https://uvadoc.uva.es/handle/10324/1215 Dpto. Bioquímica y Biología Molecular y Fisiología - Comunicaciones a congresos, conferencias, etc. 2026-04-10T22:22:03Z https://uvadoc.uva.es/handle/10324/79591 La enfermedad renal crónica (ERC) es una alteración caracterizada por un daño renal estructural y/o funcional, presente por más de 3 meses. Se proyecta que para el año 2040, será la quinta causa de muerte a nivel global. Diversos estudios han señalado que la activación de TLR4 está asociada al desarrollo de la ERC y regula el proceso de la autofagia. La hipótesis de este trabajo sostiene que la activación de TLR4 contribuye al avance de la ERC a través de la regulación de la autofagia. Para validad esta hipótesis, se ha llevado a cabo en primer lugar un análisis in silico de datos de transcriptómica de diferentes tipos de daño renal, el cual respalda la participación de la activación de TLR4 y la autofagia en el daño renal. Luego, se utilizó un modelo murino de ERC en machos y hembras por nefrectomía 5/6, donde se confirmó la presencia de daño renal y se observó un incremento en los marcadores relacionados tanto con la vía de TLR4 como con la autofagia en los ratones con ERC. Además, al comparar los resultados entre ambos sexos de ratones, se pudo descartar la hipótesis de un posible dimorfismo sexual para los mecanismos analizados en este modelo animal. Finalmente, en cultivos celulares con las células HEK293, se evaluó el impacto de la activación de TLR4 mediante la exposición a LPS, observando su efecto sobre la autofagia, para poder comprender en futuros experimentos que parte de la activación de TLR4 podría ser responsable de la desregulación de la autofagia en la ERC. En conjunto, los datos sugieren que la activación de TLR4 podría ser un factor clave en la desregulación de la autofagia asociada a la progresión de la ERC. 2024-01-01T00:00:00Z https://uvadoc.uva.es/handle/10324/79582 La enfermedad renal crónica (ERC) se define como un deterioro progresivo de los riñones presente por más de 3 meses, con una alta prevalencia a nivel mundial. Se asocia con una inflamación crónica, en la que TLR4 desempeña un papel clave produciendo mediadores inflamatorios. Además, TLR4 puede regular la autofagia, que se encuentra alterada en la patología, contribuyendo a su progresión. La hipótesis de este trabajo es que TLR4 es partícipe de la desregulación de la autofagia en la ERC, y que tanto autofagia como inflamación se regulan mutuamente. Para comprobarlo, se han hecho estudios en cultivos celulares HEK293 con TLR4 sobreexpresado, estimulados con LPS, su ligando canónico, para medir el nivel de inflamación y autofagia. Además, se utilizaron inhibidores de ambos procesos para estudiar su regulación. Los resultados demostraron que la activación de TLR4 en células HEK-TLR4 producía citoquinas inflamatorias como TNF-α, IL-6, IL-1β o P-JNK, y un aumento de p62 que se traduce en una disminución de autofagia. Además, en el estudio de la regulación de la inflamación sobre la autofagia, los resultados parecían sugerir una regulación cruzada, en la que la inhibición inflamatoria parecía reestablecer la autofagia. Sin embargo, en el estudio de la regulación de la autofagia basal sobre la inflamación no se obtuvieron datos concluyentes. Como conclusión, la activación de TLR4 parece disminuir la autofagia, y ese efecto podría estar regulada por mediadores inflamatorios como las MAPK; mientras que la autofagia basal no tiene efecto sobre la inflamación, siendo necesario continuar investigando los mecanismos de estos procesos. 2025-01-01T00:00:00Z https://uvadoc.uva.es/handle/10324/68236 OBJECTIVE: The long term aim of our work is to develop effective neuroprotective treatments for Parkinson’s disease (PD). The objective of this study was to test whether reducing endogenous alpha-synuclein expression in the substantia nigra prevented degeneration of dopamine neurons in the rat rotenone model of PD. BACKGROUND: Convergent evidence implicates both alpha-synuclein and mitochondrial dysfunction in the pathogenesis of sporadic Parkinson's disease (PD). Rats exposed chronically to rotenone, a pesticide linked epidemiologically to PD, show systemic partial mitochondrial complex I inhibition, but develop motor abnormalities that respond to dopaminergic medications and specific PD-like neuropathology, including degeneration of substantia nigra dopaminergic neurons and alpha-synuclein aggregation reminiscent of Lewy body pathology. METHODS: We inhibited expression of endogenous alpha-synuclein in the adult rat substantia nigra using a viral vector encoding a short hairpin RNA (shRNA) targeting the SNCA transcript. An isogenic control vector expressed a non-targeting shRNA. After vector transduction, we analyzed motor function, neurochemistry, and nigrostriatal histology at baseline and following chronic rotenone exposure. RESULTS: Significant knockdown of alpha synuclein in vivo did not provoke behavioral abnormalities, loss of nigral dopamine neurons or changes in the number of dendrites or density of striatal terminals, but protected nigral dopaminergic neurons from degeneration following chronic rotenone exposure. Compared with animals transduced with control vector, SNCA knockdown rescued contralateral motor function, and preserved dopaminergic neuron numbers and morphology following rotenone exposure. CONCLUSIONS: Alpha-synuclein is a critical factor in the specific vulnerability of dopaminergic neurons to systemic mitochondrial dysfunction, supporting a model in which genetic modulation of SNCA expression can determine whether environmental exposures trigger PD pathogenesis. shRNA targeting the SNCA transcript should be further evaluated as a possible neuroprotective therapy in PD. 2015-01-01T00:00:00Z https://uvadoc.uva.es/handle/10324/66159 Store-operated Ca2+ entry (SOCE) is the most important Ca2+ entry pathway in non-excitable cells. Colorectal cancer (CRC) show decreased Ca2+ store content and enhanced SOCE that correlate with cancer hallmarks and are associated to remodeling of store-operated channels (SOCs). Normal colonic cells display small, Ca2+-selective currents driven by Orai1 channels. In contrast, CRC cells display larger, non-selective currents driven by Orai1 and TRPC1 channels. Difluoromethylornithine (DFMO), a suicide inhibitor of ornithine decarboxylase (ODC), the limiting step in polyamine biosynthesis, strongly prevents CRC, particularly when combined with sulindac. We asked whether DFMO may reverse SOC remodeling in CRC. We found that CRC cells overexpress ODC and treatment with DFMO decreases cancer hallmarks including enhanced cell proliferation and apoptosis resistance. Consistently, DFMO enhances Ca2+ store content and decreases SOCE in CRC cells. Moreover, DFMO abolish selectively the TRPC1-dependent component of SOCs characteristic of CRC cells and this effect is reversed by the polyamine putrescine. Combination of DFMO and sulindac inhibit both SOC components and abolish SOCE in CRC cells. Finally, DFMO treatment inhibits expression of TRPC1 and STIM1 in CRC cells. These results suggest that polyamines contribute to Ca2+ channel remodeling in CRC and DFMO may prevent CRC by reversing channel remodeling. 2015-01-01T00:00:00Z https://uvadoc.uva.es/handle/10324/66154 Recently we have shown that colon cancer cells undergo remodeling of intracellular Ca2+ homeostasis including changes in store-operated Ca2+ entry (SOCE), store-operated currents and Ca2+ store content associated to changes in molecular players involved in SOCE (Sobradillo et al., 2014). Reversing this remodeling could contribute to protection against cancer and cancer chemoprevention. Difluoromethylornithine (DFMO) or Eflornithine is a suicide inhibitor of ornithine decarboxylase (ODC), the limiting step in the synthesis of polyamines that is considered one of the best chemopreventive compounds against colon cancer. Here we tested the effects of DFMO treatment on SOCE, SOCs, Ca2+ store content, the molecular players involved and resistance to cell death, a critical cancer hallmark. We found that ODC was largely overexpressed in colon cancer cells suggesting increased synthesis of polyamines in colon cancer cells. Short-term treatment with DFMO (500 µM, 12 h) decreased significantly SOCE and store-operated currents in colon cancer cells. DFMO had no effect on Icrac but prevented selectively the appearance of the outward component of store-operated current likely mediated by TRPC1. DFMO also increased Ca2+ store content and the fraction of cells undergoing early apoptosis induced by H2O2. At the molecular level, we found DFMO tend to decrease all molecular players involved in SOCE except Stim2 but the effects were statistically significant only for TRPC1 mRNA. In summary, inhibition of polyamine synthesis decreases SOCE and SOCs in human colon cancer cells acting probably on expression of TRPC1 and tends to increase Ca2+ store content and susceptibility to apoptosis in human colon cancer cells. Thus, reverting partially Ca2+ remodeling in colon cancer cells and likely contributing to colon cancer chemoprevention. 2016-01-01T00:00:00Z https://uvadoc.uva.es/handle/10324/66046 Store-operated Ca2+ entry (SOCE) is the most important Ca2+ entry pathway in non-excitable cells. Colorectal cancer (CRC) show decreased Ca2+ store content and enhanced SOCE that are associated to remodeling of store-operated channels (SOCs), in addition its correlate with cancer hallmarks. Ca2+ remodeling in CRC may consist of changes in expression of Ca2+ channels and pumps and are associated to interaction among different molecular players: nonselective currents driven by Orai1, Orai3 and TRPC1 channels and Stim1 and Stim2 sensors. Difluoromethylornithine (DFMO), a suicide inhibitor of ornithine decarboxylase (ODC), is a strongly preventor of CRC. We asked whether polyamine depletion could be reverse Ca2+ remodelling interactions in CRC. We found that the principal players in normal mucosa cells in SOCE are Orai1 and Stim2, TRPC1 and Stim1 also are presented in low levels. It seems that all this molecules make week interactions between them. However, store-operated channels in CRC display enhanced dual interactions between TRPC1, Stim1 and Orai1 compared to normal cells. Moreover, DFMO treatment decreases specifically the interaction between TRPC1 and Stim1. These data are consistent with previous results that looked like DFMO treatment in CRC cells, specifically affecting the suppression of TRPC1 and Stim1. These results suggest that polyamines contribute to Ca2+ channel remodelling interactions in CRC and DFMO may prevent CRC by reversing channel remodeling. 2020-01-01T00:00:00Z https://uvadoc.uva.es/handle/10324/25024 In heart cells, severa! distinct kinds of transient spatial patterns of cytoplasmic calcium ion concentration ([ Ca2 + )¡) can be observed: (1) [ Ca2 + )¡ waves, in which regions of spontaneously increased [ Ca2 + ] ; propagate at high velocity (100 ¡.im/s) through the cell; (2) Ca2 + 'sparks', which are spontaneous, non-propagating changes in [ Ca2 + ] ; that are localized in small ( == 2 ¡.im) subcellular regions; and (3) evoked [ Ca2 + )¡ transients that are elicited by electrical depolarization, in association with normal excitation-contraction (E­ C) coupling. In confocal [ Ca2 + ] ¡ images, evoked [ Ca2 + ] ; transients appear to be nearly spatially uniform throughout the cell, except during their rising phase or during small depolarizations. In contrast to [Ca2 + )¡ waves and spontaneous Ca2 + sparks, evoked [ Ca2 + ] ; transients are triggered by L-type Ca2 + channel current and they are 'controlled', in the sense that stopping the L-type Ca2 + current stops them. Despite their different characteristics, ali three types of Ca2 + transient involve Ca2 + -induced release of Ca2 + from the sarcoplasmic reticulum. Here, we address the question of how the autocatalytic process of Ca2 + -induced Ca2 + release, which can easily be understood to underlie spontaneous regenerative ('uncontrolled'), propagating [Ca2 + )¡ waves, might be 'harnessed', under other circumstances, to produce controlled changes in [ Ca2 + ]¡, as during normal excitation-contraction coupling, or changes in [ Ca2 + )¡ that do not propagate. We discuss our observations of Ca2 + waves, Ca2 + sparks and normal Ca2 + transients in heart cells and review our results on the 'gain' of Ca2 + -induced Ca2 + release. We discuss a model involving Ca2 + microdomains beneath L-type Ca2 + channels, and clusters of Ca2 + -activated Ca2 + release channels in the sarcoplasmic reticulum which may form the basis of the answer to this question 1995-01-01T00:00:00Z