El uniportador de calcio mitocondrial en cultivos de neuronas de hipocampo de rata

La captura de Ca2+ por parte de la mitocondria se debe principalmente a la apertura del Uniportador de Calcio Mitocondrial. La hipótesis del envejecimiento neuronal postula que durante el envejecimiento se produce una sobrecarga de Ca2+ mitocondrial que promueve la apoptosis celular (Fig. 7). Además, estudios previos de nuestro laboratorio han mostrado que la entrada de calcio a la mitocondria en neuronas jóvenes, maduras y envejecidas varía en respuesta a un estímulo con NMDA. Todos estos antecedentes nos han llevado a estudiar si existen cambios en la expresión del Uniportador de Calcio Mitocondrial en las neuronas con el envejecimiento. Para ello se han tomado cultivos a diferentes días de incubación, 2, 8, y 15 considerando este último un cultivo envejecido. Puesto que el cultivo de hipocampo es un cultivo mixto cuyas poblaciones dependen de la presencia de suero también se ha querido estudiar si la ausencia de suero provoca cambios en la expresión del MCU. Nuestra hipótesis es que la expresión del MCU aumenta con el envejecimiento de las neuronas en cultivo, lo que ayuda a que se produzca una sobrecarga de Ca2+ mitocondrial que en última instancia favorece la muerte celular por apoptosis. Para valorar nuestra hipótesis se han planteado los siguientes objetivos: 1. Caracterizar los tipos celulares presentes en los cultivos primarios de hipocampo de rata. 2. Estudiar la expresión del uniportador de calcio mitocondrial en neuronas envejecidas en un cultivo primario a largo plazo. 3. Valorar si existen diferencias en la expresión del uniportador de calcio mitocondrial en neuronas cultivadas en un medio sin suero respecto a neuronas cultivadas en un medio con suero, especialmente en neuronas envejecidas.Máster en Investigación Biomédic

Calcium signaling modulators: a novel pharmacological intervention to delay aging in Caenorhabditis elegans

Ca2+ is a second messenger that affects nearly every aspect or cellular life including muscle contraction, neuronal secretion and cell proliferation and differentiation. The dysregulation of the cellular toolkit that controls and maintains Ca2+ homeostasis has been linked to the physiopathology of the aging process including neurodegeneration. Caenorhabditis elegans has been proven to be an excellent model organism to study aging and neurodegeneration due to the conservation of numerous signaling pathways that have been proven to modulate aging, and the availability of several models of neurodegenerative diseases. Moreover, the interrelationship between aging and Ca2+ signaling can be studied in the worms because of their transparent cuticle that allows to perform in vivo Ca2+ dynamics studies throughout the whole life of the organisms. The metabolic pathways that are known to regulate aging in C. elegans are the so called nutrient sensing pathways. All these pathways, that are conserved in mammals, are able to respond to changes in nutrient availability that, in the end, affect the longevity of the worms. These pathways are the insulin/insulin-likegrowth factor (IGF-1) signaling pathway (IIS), the mechanistic target of rapamycin (mTOR) signaling pathway, the adenosine monophosphate-activated protein kinase (AMPK) pathway, and the sirtuins pathway. Although not much information about how intracellular Ca2+ regulates these pathways, there is some evidence that suggests that Ca2+ might be implicated in the modulation of nutrient sensing pathways activities. This thesis has focused in the study of the interrelationship between Ca2+ signaling and nutrient sensing pathways, and its possible effects in the aging process through two different pharmacological approaches: the submaximal inhibition of sarco-endoplasmic reticulum calcium-ATPase (SERCA) using 2,5-BHQ and thapsigargin, and the submaximal inhibition of the mitochondrial Na+/Ca2+ exchanger using CGP37157. SERCA refills the endoplasmic reticulum (ER) with Ca2+ up to the millimolar range being the main controller of the ER [Ca2+] level, implicated in the modulation of cytosolic Ca2+ signaling and ER-mitochondria Ca2+ transfer. In this work it has been proven that the submaximal inhibition of SERCA with 2,5-BHQ and thapsigargin induced an increase in the lifespan of C. elegans worms and that this effect was mediated by the modulation of mTOR and AMPK signaling pathways. Moreover, it was also discarded that the effect was mediated by the activation of the ER stress response. CGP37157 is a benzothiazepine with neuroprotective effects in several in vitro models of excitotoxicity involving dysregulation of intracellular Ca2+ homeostasis. CGP37157 has been used for decades as an inhibitor of the mitochondrial Na+/Ca2+ 20 exchanger (mNCX), although several off targets have been described. Throughout this thesis, the effects of CGP37157 in C. elegans healthspan, as well as its possible modulation of nutrient sensing pathways and Ca2+ dynamics, have been explored. Our results show that the treatment with CGP37157 is able to induce an increase in C. elegans life expectancy through the modulation of the mTOR and IIS signaling pathways. Moreover, it was proven that a functional electron transport chain activity was required for CGP37157 to exert its effects, and that CGP37157 treatment induced changes in intracellular Ca2+, including cytosolic and mitochondrial Ca2+ dynamics changes in two different muscular systems, the pharynx and the vulva. Finally, the changes induced by CGP37157 also caused an improvement in worm’s locomotion and muscle function delaying the sarcopenia process and improving mitochondrial integrity and organization in C. elegans body wall muscle cells. In conclusion, this work has described two novel pharmacological interventions that improve C. elegans lifespan through the modulation of Ca2+ signaling in a different manner. These results outline the possible therapeutic effects of both SERCA inhibitors and CGP37157 in the aging process, and the importance of Ca2+ signaling in the regulation and evolution of aging related physiopathology.Departamento de Bioquímica y Biología Molecular y FisiologíaDoctorado en Investigación Biomédic

Mitochondrial Ca2+ dynamics in MCU knockout C. elegans worms

Producción CientíficaMitochondrial [Ca2+] plays an important role in the regulation of mitochondrial function, controlling ATP production and apoptosis triggered by mitochondrial Ca2+ overload. This regulation depends on Ca2+ entry into the mitochondria during cell activation processes, which is thought to occur through the mitochondrial Ca2+ uniporter (MCU). Here, we have studied the mitochondrial Ca2+ dynamics in control and MCU-defective C. elegans worms in vivo, by using worms expressing mitochondrially-targeted YC3.60 yellow cameleon in pharynx muscle. Our data show that the small mitochondrial Ca2+ oscillations that occur during normal physiological activity of the pharynx were very similar in both control and MCU-defective worms, except for some kinetic differences that could mostly be explained by changes in neuronal stimulation of the pharynx. However, direct pharynx muscle stimulation with carbachol triggered a large and prolonged increase in mitochondrial [Ca2+] that was much larger in control worms than in MCU-defective worms. This suggests that MCU is necessary for the fast mitochondrial Ca2+ uptake induced by large cell stimulations. However, low-amplitude mitochondrial Ca2+ oscillations occurring under more physiological conditions are independent of the MCU and use a different Ca2+ pathway.Ministerio de Economía y Competitividad - (Proyecto BFU2017-83509-R)Fondo Europeo de Desarrollo Regional (FEDER) y Junta de Castilla y León - (Projecto VA011G18

The role of Ca2+ signaling in aging and neurodegeneration: Insights from caenorhabditis elegans models

Producción CientíficaCa2+ is a ubiquitous second messenger that plays an essential role in physiological processes such as muscle contraction, neuronal secretion, and cell proliferation or differentiation. There is ample evidence that the dysregulation of Ca2+ signaling is one of the key events in the development of neurodegenerative processes, an idea called the “calcium hypothesis” of neurodegeneration. Caenorhabditis elegans (C. elegans) is a very good model for the study of aging and neurodegeneration. In fact, many of the signaling pathways involved in longevity were first discovered in this nematode, and many models of neurodegenerative diseases have also been developed therein, either through mutations in the worm genome or by expressing human proteins involved in neurodegeneration (β-amyloid, α-synuclein, polyglutamine, or others) in defined worm tissues. The worm is completely transparent throughout its whole life, which makes it possible to carry out Ca2+ dynamics studies in vivo at any time, by expressing Ca2+ fluorescent probes in defined worm tissues, and even in specific organelles such as mitochondria. This review will summarize the evidence obtained using this model organism to understand the role of Ca2+ signaling in aging and neurodegeneration

Antioxidant, Immunomodulatory and Potential Anticancer Capacity of Polysaccharides (Glucans) from Euglena gracilis G.A. Klebs

The present study was carried out to determine the bioactivity of polysaccharides extracted from Euglena gracilis (EgPs). These were characterized by FT-IR and GC-MS. Cytotoxicity analyses (MTT) were performed on healthy human gingival fibroblast cell lines (HGF-1), obtaining an IC50 of 228.66 µg mL−1, and cell lines with anticancer activity for colon cancer (HCT-116), breast cancer (MCF-7), human leukemia (U-937, HL-60) and lung cancer (NCl-H460), showing that EgPs have anticancer activity, mainly in HTC-116 cells (IC50 = 26.1 µg mL−1). The immunological assay determined the immunomodulatory capacity of polysaccharides for the production of proinflammatory cytokines IL-6 and TNF-α in murine macrophages (RAW 264.7) and TNF-α in human monocytes (THP-1). It was observed that the EgPs had a stimulating capacity in the synthesis of these interleukins. The antioxidant capacity of polysaccharides and their biomass were analyzed using the ABTS method (18.30 ± 0.14% and (5.40 ± 0.56%, respectively, and the DPPH method for biomass (17.79 ± 0.57%). We quantitatively profiled HGF-1 proteins by liquid chromatography–tandem mass spectrometry analysis, coupled with 2-plex tandem mass tag labelling, in normal cells. In total, 1346 proteins were identified and quantified with high confidence, of which five were considered to be overexpressed. The data is available through ProteomeXchange, under identifier PXD029076.Partial funding for open access charge: Universidad de Málag

Inhibition of Sarco-Endoplasmic Reticulum Ca2+ ATPase Extends the Lifespan in C. elegans Worms

The sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) refills the endoplasmic reticulum (ER) with Ca2+ up to the millimolar range and is therefore the main controller of the ER [Ca2+] level ([Ca2+]ER), which has a key role in the modulation of cytosolic Ca2+ signaling and ER-mitochondria Ca2+ transfer. Given that both cytosolic and mitochondrial Ca2+ dynamics strongly interplay with energy metabolism and nutrient-sensitive pathways, both of them involved in the aging process, we have studied the effect of SERCA inhibitors on lifespan in C. elegans. We have used thapsigargin and 2,5-Di-tert-butylhydroquinone (2,5-BHQ) as SERCA inhibitors, and the inactive analog 2,6-Di-tert-butylhydroquinone (2,6-BHQ) as a control for 2,5-BHQ. Every drug was administered to the worms either directly in the agar or via an inclusion compound with γ-cyclodextrin. The results show that 2,6-BHQ produced a small but significant increase in survival, perhaps because of its antioxidant properties. However, 2,5-BHQ produced in all the conditions a much higher increase in lifespan, and the potent and specific SERCA inhibitor thapsigargin also extended the lifespan. The effects of 2,5-BHQ and thapsigargin had a bell-shaped concentration dependence, with a maximum effect at a certain dose and smaller or even toxic effects at higher concentrations. Our data show therefore that submaximal inhibition of SERCA pumps has a pro-longevity effect, suggesting that Ca2+ signaling plays an important role in the aging process and that it could be a promising novel target pathway to act on aging

Inhibition of RAC1 activity in cancer associated fibroblasts favours breast tumour development through IL-1β upregulation

Cancer-associated fibroblasts (CAFs) are highly abundant stromal components in the tumour microenvironment. These cells contribute to tumorigenesis and indeed, they have been proposed as a target for anti-cancer therapies. Similarly, targeting the Rho-GTPase RAC1 has also been suggested as a potential therapeutic target in cancer. Here, we show that targeting RAC1 activity, either pharmacologically or by genetic silencing, increases the pro-tumorigenic activity of CAFs by upregulating IL-1β secretion. Moreover, inhibiting RAC1 activity shifts the CAF subtype to a more aggressive phenotype. Thus, as RAC1 suppresses the secretion of IL-1β by CAFs, reducing RAC1 activity in combination with the depletion of this cytokine should be considered as an interesting therapeutic option for breast cancer in which tumour cells retain intact IL-1β signalling.

The Neuroprotector Benzothiazepine CGP37157 Extends Lifespan in C. elegans Worms

The benzothiazepine CGP37157 has shown neuroprotective effects in several in vitro models of excitotoxicity involving dysregulation of intracellular Ca2+ homeostasis. Although its mechanism of neuroprotection is unclear, it is probably related with some of its effects on Ca2+ homeostasis. CGP37157 is a well-known inhibitor of the mitochondrial Na+/Ca2+ exchanger (mNCX). However, it is not very specific and also blocks several other Ca2+ channels and transporters, including voltage-gated Ca2+ channels, plasma membrane Na+/Ca2+ exchanger and the Ca2+ homeostasis modulator 1 channel (CALHM1). In the present work, we have studied if CGP37157 could also induce changes in life expectancy. We now report that CGP37157 extends C. elegans lifespan by 10%–15% with a bell-shaped concentration-response, with high concentrations producing no effect. The effect was even larger (25% increase in life expectancy) in worms fed with heat-inactivated bacteria. The worm CGP37157 concentration producing maximum effect was measured by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and was close to the IC50 for inhibition of the Na+/Ca2+ exchanger. CGP37157 also extended the lifespan in eat-2 mutants (a model for caloric restriction), suggesting that caloric restriction is not involved in the mechanism of lifespan extension. Actually, CGP37157 produced no effect in mutants of the TOR pathway (daf15/unc24) or the insulin/insulin-like growth factor-1 (IGF-1) pathway (daf-2), indicating that the effect involves these pathways. Moreover, CGP37157 was also ineffective in nuo-6 mutants, which have a defect in the mitochondrial respiratory chain complex I. Since it has been described that neuroprotection by this compound in cell cultures is abolished by mitochondrial inhibitors, this suggests that life extension in C. elegans and neuroprotection in cell cultures may share a similar mechanism involving mitochondria

Desarrollo de estrategias de evaluación de competencias transversales en asignaturas de ingeniería mecánica y de materiales

[EN] The international accreditation for the Master and Bachelor degrees offered at our university, together with the demands of the employers, have made it clear that students’ curricula should specify not only what they have studied, but also what they are actually able to do. This work presents the results obtained within the frame of an innovative project (UPV – PIME program) on the evaluation of three generic competences that have been traditionally worked in subjects of mechanical and materials engineering: capacity for problem solving; capacity for applying knowledge in practice; and communication skills (both oral and written). Different tools for the assessment of these competences have developed with two main objectives: first, to quantify the level of achievement of the students in order to give a numerical evaluation; and second, to be used by the students as a learning material so that they can improve their capacities. These tools are based on the observation of some learning outcomes associated to these competences. Some evaluation activities have been proposed within the different subjects that allow to assess not only the specific scientific-technical competences, but also some of the generic ones.[ES] La acreditación internacional de los títulos de Grado y Máster en nuestra universidad, junto con las demandas de los empleadores, han puesto en evidencia que los planes de estudio deben especificar no sólo lo que se ha estudiado, sino también lo que se es capaz de hacer realmente. Este trabajo presenta los resultados obtenidos en el marco de un proyecto de innovación docente (programa PIME) sobre la evaluación de tres competencias transversales que se han trabajado tradicionalmente en asignaturas de ingeniería mecánica y materiales: la capacidad para la resolución de problemas; la capacidad para aplicar los conocimientos a la práctica; y habilidades de comunicación (oral y escrita). Se han desarrollado diferentes herramientas para la evaluación de estas competencias con dos objetivos principales: en primer lugar, para cuantificar el nivel de logro de los estudiantes con el fin de dar una evaluación numérica; y en segundo, como material de aprendizaje para que los estudiantes puedan mejorar sus capacidades. Estas herramientas se basan en la observación de algunos resultados de aprendizaje asociados a estas competencias. Se han propuesto algunas actividades de evaluación en las diferentes asignaturas que permiten evaluar no sólo las competencias científico-técnicas específicas, sino también algunas de las transversales.Por último, los autores quieren agradecer la ayuda económica y el apoyo institucional recibidos de la Universitat Politècnica de València a través del proyecto PIME/2014/A/012/B.Carballeira, J.; Martínez Casas, J.; Sahuquillo Navarro, O.; Sonseca Olalla, A.; Denia Guzmán, FD.; Suñer Martinez, JL.; Vila Tortosa, MP.... (2015). Desarrollo de estrategias de evaluación de competencias transversales en asignaturas de ingeniería mecánica y de materiales. En In-Red 2015 - CONGRESO NACIONAL DE INNOVACIÓN EDUCATIVA Y DE DOCENCIA EN RED. Editorial Universitat Politècnica de València. https://doi.org/10.4995/INRED2015.2015.1542OC

‘People lie’: overcoming obstacles to incorporate social science research to biodiversity conservation

Mesmo com o reconhecimento da importância da interdisciplinaridade na conservação da biodiversidade, ainda há resistência em incorporar a pesquisa em ciências sociais (PCS) ao pensamento e à prática conservacionista. As razões para tal resistência podem ser resumidas em três afirmações gerais ainda comumente atribuídas à PCS: 'tem pouca utilidade' e 'menos rigor metodológico' quando comparada à pesquisa em ciências naturais e, sobretudo, é pouco confiável porque 'as pessoas mentem'. Neste ensaio, desenvolvido a partir da experiência dos participantes de uma comunidade de prática, formada por profissionais de diversas áreas e setores relacionados à conservação, e das discussões geradas nesse espaço de aprendizado coletivo, abordamos as limitações e os equívocos por trás das afirmações acima. A PCS não é menos útil na conservação e nem tem menos rigor metodológico do que a pesquisa em ciências naturais, e quando as pessoas mentem para o pesquisador o problema não está na pesquisa em si, mas na relação entre sujeito e pesquisador. Argumentamos que à medida que os conservacionistas se familiarizam com a PCS e que os princípios de equidade e justiça são incorporados aos valores e objetivos da conservação, a importância e necessidade da PCS na conservação tornam-se óbvias, e a falta de confiança entre pesquisador e sujeitos deixa de ser uma preocupação significativa. Capacitar, integrar e apoiar são nossas recomendações básicas para pesquisadores, educadores, gestores e tomadores de decisão nas áreas de conservação, ensino, publicação e financiamento, para que a PCS cumpra plenamente seu papel na conservação.Despite the acknowledged importance of interdisciplinarity in biodiversity conservation, there is still resistance to incorporate social science research (SSR) to both conservationist thinking and practice. The reasons for such a resistance can be summarized in three general statements still commonly attributed to SSR, namely: it is of 'little use' and it has 'less methodological rigor' than research in the natural sciences and, above all, it is unreliable because 'people lie'. The current essay was developed based on the experience of participants of a community of practice (formed by professionals from different fields and sectors  associated with conservation), as well as on discussions held in this space of collective learning. It addresses the limitations and misconceptions behind the aforementioned statements. SSR is not less useful in conservation and not less methodologically rigorous than research conducted in the natural sciences. When researchers are lied to, the problem does not lie on the research itself, but on the subject-researcher relationship. We herein argue that as conservationists become more familiar with SSR, and as principles like equity and justice are incorporated to conservation values and goals, both the importance and need of SSR in conservation become obvious, making the lack of trust between researcher and subjects no longer a significant concern. Increasing capacity, integrating and supporting are our basic recommendations for researchers, educators, managers and decision-makers in the conservation, teaching, publishing and funding fields, so that SSR can fully fulfill its role in conservation