serum levels of pro-inflammatory cytokines TNF-α, and the chemokines MCP-3 and MDC. Transient transfection of human endothelial cells (HUVEC) with let-7e-5p inhibitor or mimic demonstrated a key role for this miRNA in endothelial function regulation in terms of cell adhesion and angiogenesis capacity. HUVEC transfected with let-7e-5p mimic showed a 20% increase in adhesion capacity, whereas transfection with let-7e-5p inhibitor increased the number of tube-like structures. This study pinpoints circulating miRNA expression fingerprint in NSTEMI patients, specific to the acute event and changes at 1-year follow-up. Additionally, given its involvement in modulating endothelial cell function and vascularization, altered let-7e-5p expression may constitute a therapeutic biomarker and target for ischemic heart disease.

Extracellular histones have been reported to aggravate different pathophysiological processes by increasing vascular permeability, coagulopathy, and inflammation. In the present study, we elucidate how extracellular histones (10–100 µg/mL) concentration dependently increase cytosolic reactive oxygen species (ROS) production using human umbilical vein endothelial cells (HUVECs). Furthermore, we identify cyclooxygenase (COX) and NADPH oxidase (NOX) activity as sources of ROS production in extracellular histone-treated HUVEC. This COX/NOX-mediated ROS production is also involved in enhanced NF-kB activity and cell adhesion molecules (VCAM1 and ICAM1) expression in histone-treated HUVEC. Finally, by using different toll-like receptor (TLR) antagonists, we demonstrate the role of TLR4 in CAMs overexpression triggered by extracellular histones in endothelial cells. In conclusion, our data suggest that through TLR4 signaling, extracellular histones increase endothelial cell activation, a mechanism involving increased COX- and NOX-mediated ROS. These findings increase our understanding on how extracellular histones enhance systemic inflammatory responses in diseases in which histone release occurs as part of the pathological processes.

Gene regulation; Somatic cell reprogramming; Trophectoderm Regulación de genes; Reprogramación de células somáticas; Trofoectodermo Regulació de gens; Reprogramació de cèl·lules somàtiques; Trofectoderma IL-6 has been shown to be required for somatic cell reprogramming into induced pluripotent stem cells (iPSCs). However, how Il6 expression is regulated and whether it plays a role during embryo development remains unknown. Here, we describe that IL-6 is necessary for C/EBPα-enhanced reprogramming of B cells into iPSCs but not for B cell to macrophage transdifferentiation. C/EBPα overexpression activates both Il6 and Il6ra genes in B cells and in PSCs. In embryo development, Cebpa is enriched in the trophectoderm of blastocysts together with Il6, while Il6ra is mostly expressed in the inner cell mass (ICM). In addition, Il6 expression in blastocysts requires Cebpa. Blastocysts secrete IL-6 and neutralization of the cytokine delays the morula to blastocyst transition. The observed requirement of C/EBPα-regulated IL-6 signaling for pluripotency during somatic cell reprogramming thus recapitulates a physiologic mechanism in which the trophectoderm acts as niche for the ICM through the secretion of IL-6. We thank C. Berenguer for help with B cell reprogramming and bone marrow collection; S. Nakagawa and B. Pernaute for advice on pre-implantation embryo culture and manipulation, and Kyle M. Loh for his valuable discussions; the flow cytometry and microscopy units of UPF-CRG for technical assistance; the CRG genomics core facility for sequencing and Graf laboratory members for critical discussions. Work in the laboratory of T.G. was supported by the Spanish Ministry of Economy, Industry and Competitiveness (Plan Estatal PID2019-109354GB-I00), the CRG, AGAUR (SGR 726), and a European Research Council Synergy grant (4D-Genome). M.P.-C. was supported by an FPI fellowship (BES-2016-076900). Work in the laboratory of M.S. was funded by the IRB and by grants from the Spanish Ministry of Economy co-funded by the European Regional Development Fund (SAF2017-82613-R), ERC (ERC-2014-AdG/669622), la Caixa Foundation, and Secretaria d’Universitats i Recerca del Departament d’Empresa i Coneixement of Catalonia (Grup de Recerca consolidat 2017 SGR 282).

[spa] La abrazadera deslizante eucariota (PCNA), juega un papel esencial como componente del replisoma. PCNA, de forma toroidal, rodea el DNA y ata las polimerasas y otros factores a la plantilla genómica para una síntesis rápida y procesiva. PCNA puede deslizar bidireccionalmente a lo largo del dúplex de DNA rastreando su columna vertebral mediante un mecanismo de «rueda dentada» basado en interacciones polares efímeras que mantienen la orientación de la pinza invariante en relación con la doble hélice. La mutación de residuos en esta interfaz de interacción PCNA-DNA hace desfavorable el inicio de la síntesis de DNA por Pol δ, por lo tanto, es necesaria una pinza orientada correctamente en el DNA para el ensamblaje de una holoenzima pol δ-PCNA competente en replicación. La cara del interior del anillo de PCNA, además de ser crucial para la función de PCNA como factor de procesividad durante la replicación, está altamente regulada para controlar la resistencia al daño en el DNA. Se puede modular (i) a través de la acetilación de su lisina 20, lo que estimula la reparación por recombinación homóloga, o (ii) mediante la unión de p15PAF, lo que desactiva el baipás de lesión en el DNA. p15PAF es una proteína intrínsecamente desordenada que atraviesa el canal del anillo de PCNA, uniendo su dominio PIP-box al bolsillo hidrofóbico de la cara frontal de la pinza y estableciendo contactos también con la superficie deslizante de la abrazadera para asomar su cola N-terminal por la cara trasera. Cuando dos moléculas de p15PAF ocupan dos subunidades del homotrímero de PCNA, el DNA dentro del canal del anillo se une a la subunidad que queda desocupada y no desplaza a p15PAF de la pared del anillo interno de PCNA. Cuando p15PAF está unida a PCNA, se reduce la superficie deslizante disponible de la abrazadera, así que p15PAF puede estar funcionando como un cinturón que abrocha el DNA a PCNA durante la síntesis por la polimerasa replicativa Pol δ. Esta restricción de la superficie deslizante, sin embargo, necesita ser eliminada para un baipás eficaz de la lesión del DNA por parte de la polimerasa de síntesis translesión Pol η. PCNA es estable en forma de anillo cerrado y, por lo tanto, debe cargarse activamente en las uniones cebador/plantilla del DNA, colocándose exactamente en el lugar y posición correctas para una replicación procesiva. La apertura y carga de PCNA la lleva a cabo el cargador de la pinza RFC. Una vez en el DNA, PCNA se vuelve a sellar alrededor del DNA y entonces el cargador de la pinza es expulsado. Cuando PCNA ya no es necesaria anclada en el DNA, el complejo RFC es el encargado de retirarla abriéndola y soltándola fuera de la doble hebra. Pero la flexibilidad intrínseca de PCNA hace que tenga cierta predisposición a estar en estado abierto separando dos de sus subunidades a través de su interfaz. Esto, que favorece la apertura del anillo por parte de RFC para lograr el ensamblado alrededor del DNA, puede ser un problema para mantenerla cerrada en la unión cebador/plantilla. De hecho, la estabilidad de las interfaces entre subunidades de PCNA disminuye cuando se une al DNA después de ser cargada por RFC, y dicha estabilidad solo la ve recuperada cuando p15PAF se ancla por su dominio PIP-box a sus bolsillos hidrofóbicos, grapando así las subunidades de la pinza e impidiendo su salida prematura del complejo con el DNA. Además de estabilizar la forma cerrada del anillo de PCNA, cuando p15PAF está anclada a su cara frontal, impide que RFC se aproxime, se una a ella y la desenganche de la unión cebador/plantilla., [eng] The eukaryotic sliding clamp (PCNA) is an essential replisome's component. PCNA, with a toroidal shape, surrounds DNA and binds polymerases and other factors to the genomic template for rapid and processive synthesis. PCNA can slide bi-directionally along the DNA duplex using a "cogwheel" mechanism based on ephemeral polar interactions that maintain the orientation of the clamp invariant relative to the double helix. However, mutations in the PCNA-DNA interaction interface render unfavourable the initiation of DNA synthesis by Pol δ. Therefore, a correctly oriented clamp on the DNA is necessary to assemble a competent pol δ-PCNA holoenzyme. Tight regulation of the inner face of PCNA, which is crucial for PCNA function as a processivity factor during replication, controls the DNA damage resistance. The inner PCNA ring face can be regulated (i) through acetylation of its lysine 20, which stimulates repair by homologous recombination, or (ii) by p15PAF binding, which deactivates the bypass of DNA damage. p15PAF is an intrinsically disordered protein that crosses the channel of the PCNA ring, attaching its PIP-box domain to the hydrophobic pocket on the front face of the clamp and establishing contacts with the sliding surface to show its N-terminal tail through the rear face. When two p15PAF molecules occupy two subunits of the PCNA homotrimer, the DNA within the ring channel binds to the unoccupied subunit and does not displace p15PAF from the inner ring wall of PCNA. When p15PAF is bound to PCNA, the available slip surface of the clamp is reduced, so p15PAF may be functioning as a belt that binds DNA to PCNA during synthesis by the replicative polymerase Pol δ. This sliding surface restriction, however, needs to be removed for efficient bypass of DNA damage by the translesion synthesis polymerase Pol η. PCNA is a stable closed ring and must be actively loaded onto the primer/template junctions of DNA, getting precisely in the right place and position for processive replication. RFC clamp loader opens and loads PCNA onto the DNA. Once in the DNA, PCNA reseals around the DNA and RFC is then ejected. When PCNA is no longer needed around the DNA, the RFC complex opens and unloads it. But the local flexibility of PCNA's subunits interfaces makes it have a certain predisposition to be in the open state. This local flexibility, which favours the RFC opening of the ring to achieve assembly around the DNA, can be a problem in keeping it closed at the primer/template junction. Furthermore, the stability of the interfaces between PCNA subunits decreases further when it binds to DNA. Interestingly, the PCNA homotrimer interfaces stability recovers when p15PAF is anchored by its PIP-box domain to PCNA's hydrophobic pockets, thus stapling the clamp subunits and preventing their premature exit from the complex with DNA. In addition, when p15PAF is anchored to PCNA front face, it prevents RFC from approaching, binding to, and unloading PCNA from the primer/template junction.

Transició epitelial-mesenquimal; Carcinoma de mama metaplàstic Transición epitelio-mesenquimatosa; Carcinoma de mama metaplasico Epithelial-mesenchymal transition; Metaplastic breast carcinoma Metaplastic breast carcinoma (MBC) is a heterogeneous group of infrequent triple negative (TN) invasive carcinomas with poor prognosis. MBCs have a different clinical behavior from other types of triple negative breast cancer (TNBC), being more resistant to standard chemotherapy. MBCs are an example of tumors with activation of epithelial–mesenchymal transition (EMT). The mechanisms involved in EMT could be responsible for the increase in the infiltrative and metastatic capacity of MBCs and resistance to treatments. In addition, a relationship between EMT and the immune response has been seen in these tumors. In this sense, MBC differ from other TN tumors showing a lower number of tumor-infiltrating lymphocytes (TILS) and a higher percentage of tumor cells expressing programmed death-ligand 1 (PD-L1). A better understanding of the relationship between the immune system and EMT could provide new therapeutic approaches in MBC. This review was funded by grants from the Instituto de Salud Carlos III (ISCIII) (PI19/01331) and CIBERONC (CB16/12/00316 and CB16/12/00449), co-financed by the European Development Regional Fund. ‘A way to achieve Europe’ (FEDER), and by the Spanish Association Against Cancer Scientific Foundation (Grupos Coordinados Traslacionales aecc 2018).

[eng] Our knowledge on the molecular basis behind physiological angiogenesis has significantly expanded in the last two decades. This progress also led to improvement in understanding of many vascular-related diseases in which angiogenesis is pathologically altered. Among many molecular regulators of angiogenesis, a family of lipid kinases called phosphatidylinositol 3-kinases (PI3Ks) occupies an important position in controlling endothelial cell functions. Indeed, numerous studies showed that endothelial cells are highly sensitive to fluctuations in the levels of phospholipids generated by these enzymes. Although highly conservative, the eight PI3K isoforms can produce three different types of phospholipids and this phenomenon formed the basis of the division into three different classes. Two members, ass I PI3Kα and ass II PI3K-C2α, are essential for proper vascular development. Moreover, somatic activating mutations in the gene encoding PI3Kα (PIK3CA) were found to cause 25% of venous malformations – a non-malignant, painful and mainly pediatric vascular disease for which the treatment options are limited. The vascular function of other isoforms, in particular class II PI3K-C2β, remains enigmatic. This is surprising given that this isoform is also express in cultured endothelial cells. This thesis is composed of two principal objectives objectives which together have been conceived to increase our knowledge on PI3K signaling in the endothelium. In the first part I evaluated the therapeutic efficacy of pan-AKT inhibitor, miransertib, in Pik3ca-driven vascular malformations using a preclinical mouse model. I showed that miransertib significantly prevents and reverts Pik3ca associated vascular hyperplasia through inhibition of endothelial cell proliferation. My results provide rationale for the therapeutic intervention of miransertib in treating patients with vascular malformations. The second part of the thesis studies the impact of PI3K-C2β isoform on blood vessel expansion and endothelial cell biology. Using both in vivo and in vitro models, I demonstrated for the first time that PI3K-C2β regulates retinal vascularity and vessel width, most likely as result of elevated vascular mTORC1 activity. Moreover, PI3K-C2β kinase inactivation led to increased collagen IV deposition and more stable vascular connections. In parallel, we showed that blood vessel-associated pericytes express high levels of PI3K-C2β and that its loss of function alters their morphology. Finally, we addressed the role of PI3K-C2β in the pathological neoangiogenesis associated with oxygen-induced retinopathy.

Càncer; Immunoteràpia; Radioteràpia Cáncer; Inmunoterapia; Radioterapia Cancer; Immunotherapy; Radiotherapy Advances in immunotherapy have achieved remarkable clinical outcomes in tumors with low curability, but their effects are limited, and increasing evidence has implicated tumoral and non-tumoral components of the tumor microenvironment as critical mediators of cancer progression. At the same time, the clinical successes achieved with minimally invasive and optically-guided surgery and image-guided and ablative radiation strategies have been successfully implemented in clinical care. More effective, localized and safer treatments have fueled strong research interest in radioimmunotherapy, which has shown the potential immunomodulatory effects of ionizing radiation. However, increasingly more observations suggest that immunosuppressive changes, metabolic remodeling, and angiogenic responses in the local tumor microenvironment play a central role in tumor recurrence. In this review, we address challenges to identify responders vs. non-responders to the immune checkpoint blockade, discuss recent developments in combinations of immunotherapy and radiotherapy for clinical evaluation, and consider the clinical impact of immunosuppressive changes in the tumor microenvironment in the context of surgery and radiation. Since the therapy-induced modulation of the tumor microenvironment presents a multiplicity of forms, we propose that overcoming microenvironment related resistance can become clinically relevant and represents a novel strategy to optimize treatment immunogenicity and improve patient outcome. This work was supported by grants from the Instituto de Salud Carlos III (ISCIII): PI15/01262 (ML) and CP03/00101 (ML), co-financed by the European Regional Fund (ERDF) and AECC (Spanish Association of Cancer Research) Founding Ref. GC16173720CARR (ML). AS-G was granted by VHIR fellowships.

In this Primer, Sunyer and Trepat introduce durotaxis, the mode of migration by which cells follow gradients of extracellular matrix stiffness.

Apoptosi; Motoneurones; Atròfia muscular espinal Apoptosis; Motoneuronas; Atrofia muscular espinal Apoptosis; Motoneurons; Spinal muscular atrophy Spinal Muscular Atrophy (SMA) is a severe neuromuscular disorder caused by loss of the Survival Motor Neuron 1 gene (SMN1). Due to this depletion of the survival motor neuron (SMN) protein, the disease is characterized by the degeneration of spinal cord motoneurons (MNs), progressive muscular atrophy, and weakness. Nevertheless, the ultimate cellular and molecular mechanisms leading to cell loss in SMN-reduced MNs are only partially known. We have investigated the activation of apoptotic and neuronal survival pathways in several models of SMA cells. Even though the antiapoptotic proteins FAIM-L and XIAP were increased in SMA MNs, the apoptosis executioner cleaved-caspase-3 was also elevated in these cells, suggesting the activation of the apoptosis process. Analysis of the survival pathway PI3K/Akt showed that Akt phosphorylation was reduced in SMA MNs and pharmacological inhibition of PI3K diminished SMN and Gemin2 at transcriptional level in control MNs. In contrast, ERK phosphorylation was increased in cultured mouse and human SMA MNs. Our observations suggest that apoptosis is activated in SMA MNs and that Akt phosphorylation reduction may control cell degeneration, thereby regulating the transcription of Smn and other genes related to SMN function. This work was supported by grants from Instituto de Salud Carlos III, Fondo de Investigaciones Sanitarias, Unión Europea, Fondo Europeo de Desarrollo Regional (FEDER) “Una manera de hacer Europa” (PI17/00231 and PI20/00098) to RMS and AG; CERCA Program/Generalitat de Catalunya; and Spanish Agency of Research (Agencia Estatal de Investigacion-PID2019-107286RB-I00) and CIBERNED to JXC. AS holds a fellowship from Universitat de Lleida and SdF holds a fellowship from “Ajuts de Promoció de la Recerca en Salut” (IRBLleida-Diputació de Lleida). We thank Elaine Lilly, PhD, for English language revision of the paper.

Treballs Finals de Grau de Farmàcia, Facultat de Farmàcia i Ciències de l'Alimentació, Universitat de Barcelona, 2019. Tutor/a: Helena Colom Codina, The applications of Busulfan as a chemotherapeutic agent has been disappearing over the years as better therapeutic alternatives have been released. Nowadays it is mainly used for some myeloproliferative diseases and the conditioning process previous to Hematopoietic Stem Cell Transplantation. During this project, data was collected from 98 patients taking Busulfan for the aforementioned conditioning, which were further used to develop a population pharmacokinetic model aiming to predict plasma concentrations of the drug in patients. A one-compartment model best described the data, which adjusted its interindividual variability for clearance (CL/F) with a Typical Value of 11.0 L/h, and for the distribution volume (V/F) with a Typical Value of 42.7 L. Both parameters showed a better fit to the data by the application of the Adjusted Ideal Body Weight covariate to explain the residual error. The final model went through two internal validation processes and will be used henceforth in the clinical pharmacokinetic unit of the Catalan Oncology Institute targeting those patients with potential to achieve toxic concentrations and avoid adverse events derived from its use.

[eng] Vesicle trafficking is fundamental to distribute correctly lipids and proteins among cellular compartments. A complex network of interconnected pathways transport vesicles between organelles to satisfy the cellular demand. Multisubunit Tethering Complexes (MTCs) form a group of 9 protein assemblies that are conserved from yeast to human and that are essential for intracellular trafficking: Exocyst, Dsl1, COG, GARP, HOPS, Corvet, TRAPPI, TRAPPII and TRAPPIII. Each MTC recognizes a specific type of vesicle and tether it to the appropriate acceptor membrane, providing specificity and directionality to the trafficking. My research aimed to do a comparative analysis among all MTCs to shed light on the mechanism of action of these complexes. In my group we have developed PICT (Protein interaction from Imaging of Complexes after Translocation), a high sensitivity technique that allows us to study interactions between two proteins using live-cell imaging. We first conducted an in silico genome-wide screen to identify those proteins that could be functionally related to MTCs. We then used PICT for the protein interactions screen with the proteins selected in the in silico analysis. Given the high sensitivity of the assay and that the experiments were done directly in living cells, this method was prompt to detect interactions that have been overlooked by previous analysis, such as indirect, transient or low abundant interactions. I found that MTCs establish a network of interactions with P4-ATPases, lipid flippases that translocate phospholipids to the cytosolic leaflet of the membranes. P4-ATPases maintain the asymmetric distribution of phospholipids and they also play essential roles in vesicle trafficking. In yeast, there are 5 different P4-ATPases: Dnf1, Dnf2, Dnf2, Drs2 and NeoI. Dnf1 and Drs2 are the ones that concentrate most of the interactions with MTCs, mainly GARP and the TRAPPs. Up to date, no study had observed that MTCs and P4-ATPases might function together. In this Thesis I investigated the molecular mechanism that mediates the interplay in this network of essential components for the trafficking machinery. I found that GARP is responsible to transport Dnf1 from late endosomes to trans-Golgi network during the recycling of Dnf1 to the plasma membrane, a transport step that is essential to maintain the correct distribution of PE in the plasma membrane. Furthermore, I have found that Drs2 is required for the proper functioning of the Cytoplasm-to-Vacuole Targeting (Cvt) pathway, a model historically used to study selective autophagy. When the Cvt pathway is triggered, both TRAPIII and a transmembrane protein named Atg9 reach the phagophore assembly site (PAS) to start the process. The results of my thesis show that, in response to a temperature decrease, the cell induces the binding between TRAPPIII and Drs2 and that this interaction is required to sustain the adequate transport of Atg9 to the PAS. In line with this, I have seen Drs2 is fundamental for the biogenesis of vesicles loaded with Atg9 from endocytic compartments. Altogether, this data evidences a new function of this flippase in the Cvt pathway that might have important implications in other types of autophagy. Additionally, I identified a short motif in the N-terminal tail of Drs2 that is required for the interaction TRAPPIII-Drs2. This is a motif highly conserved among P4-ATPases and other proteins that bind MTCs, what suggests that it might have a more general role in regulating the trafficking of vesicles that remains to be elucidated.

[eng] Transportome alterations have been associated with oncogenesis. The loss of human Concentrative Nucleoside Transporter 1 (hCNT1) during carcinogenesis seems to be a relatively common event in tumors and might by itself contributing to oncogenesis. In fact, hCNT1 can play a dual role in cell biology being a nucleoside and nucleoside-derived drug transporter but also an important player in the modulation of a variety of cellular functions. Accordingly, we have previously reported that hCNT1 function is able to induce physiological changes that are relevant to tumor biology in a transport independent-manner. These observations argued in favor of hCNT1 being a transceptor protein. Thus, in this thesis we focused our study on the role of hCNT1 in cell physiology beyond the mere nucleoside salvage. We found that hCNT1 is able to induce antiproliferative effects in different tumor backgrounds, and its restitution in Hepatocellular carcinoma (HCC) derived cell lines alters several different signaling cascades that are important to many physiological functions of the cell, including cell survival and cell migration. Moreover, hCNT1-induced cell death seems to be triggered by an increase in intracellular Ca2+ levels, which might be related to the interaction of the Ca2+-binding protein S100A11 with hCNT1.

Sugammadex, a γ-cyclodextrin that encapsulates selectively steroidal neuromuscular blocking agents, such as rocuronium or vecuronium, has changed the face of clinical neuromuscular pharmacology. Sugammadex allows a rapid reversal of muscle paralysis. Sugammadex appears to be safe and well tolerated. Its blood-brain barrier penetration is poor (< 3% in rats), and thus no relevant central nervous toxicity is expected. However the blood brain barrier permeability can be altered under different conditions (i.e. neurodegenerative diseases, trauma, ischemia, infections, or immature nervous system). Using MTT, confocal microscopy, caspase-3 activity, cholesterol quantification and Western-blot we determine toxicity of Sugammadex in neurons in primary culture. Here we show that clinically relevant sugammadex concentrations cause apoptotic/necrosis neuron death in primary cultures. Studies on the underlying mechanism revealed that sugammadex-induced activation of mitochondria- dependent apoptosis associates with depletion of neuronal cholesterol levels. Furthermore SUG increase CytC, AIF, Smac/Diablo and CASP-3 protein expression in cells in culture. Potential association of SUG-induced alteration in cholesterol homeostasis with oxidative stress and apoptosis activation occurs. Furthermore, resistance/sensitivity to oxidative stress differs between neuronal cell types.

[spa] La adherencia a la Dieta Mediterránea se ha asociado a una disminución de enfermedades de alta prevalencia como el cáncer colorrectal, y este hecho se ha relacionado con el consumo moderado de vino y aceite de oliva, que son dos alimentos característicos de la Dieta Mediterránea. Ambos alimentos son una mezcla compleja de numerosos componentes con estructura y características fisicoquímicas diversas, entre los que destacan los polifenoles. Los estilbenos y los tirosoles son polifenoles que se encuentran presentes en pocos alimentos, entre los que se incluyen el vino y el aceite de oliva. El objetivo de este trabajo ha sido estudiar el papel de componentes bioactivos del vino y el aceite de oliva en el control del crecimiento de células epiteliales intestinales. Para ello, como modelo experimental se utilizó una línea celular procedente de un adenocarcinoma humano denominada Caco-2. El resveratrol (RV), que es un polifenol representativo del vino, es capaz de regular el crecimiento de las células epiteliales intestinales in vitro a concentraciones que no se alcanzan en plasma tras un consumo moderado de vino, dado que esta molécula se metaboliza muy rápidamente. En este sentido, los resultados muestran que los metabolitos sulfatados y glucuronidados del RV mantienen la capacidad antioxidante de este, así como la de inhibir el crecimiento y de inducir apoptosis. El trans-piceido, que es el glucósido del RV presente en concentraciones elevadas en el vino, presenta un efecto antioxidante, una actividad antimitogénica y pro-apoptótica sobre las células Caco-2 similar a la del RV. Además se observó que la capacidad antioxidante del RV está relacionada con la presencia de grupos hidroxilo en posición 3, 5 y 4’. Aunque es este último hidroxilo el determinante de dicha actividad antioxidante. La sustitución de estos hidroxilos por grupos cloro o metoxi, a pesar de reducir su actividad antioxidante, conserva sus efectos biológicos sobre el crecimiento de las células Caco-2. Los ácidos grasos monoinsaturados, y en especial el ácido oleico, que es el ácido graso mayoritario del aceite de oliva, inducen el crecimiento de las Caco-2, probablemente a través de la síntesis de metabolitos por la vía de las lipoxigenasas. Algunos ácidos grasos poliinsaturados, como el eicosapentaenoico y el docosahexaenoico, presentaron un efecto dual, ya que a baja concentración indujeron la proliferación celular, pudiendo estar implicados en este efecto las prostaglandinas y los ácidos hidroxieicosapentaenoicos. Mientras que una concentración alta de estos ácidos grasos poliinsaturados tuvo un efecto opuesto sobre el crecimiento celular e indujo apoptosis. Es interesante señalar que el efecto mitogénico del ácido oleico fue revertido por diversos componentes minoritarios del aceite de oliva como el hidroxitirosol, la oleuropeina, el escualeno, el ácido maslínico y el pinoresinol. Algunos de los cuales inhibieron la liberación de ácido araquidónico y la síntesis de eicosanoides, elementos implicados en el control del crecimiento de las células epiteliales intestinales. En conclusión, la cantidad y el tipo de ácidos grasos consumidos junto con los componentes minoritarios del vino y del aceite de oliva, así como sus metabolitos, pueden tener un importante papel en el control de la cascada del ácido araquidónico y de la proliferación de células epiteliales intestinales Caco-2., [eng] Adherence to Mediterranean Diet has been associated with colorectal cancer prevention, this fact has been linked to moderate consumption of wine and olive oil, two characteristic foods of Mediterranean Diet. Both are a complex mixture of components among which polyphenols stand out. Stilbenes and tyrosols are polyphenols present in wine and olive oil. The objective of this work was to study the effect of bioactive components of wine and olive oil on intestinal epithelial cell growth. For this purpose, Caco-2 cells ware used as experimental model. Resveratrol (RV), a polyphenol representative of wine, is able to regulate intestinal epithelial cells growth in vitro and sulphated and glucuronidated RV metabolites maintain their antioxidant, antiproliferative and apoptotic activity. Trans-piceid is the RV aglycone more present in wine, has antioxidant, anti-mitotic and pro-apoptotic effect similar to that of RV. The antioxidant capacity of RV is related to the presence of hydroxyl groups in 3, 5 and 4 'position, been the last determinant for this activity. The substitution of these hydroxyls for chloro or methoxy groups, despite reducing their antioxidant activity, retains its biological effects on Caco-2 cells growth. Monounsaturated fatty acids, and especially oleic acid, which is the major fatty acid in olive oil, induce Caco-2 cell growth, probably through the synthesis of metabolites from lipoxygenase pathway. Some polyunsaturated fatty acids (PUFA), such as eicosapentaenoic and docosahexaenoic, had a dual effect, since at low concentration they induced cell proliferation, while at high concentration these PUFA had an opposite effect on cell growth and induced apoptosis. Is interesting to note that the mitogenic effect of oleic acid was reversed by some minor components of olive oil such as hydroxytyrosol, oleuropein, squalene, maslinic acid and pinoresinol. Some of which inhibited the release of arachidonic acid and the synthesis of eicosanoids, elements involved in the control of intestinal epithelial cells growth. In conclusion, the amount and type of fatty acids consumed together with minor components of wine and olive oil, as well as their metabolites, may play an important role in arachidonic acid cascade regulation and intestinal epithelial Caco-2 cell proliferation.

The MC1R gene plays a crucial role in pigmentation synthesis. Loss-of-function MC1R variants, which impair protein function, are associated with red hair color (RHC) phenotype and increased skin cancer risk. Cultured cutaneous cells bearing loss-of-function MC1R variants show a distinct gene expression profile compared to wild-type MC1R cultured cutaneous cells. We analysed the gene signature associated with RHC co-cultured melanocytes and keratinocytes by Protein-Protein interaction (PPI) network analysis to identify genes related with non-functional MC1R variants. From two detected networks, we selected 23 nodes as hub genes based on topological parameters. Differential expression of hub genes was then evaluated in healthy skin biopsies from RHC and black hair color (BHC) individuals. We also compared gene expression in melanoma tumors from individuals with RHC versus BHC. Gene expression in normal skin from RHC cutaneous cells showed dysregulation in 8 out of 23 hub genes (CLN3, ATG10, WIPI2, SNX2, GABARAPL2, YWHA, PCNA and GBAS). Hub genes did not differ between melanoma tumors in RHC versus BHC individuals. The study suggests that healthy skin cells from RHC individuals present a constitutive genomic deregulation associated with the red hair phenotype and identify novel genes involved in melanocyte biology.

Voltage-dependent K+ channels (Kv) are involved in the proliferation and differentiation of mammalian cells, since Kv antagonists impair cell cycle progression. Although myofibers are terminally differentiated, some myoblasts may re-enter the cell cycle and proliferate. Since Kv1.3 and Kv1.5 expression is remodeled during tumorigenesis and is involved in smooth muscle proliferation, the purpose of this study was to analyze the expression of Kv1.3 and Kv1.5 in smooth muscle neoplasms. In the present study, we examined human samples of smooth muscle tumors together with healthy speci­mens. Thus, leiomyoma (LM) and leiomyosarcoma (LMS) tumors were analyzed. Results showed that Kv1.3 was poorly expressed in the healthy muscle and indolent LM specimens, whereas aggressive LMS showed high levels of Kv1.3 expression. Kv1.5 staining was correlated with malignancy. The findings show a remodeling of Kv1.3 and Kv1.5 in human smooth muscle sarcoma. A correlation of Kv1.3 and Kv1.5 expression with tumor aggressiveness was observed. Thus, our results indicate Kv1.5 and Kv1.3 as potential tumorigenic targets for aggressive human LMS.

Voltage dependent potassium channels are a group of plasma membrane ion channels with a key role in the immune system as the predominant ion channels controlling the resting membrane potential and tuning intracellular Ca2+ signaling in lymphocytes, monocytes, macrophages, and dendritic cells. Leukocytes present a limited Kv repertoire, including Kv1.3 and Kv1.5 channel isoforms. Kv1.3 is expressed in the immune system, and the blockade of this channel is associated with selective inhibition of T cell activation and proliferation. A functional Kv channel is an oligomeric complex composed of pore-forming and ancillary subunits. The KCNE gene family (KCNE1-5) is a novel group of modulatory Kv channel elements expressed in several tissues including leukocytes. KCNE peptides are small single spanning membrane proteins known to modulate Kv channels trafficking and biophysical properties. The hypothesis of the present PhD thesis entitled “Kv1.3 and Kv1.5 channels in leukocytes” was that changes in the channelosome composition by modulating the heterooligomeric combinations of the Kv1.3 channelosome control physiological and neoplastic cell growth as well as leukocyte responses. Evidence suggests that Kv channels are involved in cell differentiation and cell cycle control (because non-specific drugs, such as 4-AP and TEA, impaire proliferation), and they are also known to be remodeled during carcinogenesis. Thus, we elucidated the role of Kv1.3 and Kv1.5 channels in cell growth and their relationship with cancer, in models such as B lymphocytes and lymphomas (non-Hodgkin lymphomas), pancreatic ductal adenocarcinoma (PDAC) and glioblastomas. In spite of its significance, the mechanisms that regulate Kv1.3 and its role in the T cell activation are not well known. To that end, we analyzed the expression of KCNEs ancillary subunits upon different states of activation and proliferation of leukocytes (macrophages, T and B lymphocytes). In addition, recent data from our laboratory demonstrate that KCNE4, acting as a dominant negative ancillary subunit, physically interacts with Kv1.3 inhibiting K+ currents and retaining the channel intracellularly. Therefore, we studied the Kv1.3 modulation by the auxiliary subunit KCNE4 in leukocytes.

Contractile tension of alveolar epithelial cells plays a major role in the force balance that regulates the structural integrity of the alveolar barrier. The aim of this work was to study thrombin-induced contractile forces of alveolar epithelial cells. A549 alveolar epithelial cells were challenged with thrombin, and time course of contractile forces was measured by traction microscopy. The cells exhibited basal contraction with total force magnitude 55.0 ± 12.0 nN (mean ± SE, n = 12). Traction forces were exerted predominantly at the cell periphery and pointed to the cell center. Thrombin (1 U/ml) induced a fast and sustained 2.5-fold increase in traction forces, which maintained peripheral and centripetal distribution. Actin fluorescent staining revealed F-actin polymerization and enhancement of peripheral actin rim. Disruption of actin cytoskeleton with cytochalasin D (5 μM, 30 min) and inhibition of myosin light chain kinase with ML-7 (10 μM, 30 min) and Rho kinase with Y-27632 (10 μM, 30 min) markedly depressed basal contractile tone and abolished thrombin-induced cell contraction. Therefore, the contractile response of alveolar epithelial cells to the inflammatory agonist thrombin was mediated by actin cytoskeleton remodeling and actomyosin activation through myosin light chain kinase and Rho kinase signaling pathways. Thrombin-induced contractile tension might further impair alveolar epithelial barrier integrity in the injured lung.

Una de les aproximacions que es fa servir en recerca biomèdica per assajar fàrmacs és analitzar els efectes que tenen sobre el metabolisme cel·lular amb cèl·lules humanes en cultiu. El metabolisme i la fisiologia de les cèl·lules humanes és raonablement similar al d'uns organismes molt més simples, els llevats. Aquesta simplicitat fa que sigui molt més fàcil i econòmic mantenir-los al laboratori, per la qual cosa resultaria molt avantatjós que es poguessin utilitzar en aquests estudis. ¿Fins a quin punt, però, els gens del llevat realitzen la mateixa funció que els equivalents humans?

Summary Neurons obtained directly from human somatic cells hold great promise for disease modeling and drug screening. Available protocols rely on overexpression of transcription factors using integrative vectors and are often slow, complex, and inefficient. We report a fast and efficient approach for generating induced neural cells (iNCs) directly from human hematopoietic cells using Sendai virus. Upon SOX2 and c-MYC expression, CD133-positive cord blood cells rapidly adopt a neuroepithelial morphology and exhibit high expansion capacity. Under defined neurogenic culture conditions, they express mature neuronal markers and fire spontaneous action potentials that can be modulated with neurotransmitters. SOX2 and c-MYC are also sufficient to convert peripheral blood mononuclear cells into iNCs. However, the conversion process is less efficient and resulting iNCs have limited expansion capacity and electrophysiological activity upon differentiation. Our study demonstrates rapid and efficient generation of iNCs from hematopoietic cells while underscoring the impact of target cells on conversion efficiency., Graphical Abstract, Highlights • Expandable iNCs are generated from CB cells by SeV at high efficiency • CB-iNCs differentiate into functional neurons in vitro • CB stem/progenitor cells are more amenable to neural conversion than PB-MNCs, Giorgetti, Sanchez-Pernaute and colleagues report a fast and efficient approach for generating functional induced neurons from both cord blood and peripheral blood cells using SOX2 and c-MYC Sendai virus. However, the reprogramming of adult peripheral blood cells is less efficient. This study provides additional evidence that the origin and the nature of target cells may impact the outcome of the conversion process.

[cat] Els organismes multicel·lulars estan constituïts per diferents tipus cel·lulars ordenats d’una certa manera, formant teixits amb funcions específiques. L’organització de cèl·lules de tipus diferents pot donar lloc a patrons espacio-temporals Aquesta Tesi es basa en l’estudi de com a partir d’un teixit de cèl·lules equivalents — estat homogeni precursor — s’estableixen patrons ordenats de tipus cel·lulars diferents. En particular, ens hem centrat en l’estudi d’un tipus de patrons que sorgeixen en teixits animals que tenen dos tipus cel·lulars i que presenten un ordre fi en el teixit, i.e. de longitud d’ona de poques cèl·lules. Aquest tipus de patrons són formats degut al efecte de la inhibició lateral. La inhibició lateral és un fenomen en el qual cèl·lules precursores equivalents intenten adoptar un cert estat o destí cel·lular per a diferenciar-se en un tipus cel·lular en particular, i al mateix temps inhibeixen a les seves cèl·lules veïnes que adquireixin aquest mateix estat. Aquest procés dinàmic dóna lloc a un patró fi, on les cèl·lules que han finalment adoptat l’estat desitjat vénen rodejades per cèl·lules que són inhibides, i que acabaran diferenciant-se en un tipus cel·lular diferent. Aquest tipus de patró es troba en una àmplia varietat de teixits animals, com ara en la retina ,i en l’oïda interna de vertebrats, i en l’ull de la mosca Drosophila, [eng] Multicellular organisms are constituted by different kinds of cells which are arranged in a particular way, forming tissues with specific functions. The organization of these different cells can give rise to regular spatiotemporal patterns. In this Thesis we evaluate from a theoretical perspective the effects of different regulatory elements of the Notch signaling pathway in lateral inhibition patterning. These new elements under study are motivated by recent experimental observations. For studying them, we reformulate a phenomenological model proposed by Collier and colleagues (1996). Our modeling approach is based on coupled ordinary differential equations in hexagonal and irregular bidimensional lattices. We use both deterministic and stochastic approaches. We analyze the pattern formation capabilities of our proposed models by using different analytical tools and integrate numerically our dynamical equations. We focus on four main topics. In the first topic we study how a neurogenic differentiation wavefront in the embryonic vertebrate retina depends on the state of the invaded tissue. Our results show that the properties (pattern formed, shape and velocity) of progressing fronts of lateral inhibition depend crucially on the presence of ligand ahead of the differentiation front. We find similar results in a planar growing wavefront that would mimic morphogenetic furrow progression in embryonic Drosophila eye. Hence, our results point to a mechanism for neurogenic front regulation, and to a potential new design principle. In the second topic, we study the effects of a diffusible ligand in the context of lateral inhibition. We show that the diffusible ligand per se combined with its inhibition by Notch is not able to generate a pattern. Our results indicate that diffusible ligand with the classical lateral inhibition circuit softens and destroys the lateral inhibition pattern. At intermediate diffusion rates, diffusion can help to create perfect patterns. The third topic focuses on the study of receptor-ligand interactions within the same cell, what is called cis-interactions. We study the effect of Notch signal-productive cis-interactions in combination with another signaling source in two different situations: (i) in a multicellular scenario, where the other signaling source would be provided by the trans-interactions, and (ii) in a single-cell scenario in which a basal ligand-independent signaling source would be provided. In both situations, we predict that cis-interactions can drive cis-inhibition - i.e. an effective depletion of the signal production rate - at weak cis-signaling rates when acting together with a stronger signaling source, e.g. trans-interactions or with a ligand-independent signaling source. Our work also shows that cis-inhibition in the single-cell system together with a basal signal production can drive bistability. In the multicellular case, we observe that by increasing the amount of cis-interactions in the cis-inhibition scenario the proportion of high-Delta fated cells in a tissue gradually increases. In the fourth topic we study the case of hair cell differentiation in the embryonic chick inner ear. In this context, Notch pathway operates in two opposite modes with two different ligands: first, lateral induction through Jag1 ligand and afterwards, lateral inhibition through Dl1 ligand. We predict that relative signaling rates (or strengths) by Jag1 and Dl1 when bound to Notch are critical for the transit of operating modes. Also, we predict that in the lateral inhibition stage, competition between Dl1 and Jag1 ligands arise. This competition introduces an extra intercellular mutual inhibitory feedback loop, contributing to lateral inhibition. Overall, this Thesis presents new theoretical results and predictions on pattern formation in the context of lateral inhibition mediated by Notch signaling.

Background: Prostaglandin E2 (PGE(2)), the main metabolite of cyclooxygenase (COX), is a well-known anti-fibrotic agent. Moreover, myofibroblasts expressing alpha-smooth muscle actin (alpha-SMA), fibroblast expansion and epithelial-mesenchymal transition (EMT) are critical to the pathogenesis of idiopathic pulmonary fibrosis (IPF). Our aim was to investigate the expression of COX-2 and PGE(2) in human lung myofibroblasts and establish whether fibroblast-myofibroblast transition (FMT) and EMT are associated with COX-2 and PGE(2) down-regulation. Methods: Fibroblasts obtained from IPF patients (n = 6) and patients undergoing spontaneous pneumothorax (control, n = 6) and alveolar epithelial cell line A549 were incubated with TGF-beta 1 and FMT and EMT markers were evaluated. COX-2 and alpha-SMA expression, PGE(2) secretion and cell proliferation were measured after IL-1 beta and PGE(2) incubation. Results: Myofibroblasts from both control and IPF fibroblast cultures stimulated with IL-1 beta showed no COX-2 expression. IPF fibroblasts showed increased myofibroblast population and reduced COX-2 expression in response to IL-1 beta. TGF-beta 1 increased the number of myofibroblasts in a time-dependent manner. In contrast, TGF-beta 1 induced slight COX-2 expression at 4 h (without increase in myofibroblasts) and 24 h, but not at 72 h. Both IPF and control cultures incubated with TGF-beta 1 for 72 h showed diminished COX-2 induction, PGE(2) secretion and alpha-SMA expression after IL-1 beta addition. The latter decreased proliferation in fibroblasts but not in myofibroblasts. A549 cells incubated with TGF-beta 1 for 72 h showed down-regulated COX-2 expression and low basal PGE(2) secretion in response to IL-1 beta. Immuno-histochemical analysis of IPF lung tissue showed no COX-2 immuno-reactivity in myofibroblast foci. Conclusions: Myofibroblasts are associated with COX-2 down-regulation and reduced PGE(2) production, which could be crucial in IPF development and progression.

Pyruvate dehydrogenase kinase 4 (PDK4) inhibition by nuclear factor-κB (NF-κB) is related to a shift towards increased glycolysis during cardiac pathological processes such as cardiac hypertrophy and heart failure. The transcription factors estrogen-related receptor-α (ERRα) and peroxisome proliferator-activated receptor (PPAR) regulate PDK4 expression through the potent transcriptional coactivator PPARγ coactivator-1α (PGC-1α). NF-κB activation in AC16 cardiac cells inhibit ERRα and PPARβ/δ transcriptional activity, resulting in reduced PGC-1α and PDK4 expression, and an enhanced glucose oxidation rate. However, addition of the NF-κB inhibitor parthenolide to these cells prevents the downregulation of PDK4 expression but not ERRα and PPARβ/δ DNA binding activity, thus suggesting that additional transcription factors are regulating PDK4. Interestingly, a recent study has demonstrated that the transcription factor E2F1, which is crucial for cell cycle control, may regulate PDK4 expression. Given that NF-κB may antagonize the transcriptional activity of E2F1 in cardiac myocytes, we sought to study whether inflammatory processes driven by NF-κB can downregulate PDK4 expression in human cardiac AC16 cells through E2F1 inhibition. Protein coimmunoprecipitation indicated that PDK4 downregulation entailed enhanced physical interaction between the p65 subunit of NF-κB and E2F1. Chromatin immunoprecipitation analyses demonstrated that p65 translocation into the nucleus prevented the recruitment of E2F1 to the PDK4 promoter and its subsequent E2F1-dependent gene transcription. Interestingly, the NF-κB inhibitor parthenolide prevented the inhibition of E2F1, while E2F1 overexpression reduced interleukin expression in stimulated cardiac cells. Based on these findings, we propose that NF-κB acts as a molecular switch that regulates E2F1-dependent PDK4 gene transcription.

La función de las caveolas y de la caveolina in vivo ha sido motivo de controversia, de hecho se han implicado en procesos de endocitosis y transcitosis, transporte de colesterol y ácidos grasos, regulación de procesos de transducción de señales y tumorigénesis (Liu et al., 2002). Sin embargo, la generación de los ratones KO de caveolina-1 (Drab et al., 2001; Razani et al., 2001a) mostraba que estos ratones eran perfectamente viables y fértiles, aunque estudios posteriores relacionaban a caveolina-1 con la homeostasis lipídica (Razani et al., 2002a).A partir de estos antecedentes, el objetivo general de esta tesis ha sido: · Definir la función de las caveolas/ caveolina-1 en la fisiología del adipocitoNos planteamos así, nuestro primer objetivo:1- Obtención de líneas 3T3L1 deficientes en caveolina-1 mediante silenciamiento génico posttranscripcional.2- Analizar la presencia de caveolas y la distribución de la caveolina-1 remanente en las membranas de los adipocitos y estudiar la estructura y composición de los rafts lipídicos/caveolas en estos adipocitos deficientes en caveolina-1.3- Analizar la diferenciación adipocitaria de los preadipocitos 3T3L1 deficientes en caveolina-1. 4- Analizar la acción de la insulina en los adipocitos deficientes en caveolina-1, 5- Estudiar el papel de caveolina-1 en el metabolismo lipídico del adipocito.A partir de los objetivos planteados conseguimor obtener las siguientes conclusiones en el desarrollo de esta tesis:1. Se ha conseguido la transducción eficiente de adipocitos 3T3L1 maduros usando estas partículas lentivirales que expresan transgenes, aunque no se ha logrado transducir los adipocitos maduros eficazmente en el caso de los lentivirus que expresan moléculas de siRNA.2. Los preadipocitos 3T3L1 son eficientemente transducidos usando las partículas lentivirales que permiten la expresión tanto de transgenes como de moléculas de siRNA. Mediante esta técnica hemos generado líneas estables 3T3L1 que expresan moléculas de siRNA de caveolina-1 y que presentan una reducción en la expresión de la proteína caveolina-1 de más del 93%.3. La disminución de la proteína caveolina-1 en más de un 93% en adipocitos 3T3L1 provoca una disminución equivalente en el número de caveolas en la membrana plasmática.4. La deficiencia en caveolina-1 en los adipocitos 3T3L1 no afecta a la localización de los marcadores proteicos y lipídicos característicos de los rafts lipídicos, que continúan recuperándose en las fracciones ligeras de los extractos celulares durante el aislamiento bioquímico de los rafts lipídicos. 5. La deficiencia en caveolina-1 y caveolas no afecta a la diferenciación de los preadipocitos 3T3L1 a adipocitos maduros, ni tampoco a las características morfológicas y el contenido lipídico (triacilglicéridos y colesterol) de los adipocitos maduros. 6. Los adipocitos 3T3L1 con una disminución del 93% en la expresión de caveolina-1, presentan una reducción del 50% en la expresión de las proteínas GLUT4 y del 40% en el caso del receptor de insulina, sin que este hecho responda a una menor cantidad de sus RNA mensajeros, sino a una disminución en la vida media de estas proteínas. Este dato sugiere que caveolina-1 participe en la estabilidad de estas proteínas.7. El déficit de caveolina-1 en los adipocitos 3T3L1 no afecta a las vías de señalización de la insulina (dependiente de PI3K y dependiente de Cbl-TC10) ni tampoco a la translocación de GLUT4 a la membrana plasmática inducida por la insulina ni a la estimulación del transporte de glucosa. 8. La presencia de caveolas/caveolina-1 no es necesaria para la correcta internalización de GLUT4 durante la reversión de la acción de la insulina.9. Las caveolas/caveolina-1 tampoco son necesarias para la acción de la insulina inhibiendo o activando la lipólisis y la lipogénesis en los adipocitos 3T3L1, respectivamente.10. La deficiencia de caveolina-1 y caveolas no altera el transporte de ácido palmítico a través de la membrana plasmática de adipocitos 3T3L1.Esta deficiencia tampoco altera la activación lipolítica por los efectores beta-adrenérgicos: Isoproterenol 10mu-M e IBMX 0,5mM en los adipocitos 3T3L1.PALABRAS CLAVE: Cavelonia-1, Caveola, Adipocito, Insulina, GLUT4, Receptor de insulina, Caveolae are a specialized type of lipid rafts that are stabilized by oligomers of caveolin protein. Caveolae are particularly enriched in adipocytes. Here we analyzed the effects of caveolin-1 knockdown and caveolae ablation on adipocyte function. To this end, we obtained several multiclonal mouse 3T3-L1 cell lines with a reduced expression of caveolin-1 (95% reduction) by a small interfering RNA approach using lentiviral vectors. Control cell lines were obtained by lentiviral infection with lentiviral vectors encoding appropriate scrambled RNAs. Caveolin-1 knockdown adipocytes showed a drastic reduction in the number of caveolae (95% decrease) and cholera toxin-labeling was reorganized in dynamic plasma membrane microdomains. Caveolin-1 depletion caused a specific decrease in GLUT4 glucose transporter and insulin receptor protein levels. This reduction was not the result of a generalized defect in adipocyte differentiation or altered gene expression but was explained by faster degradation of these proteins. Caveolin-1 knockdown adipocytes showed reductions in insulin-stimulated glucose transport, insulin-triggered GLUT4 recruitment to the cell surface and insulin receptor activation. In all, our data indicate that caveolin-1 loss-of-function reduces maximal insulin response through lowered stability and diminished expression of insulin receptors and GLUT4. We propose that caveolin-1/caveolae control insulin action in adipose cells.

El procesamiento y la presentación de antígenos es un mecanismo clave en el desarrollo de la respuesta inmunitaria. En el presente trabajo, se ha estudiado la regulación de los genes tap1 y lmp2. Estos genes codifican proteínas cuyas funciones son vitales para la presentación antigénica de proteínas endógenas a través del MHC de clase I. La proteína lmp2 forma parte de un complejo multiproteico conocido como proteosoma, cuya función es la degradación de proteínas citosólicas dando lugar a la formación de péptidos. La proteína tap1 está implicada en el transporte de los péptidos al interior retículo endoplasmático, para su posterior carga en las moléculas de histocompatibilidad de clase I. Ambas proteínas son codificadas por genes que comparten una misma región promotora en sentidos opuestos.Los resultados confirman que la inducción de la expresión de los genes murinos tap1 y lmp2 mediada por el IFN-gamma tiene lugar en células de diferentes linajes y ocurre entre una y tres horas post-activación. El análisis de otros estímulos inductores de la expresión de tap1 y lmp2 demostró patrones de expresión diferentes del obtenido con el IFN-gamma. El LPS induce la expresión de tap1 y lmp2 a las seis horas de activación y el TNF-alfa a las 24 horas. En nuestros experimentos, observamos, en macrófagos derivados de médula ósea mantenidos en medio de cultivo en ausencia de suero (situación de estrés), una inducción de la expresión de los genes tap1 y lmp2. Esta inducción tiene lugar aproximadamente a las 24 horas del estímulo, siguiendo un patrón de inducción semejante al observado tras la activación con el TNF-alfa.Utilizando cicloheximida, un inhibidor de la síntesis proteica, se comprobó que la inducción de tap1 y lmp2 por el IFN-gamma no es dependiente de la síntesis proteica. Los tránscritos de ARN son altamente estables, ya que sus niveles no decaen de forma rápida en presencia de actinomicina D, que actúa bloqueando la transcripción. La elevada estabilidad de los ARNm indica que el IFN-gamma no modifica la vida media del ARN, sino que actúa a nivel de la transcripción de los dos genes.Se determinó utilizando ratones knock out para STAT1 que este factor de transcripción está implicado en la señal de transducción por el IFN-gamma, el LPS y el TNF-alfa. Sin embargo, la expresión de tap1 y lmp2 inducida por el IFN-alfa/beta es independiente de STAT1. Las cajas reguladoras GAS e IRF-1 son esenciales en la inducción de la expresión de tap1 y lmp2 mediada por el IFN-gamma. En cambio, sólo la caja GAS está implicada en la inducción de ambos genes por el LPS. Finalmente, las cajas IRF-1 y NFB son esenciales en el caso de la activación mediada por el TNF-alfa.Los estímulos inhibidores TGF-beta y los glucocorticoides, inhiben la expresión de los genes tap1 y lmp2 inducida por el IFN-. Nuestros resultados demuestran también que la expresión de PU.1 en los fibroblastos murinos, mediante la transfección de un vector de expresión que codifica para esta proteína, inhibe la expresión de los genes tap1 y lmp2 inducida por IFN-gamma. Posiblemente, debido a la localización de una caja PU.1 dentro de la caja GAS, el factor de transcripción PU.1 puede afectar a la transcripción de los ARNm para tap1 y lmp2 En resumen, en este trabajo hemos determinado la regulación de los genes tap1 y lmp2 implicados en la presentación antigénica a través de las moléculas del MHC de clase I. Existen citocinas que son capaces de inducir mientras que otras inhiben la expresión de tap1 y lmp2. Las vías de señalización comparten elementos comunes como STAT1, así como regiones promotoras implicadas en la regulación génica.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited., Local neocortical circuits are characterized by stereotypical physiological and structural features that subserve generic computational operations. These basic computations of the cortical microcircuit emerge through the interplay of neuronal connectivity, cellular intrinsic properties, and synaptic plasticity dynamics. How these interacting mechanisms generate specific computational operations in the cortical circuit remains largely unknown. Here, we identify the neurophysiological basis of both the rate of change and anticipation computations on synaptic inputs in a cortical circuit. Through biophysically realistic computer simulations and neuronal recordings, we show that the rate-of-change computation is operated robustly in cortical networks through the combination of two ubiquitous brain mechanisms: short-term synaptic depression and spike-frequency adaptation. We then show how this rate-of-change circuit can be embedded in a convergently connected network to anticipate temporally incoming synaptic inputs, in quantitative agreement with experimental findings on anticipatory responses to moving stimuli in the primary visual cortex. Given the robustness of the mechanism and the widespread nature of the physiological machinery involved, we suggest that rate-of-change computation and temporal anticipation are principal, hard-wired functions of neural information processing in the cortical microcircuit., AC is supported by the Researcher Stabilization Program of the Health Department of the Generalitat de Catalunya.

[spa] Los túbulos epiteliales del hepatopáncreas de Palaemonetes zariquieyi presentan externamente fibras musculares con disposición circular y perpendicular posibilitando la existencia de movimientos persistálticos en la glándula. En la capa epitelial, formada por células prismáticas y polarizadas, hemos tipificado cinco modelos celulares: 1) Células E (embrionarias) situadas mayoritariamente en la región distal de los túbulos presentan frecuentes mitosis. 2) Células R (absorbentes) presentan en su citoplasma numerosas gotas lipídicas y gránulos de glucógeno, con una función eminentemente de reserva. Estas células R intervienen en la absorción y metabolismo lipídico, detectándose en el interior de los sáculos golgianos la formación de partículas de naturaleza lipoproteica. En la región basal se localiza un sistema membranoso túbulo-laminar, que adopta un aspecto reticular laberintico. Al aplicar trazadores del espacio intercelular (técnica de nitrato de lantano, o del ácido tánico) se detecta la continuidad del sistema membranoso con el espacio intercelular. 3) Células F (fibrilares): especializadas en la síntesis y secreción proteica presentan el retículo endoplasmático rugoso muy desarrollado. El complejo de Golgi presenta habitualmente su cara de formación hiperdilatada. 4) Células B (vacuoladas): de citoplasma vacuolado presentan una elevada actividad pinocítica y una digestión intracelular. 5) Células endocrinas: de situación basal presentan un núcleo lobulado y gránulos de secreción electrodensos. Se ha realizado un estudio morfométrico de los cuatro tipos celulares mayoritarios (células embrionarias, células absorbentes, células fibrilares, células vacuoladas) y una cuantificación de los mismos en diversos tramos del túbulo hepatopancreático.

To determine the effect of aging on IFN-γ‐induced MHC class II antigen expression, we produced bone marrow‐derived macrophages in vitro. In these conditions, we analyzed the effect of aging on the genomic expression of macrophages without the influence of other cell types that may be affected by aging. Although macrophages from young and aged mice showed an identical degree of differentiation, after incubation with IFN-γ, the expression at the cell surface of the IA complex and the levels of IAβ protein and mRNA were lower in aged macrophages. Moreover, the transcription of the IAβ gene was impaired in aged macrophages. The amount of transcription factors that bound to the W and X, but not to the Y, boxes of the IAβ promoter gene was lower in aged macrophages. Similar levels of CIITA mRNA were found after IFN-γ treatment of both young and aged macrophages. This shows that neither the initial cascade that starts after the interaction of IFN-γ with the receptor nor the second signals involved in the expression of CIITA are impaired in aged macrophages. These data indicate that aging is associated with low levels of MHC class II gene induction by IFN-γ because of impaired transcription. J. Clin. Invest. 107:485‐493 (2001).

Normal pigmentation depends on the uniform distribution of melanin-containing vesicles, the melanosomes, in the epidermis. Griscelli syndrome (GS) is a rare autosomal recessive disease, characterized by an immune deficiency and a partial albinism that has been ascribed to an abnormal melanosome distribution. GS maps to 15q21 and was first associated with mutations in the myosin-V gene. However, it was demonstrated recently that GS can also be caused by a mutation in the Rab27a gene. These observations prompted us to investigate the role of Rab27a in melanosome transport. Using immunofluorescence and immunoelectron microscopy studies, we show that in normal melanocytes Rab27a colocalizes with melanosomes. In melanocytes isolated from a patient with GS, we show an abnormal melanosome distribution and a lack of Rab27a expression. Finally, reexpression of Rab27a in GS melanocytes restored melanosome transport to dendrite tips, leading to a phenotypic reversion of the diseased cells. These results identify Rab27a as a key component of vesicle transport machinery in melanocytes.

We have compared by immunocytochemistry and immunoblotting the expression and distribution of adhesion molecules participating in cell-matrix and cell-cell interactions during embryonic development and regeneration of rat liver. Fibronectin and the fibronectin receptor, integrin alpha 5 beta 1, were distributed pericellularly and expressed at a steady level during development from the 16th day of gestation and in neonate and adult liver. AGp110, a nonintegrin fibronectin receptor was first detected on the 17th day of gestation in a similar, nonpolarized distribution on parenchymal cell surfaces. At that stage of development haemopoiesis is at a peak in rat liver and fibronectin and receptors alpha 5 beta 1 and AGp110 were prominent on the surface of blood cell precursors. During the last 2 d of gestation (20th and 21st day) hepatocytes assembled around lumina. AGp110 was initially depolarized on the surface of these acinar cells but then confined to the lumen and to newly-formed bile canaliculi. At birth, a marked increase occurred in the canalicular expression of AGp110 and in the branching of the canalicular network. Simultaneously, there was enhanced expression of ZO-1, a protein component of tight junctions. On the second day postpartum, presence of AGp110 and of protein constituents of desmosomes and intermediate junctions, DGI and E-cadherin, respectively, was notably enhanced in cellular fractions insoluble in nonionic detergents, presumably signifying linkage of AGp110 with the cytoskeleton and assembly of desmosomal and intermediate junctions. During liver regeneration after partial hepatectomy, AGp110 remained confined to apical surfaces, indicating a preservation of basic polarity in parenchymal cells. A decrease in the extent and continuity of the canalicular network occurred in proliferating parenchyma, starting 24 h after resection in areas close to the terminal afferent blood supply of portal veins and spreading to the rest of the liver within the next 24 h. Distinct acinar structures, similar to the ones in prenatal liver, appeared at 72 h after hepatectomy. Restoration of the normal branching of the biliary tree commenced at 72 h. At 7 d postoperatively acinar formation declined and one-cell-thick hepatic plates, as in normal liver, were observed.