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[eng] With the most absolute purpose of understanding what the “simple” concept of “growth regulation” implies, the present thesis is a compilation of original works that aims to deepen in how the growth and metabolism of fish, and most concretely, its muscle, behaves in different metabolically challenging conditions. Hopefully, the knowledge generated in these works and integrated in this multi-perspective thesis, can contribute to somehow help to improve the Aquaculture sector in the Mediterranean Sea and abroad. This ambitious goal is structured in different objective: 1) To establish the regulation of the ghrelinergic system in bream through the fasting and feedback model: a) Characterization of the sequences of Preproghrelin, Ghsr1a and Ghsr1b in gilthead sea bream (Article 1). b) Characterizing Preproghrelin and its two receptors in acute and long-term feeding responses and its interaction with the Gh/Igf1 axis (Article 1). 2) To deepen in the beneficial effects of exercise on growth and its modulation through changes in the dietary protein/lipid ratio: a) Determining how this dietary modification alters different growth parameters, muscular proximal composition and reserves turnover (Article 3). b) Determining how the mitochondrial proteins in red and white muscle and in the liver respond to this dietary modification in order to infer changes in the nutritional efficiency and growth improvement (Article 3). 3) To make a comparative study about the response of the main musculoskeletal growth regulation systems under two different challenging experimental models, the fasting and refeeding and the sustained exercise under two different diets. a) Studying the gene expression of specific proteins of the ontogenetic and growth pathways (myogenesis and osteogenesis), as well as the degradation pathways (proteolysis and osteoclastogenesis) in white muscle and bone (Articles 2 and 4). b) Determining its effects on white muscle growth (myogenesis/proteolysis balance) and meat quality (Article 4). 4) To complete the characterization of the new Myomaker and Myomixer fusogens in rainbow trout and gilthead sea bream and its involvement in the myogenic process in vivo and in vitro and in the in vivo muscle regeneration. a) Characterization of the sequence of Myomixer in rainbow trout (Article 5) and Muomaker and Myomixer in gilthead sea bream (Article 6). b) To study the involvement of Myomaker (in gilthead sea bream) and Myomixer (in rainbow trout and gilthead sea bream) in the myogenic process in vivo and in vitro as well as during the muscle regeneration in vivo (Article 5 and 6).

Trypanosoma cruzi, the etiological agent of Chagas disease in humans, infects a wide variety of vertebrates. Trypomastigotes, the parasite infective forms, invade mammalian cells by a still poorly understood mechanism. Adhesion of tissue culture- derived trypomastigotes to the extracellular matrix (ECM) prior to cell invasion has been shown to be a relevant part of the process. Changes in phosphorylation, S-nitrosylation, and nitration levels of proteins, in the late phase of the interaction (2 h), leading to the reprogramming of both trypomastigotes metabolism and the DNA binding profile of modified histones, were described by our group. Here, the involvement of calcium signaling at a very early phase of parasite interaction with ECM is described. Increments in the intracellular calcium concentrations during trypomastigotes-ECM interaction depends on the Ca2+ uptake from the extracellular medium, since it is inhibited by EGTA or Nifedipine, an inhibitor of the L-type voltage gated Ca2+ channels and sphingosine-dependent plasma membrane Ca2+ channel, but not by Vanadate, an inhibitor of the plasma membrane Ca2+-ATPase. Furthermore, Nifedipine inhibits the invasion of host cells by tissue culture- derived trypomastigotes in a dose-dependent manner, reaching 95% inhibition at 100 µM Nifedipine. These data indicate the importance of both Ca2+ uptake from the medium and parasite-ECM interaction for host-cell invasion. Previous treatment of ECM with protease abolishes the Ca2+ uptake, further reinforcing the possibility that these events may be connected. The mitochondrion plays a relevant role in Ca2+ homeostasis in trypomastigotes during their interaction with ECM, as shown by the increment of the intracellular Ca2+ concentration in the presence of Antimycin A, in contrast to other calcium homeostasis disruptors, such as Cyclopiazonic acid for endoplasmic reticulum and Bafilomycin A for acidocalcisome. Total phosphatase activity in the parasite decreases in the presence of Nifedipine, EGTA, and Okadaic acid, implying a role of calcium in the phosphorylation level of proteins that are interacting with the ECM in tissue culture- derived trypomastigotes. In summary, we describe here the increment of Ca2+ at an early phase of the trypomastigotes interaction with ECM, implicating both nifedipine-sensitive Ca2+ channels in the influx of Ca2+ and the mitochondrion as the relevant organelle in Ca2+ homeostasis. The data unravel a complex sequence of events prior to host cell invasion itself.

Objective: We aimed to determine the effect of different low-temperature range interventions at different time-points in a rat model of pressure injury (PI) produced by Ischemia/Reperfusion (I/R) injury. Methods: Sprague-Dawley rats were randomly assigned to blank control, injury control, and temperature intervention groups. Rats in the injury control and temperature intervention groups (involving exposure to different temperature range at different time-points) were subjected to three cycles of I/R injury with 2-h ischemia and 0.5-h reperfusion to induce PI. Results: The muscle tissues exhibited degenerative changes after compression. Low temperature intervention of 16–18°C in the ischemia period resulted in the lowest degree of tissue damage and significantly decreased levels of Bcl-2-associated X protein (Bax), caspase-9, and caspase-3. Moreover, it resulted in the highest expression level of B-cell lymphoma 2 (Bcl-2) and lowest expression levels of Bax, caspase-9, and caspase-3 in muscle tissues among all intervention groups. Conclusion: Low-temperature intervention at 16–18°C during the ischemia period showed optimal effects on the expressions of apoptotic factors during the development of PI with I/R-induced tissue damage. RESUMO Objetivo: Nosso objetivo foi determinar o efeito de diferentes intervenções na faixa de baixa temperatura em diferentes pontos do tempo em um modelo de lesão por pressão (IP) de rato produzida por lesão de isquemia/reperfusão (I/R). Métodos: Ratos Sprague-Dawley foram aleatoriamente designados para grupos de controle em branco, controle de lesão e intervenção por temperatura. Ratos nos grupos de controle de lesão e intervenção de temperatura (envolvendo exposição a diferentes faixas de temperatura em diferentes momentos) foram submetidos a três ciclos de lesão de I/R com isquemia de 2 h e reperfusão de 0,5 h para induzir IP. Resultados: Os tecidos musculares exibiram alterações degenerativas após a compressão. A intervenção em baixa temperatura de 16–18°C no período de isquemia resultou no menor grau de dano ao tecido e diminuiu significativamente os níveis de proteína X associada a Bcl-2 (Bax), caspase-9 e caspase-3. Além disso, resultou no nível de expressão mais alto de linfoma de células B 2 (Bcl-2) e níveis de expressão mais baixos de Bax, caspase-9 e caspase-3 em tecidos musculares entre todos os grupos de intervenção. Conclusão: A intervenção em baixa temperatura de 16–18°C durante o período de isquemia mostrou efeitos ótimos nas expressões de fatores apoptóticos durante o desenvolvimento de IP com dano tecidual induzido por I/R. RESUMEN Objetivo: Nuestro objetivo fue determinar el efecto de diferentes intervenciones de rango de temperatura baja en diferentes puntos de tiempo en un modelo de rata de lesión por presión (IP) producida por lesión por isquemia/reperfusión (I/R). Métodos: Se asignaron aleatoriamente ratas Sprague-Dawley a grupos de control en blanco, control de lesiones e intervención de temperatura. Las ratas en los grupos de control de lesiones e intervención de temperatura (que implican exposición a diferentes rangos de temperatura en diferentes puntos de tiempo) se sometieron a tres ciclos de lesión I/R con isquemia de 2 h y reperfusión de 0,5 h para inducir IP. Resultados: Los tejidos musculares presentaron cambios degenerativos después de la compresión. La intervención a baja temperatura de 16–18°C en el período de isquemia resultó en el grado más bajo de daño tisular y niveles significativamente reducidos de proteína X asociada a Bcl-2 (Bax), caspasa-9 y caspasa-3. Además, dio como resultado el nivel de expresión más alto de linfoma de células B 2 (Bcl-2) y los niveles de expresión más bajos de Bax, caspasa-9 y caspasa-3 en los tejidos musculares entre todos los grupos de intervención. Conclusión: La intervención a baja temperatura a 16–18°C durante el período de isquemia mostró efectos óptimos sobre la expresión de factores apoptóticos durante el desarrollo de IP con daño tisular inducido por I/R.

The optimization of conditions to promote the stemness of pluripotent cells in vitro is instrumental for their use in advanced therapies. We show here that exposure of human iPSCs and human ESCs to low concentrations of the chemical NO donor DETA/NO leads to stabilization of hypoxia-inducible factors (HIF-1α and HIF-2α) under normoxia, with this effect being dependent on diminished Pro 402 hydroxylation and decreased degradation by the proteasome. Moreover, the master genes of pluripotency, NANOG and OCT-4, were upregulated. NO also induces a shift in the metabolic profile of PSCs, with an increased expression of hypoxia response genes in glycolysis. Furthermore, a reduction in the mitochondrial membrane potential with lower oxygen consumption and increased expression of mitochondrial fusion regulators, such as DRP1, was observed. The results reported here indicate that NO mimics hypoxia response in human PSCs and enhances their stemness properties when cultured under normoxic conditions.

Mitochondrial diseases constitute an important group of hereditary metabolic disorders with heterogeneous clinical and genetic expression. Therefore, this article aims to characterize the scientific production on mitochondrial diseases. Therefore, an integrative literature review on the subject was carried out. The information obtained was analyzed in the light of Cooper (1984). The category that emerged in the data collection and structured the discussion was: conceptual and critical contributions about mitochondrial diseases. The results show the concentration of studies on mitochondrial diseases in southeastern Brazil. In addition, research has generally highlighted that because mitochondria participate in various cellular processes, it becomes an essential component of the eukaryotic cell and that mutations that compromise its functioning can cause severe damage to the cell, causing so-called diseases mitochondrial diseases, which are the largest known group of inherited diseases, in addition to being related to neurodegenerative diseases. Las enfermedades mitocondriales constituyen un grupo importante de trastornos metabólicos hereditarios con expresión clínica y genética heterogénea. Por tanto, este artículo tiene como objetivo caracterizar la producción científica sobre enfermedades mitocondriales. Por tanto, se realizó una revisión integradora de la literatura sobre el tema. La información obtenida fue analizada a la luz de Cooper (1984). La categoría que surgió en la recolección de datos y estructuró la discusión fue: contribuciones conceptuales y críticas sobre las enfermedades mitocondriales. Los resultados muestran la concentración de estudios sobre enfermedades mitocondriales en el sureste de Brasil. Además, la investigación ha destacado en general que debido a que las mitocondrias participan en diversos procesos celulares, se convierte en un componente esencial para la célula eucariota y que las mutaciones que comprometen su funcionamiento pueden causar daños severos a la célula, provocando las llamadas enfermedades enfermedades mitocondriales, que son el grupo más grande conocido de enfermedades hereditarias, además de estar relacionado con enfermedades neurodegenerativas. As doenças mitocondriais constituem um importante grupo de doenças hereditárias do metabolismo de expressão clínica e genética heterogênea. Assim sendo, este artigo tem como objetivo caracterizar a produção científica sobre as doenças mitocondriais. Para tanto, foi realizada uma revisão integrativa da literatura sobre o tema. As informações obtidas foram analisadas a luz do Cooper (1984). A categoria que emergiu na coleta de dados e estrututou a discussão foi: contribuições conceituais e críticas acerca das doenças mitocondriais. Os resultados evidenciam a concentração dos estudos sobre as doenças mitocondriais na região sudeste do Brasil. Além disso, as pesquisas de um modo geral ressaltaram que em virtude da mitocôndria participar de vários processos celulares, ela torna-se um componente essencial para a célula eucariótica e que mutações que comprometam seu funcionamento podem gerar danos severos à célula, causando as chamadas doenças mitocondriais, que são o maior grupo de doenças hereditárias conhecidas, além de estarem relacionadas com doenças neurodegenerativas.

Abnormal trinucleotide expansions cause rare disorders that compromise quality of life and, in some cases, lifespan. In particular, the expansions of the CGG-repeats stretch at the 5'-UTR of the Fragile X Mental Retardation 1 (FMR1) gene have pleiotropic effects that lead to a variety of Fragile X-associated syndromes: the neurodevelopmental Fragile X syndrome (FXS) in children, the late-onset neurodegenerative disorder Fragile X-associated tremor-ataxia syndrome (FXTAS) that mainly affects adult men, the Fragile X-associated primary ovarian insufficiency (FXPOI) in adult women, and a variety of psychiatric and affective disorders that are under the term of Fragile X-associated neuropsychiatric disorders (FXAND). In this review, we will describe the pathological mechanisms of the adult "gain-of-function" syndromes that are mainly caused by the toxic actions of CGG RNA and FMRpolyG peptide. There have been intensive attempts to identify reliable peripheral biomarkers to assess disease progression and onset of specific pathological traits. Mitochondrial dysfunction, altered miRNA expression, endocrine system failure, and impairment of the GABAergic transmission are some of the affectations that are susceptible to be tracked using peripheral blood for monitoring of the motor, cognitive, psychiatric and reproductive impairment of the CGG-expansion carriers. We provided some illustrative examples from our own cohort. Understanding the association between molecular pathogenesis and biomarkers dynamics will improve effective prognosis and clinical management of CGG-expansion carriers. Yes

Classically considered a by-product of anaerobic metabolism, lactate is now viewed as a fundamental fuel for oxidative phosphorylation in mitochondria, and preferred over glucose by many tissues. Lactate is also a signaling molecule of increasing medical relevance. Lactate levels in the blood can increase in both normal and pathophysiological conditions (e.g., hypoxia, physical exercise, or sepsis), however the manner by which these changes are sensed and induce adaptive responses is unknown. Here we show that the carotid body (CB) is essential for lactate homeostasis and that CB glomus cells, the main oxygen sensing arterial chemoreceptors, are also lactate sensors. Lactate is transported into glomus cells, leading to a rapid increase in the cytosolic NADH/NAD+ ratio. This in turn activates membrane cation channels, leading to cell depolarization, action potential firing, and Ca2+ influx. Lactate also decreases intracellular pH and increases mitochondrial reactive oxygen species production, which further activates glomus cells. Lactate and hypoxia, although sensed by separate mechanisms, share the same final signaling pathway and jointly activate glomus cells to potentiate compensatory cardiorespiratory reflexes., This research was supported by the Spanish Ministries of Science and Innovation and Health (SAF2016-74990-R), and the European Research Council (ERC Advanced Grant PRJ201502629). H.T.-T. received a predoctoral fellowship (FPI program) from the Spanish Government.

[spa] Las mitofusinas 1 y 2 son unas GTPasas que se encuentran en la membrana mitocondrial externa, donde llevan a cabo el proceso de fusión mitocondrial. La existencia de mutaciones en Mfn2 es la principal causa de la enfermedad de Charcot- Marie-Tooth tipo 2A, un grupo de distintas neuropatías caracterizadas por degeneración axonal. Por otro lado, otras alteraciones en Mfn2 también han sido descritas en múltiples enfermedades neurodegenerativas crónicas, en episodios traumáticos agudos como accidentes cerebrovasculares e incluso otras enfermedades no neurológicas como las cardiometabólicas. Un gran número de estudios han demostrado que Mfn2 está relacionada con la regulación del metabolismo y la bioenergética mitocondrial, ya que su supresión afecta negativamente ambos procesos y su sobreexpresión los estimula. Dado el papel del metabolismo mitocondrial en la fisiopatología de estas enfermedades, entender el mecanismo por el que Mfn2 regula la bioenergética mitocondrial resulta de gran importancia. En esta tesis se demostró que la activación de la bioenergética mitocondrial por parte de Mfn2 requiere su localización en el retículo endoplasmático (RE) y es independiente a su función como proteína de fusión mitocondrial. A diferencia de Mfn1, Mfn2 se encuentra presente en el RE, concretamente en las membranas asociadas con las mitocondrias. Los contactos entre ambos orgánulos son de vital importancia en las células, ya que, entre otras funciones esenciales, crean micro-dominios que permiten la transferencia de Ca2+ del RE hacia las mitocondrias. Por su parte, el Ca2+ captado por las mitocondrias estimula diferentes enzimas del metabolismo mitocondrial y complejos encargados de mantener la bioenergética mitocondrial. En esta tesis, se observó que células carentes de Mfn2 también manifiestan defectos en la homeostasis del Ca2+ y en el mantenimiento de los contactos RE-mitocondrias. Mediante el uso de un enlazador artificial que mimetiza la función enlazadora de Mfn2 o diferentes formas de Mfn2 dirigidas a ambos orgánulos, se determinó que Mfn2 actúa como complejo enlazador de RE y mitocondrias, propiciando la formación de contactos óptimos para la transferencia de Ca2+ del RE a las mitocondrias. A su vez, el restablecimiento de la captación de Ca2+ por las mitocondrias gracias a la función enlazadora de Mfn2 fue capaz de estimular la bioenergética mitocondrial, definiendo de este modo un mecanismo por el cual Mfn2 regula la bioenergética mitocondrial a través de los contactos RE-mitocondria. La relevancia fisiológica de estos resultados se mostró en dos procesos neuronales. Por un lado, la falta de Mfn2 provocó alteraciones en el crecimiento neurítico y la formación de espinas dendríticas durante el periodo de desarrollo neuronal en modelos in vivo de ratón knockout para Mfn2. Estas alteraciones, fueron rescatadas in vitro mediante el enlazador artificial de RE-mitocondrias. Usando distintas formas de Mfn2 dirigidas específicamente a RE o mitocondria, se determinó que el mecanismo por el cual Mfn2 regula la bioenergética a través de los contactos RE-mitocondrias podría ser esencial durante el desarrollo neuronal para un correcto crecimiento y ramificación de sus neuritas. Por otro lado, el restablecimiento de los contactos RE-mitocondria protegió contra la excitotoxicidad. Mfn2 destaca también por su capacidad neuroprotectora frente diferentes tipos de lesiones como daño al ADN, estrés oxidativo o privación de iones K+. Frente a una sobreactivación persistente de los receptores de NMDA, acontece un aumento continuo y acusado de Ca2+ intracelular que desencadena en muerte neuronal. Durante este proceso, los niveles de Mfn2 decrecen, hecho que correlaciona con una mayor predisposición neuronal a la apoptosis. En este estudio, se determinó que la función enlazadora de Mfn2 podría estar relacionada con el reclutamiento de Bax hacia las mitocondrias, hecho típicamente definitorio de un proceso apoptótico. De este modo, se propone este mecanismo como una potencial diana terapéutica en procesos patológicos como los accidentes cerebrovasculares isquémicos, en los cuales aparecen eventos excitotóxicos y los niveles de Mfn2 se ven reducidos.

Blastocladiella emersonii is an early diverging fungus of the phylum Blastocladiomycota. During the life cycle of the fungus, mitochondrial morphology changes significantly, from a fragmented form in sessile vegetative cells to a fused network in motile zoospores. In this study, we visualize these morphological changes using a mitochondrial fluorescent probe and show that the respiratory capacity in zoospores is much higher than in vegetative cells, suggesting that mitochondrial morphology could be related to the differences in oxygen consumption. While studying the respiratory chain of the fungus, we observed an antimycin A and cyanide-insensitive, salicylhydroxamic (SHAM)-sensitive respiratory activity, indicative of a mitochondrial alternative oxidase (AOX) activity. The presence of AOX was confirmed by the finding of a B. emersonii cDNA encoding a putative AOX, and by detection of AOX protein in immunoblots. Inhibition of AOX activity by SHAM was found to significantly alter the capacity of the fungus to grow and sporulate, indicating that AOX participates in life cycle control in B. emersonii.

This study was funded by grants from the Ministerio de Economia, Industria y Competitividad y por el Fondo de Desarrollo Regional FEDER, Spain nº SAF2013-49019, SAF2017-85903-P, and from the Consejería de Innovación, Ciencia y Empresa, Junta de Andalucía (P07- CTS- 03135, P10- CTS- 5784, and CTS- 101), Spain. J.F. and L.M. have FPU fellowships from the Ministerio de Educación Cultura y Deporte, Spain. C.R.S. was a schorlarship holder from the Plan Propio de Investigación of the University of Granada., We wish to thank Michael O’Shea for proofreading the paper., Metabolic reprogramming, which is characteristic of cancer cells that rapidly adapt to the hypoxic microenvironment and is crucial for tumor growth and metastasis, is recognized as one of the major mechanisms underlying therapeutic resistance. Mitochondria, which are directly involved in metabolic reprogramming, are used to design novel mitochondria-targeted anticancer agents. Despite being targeted by melatonin, the functional role of mitochondria in melatonin's oncostatic activity remains unclear. In this study, we aim to investigate the role of melatonin in mitochondrial metabolism and its functional consequences in head and neck cancer. We analyzed the effects of melatonin on head and neck squamous cell carcinoma (HNSCC) cell lines (Cal-27 and SCC-9), which were treated with 100, 500, and 1500 mu M of melatonin for 1, 3, and 5 days, and found a connection between a change of metabolism following melatonin treatment and its effects on mitochondria. Our results demonstrate that melatonin induces a shift to an aerobic mitochondrial metabolism that is associated with changes in mitochondrial morphology, function, fusion, and fission in HNSCC. We found that melatonin increases oxidative phosphorylation (OXPHOS) and inhibits glycolysis in HNSCC, resulting in increased ROS production, apoptosis, and mitophagy, and decreased cell proliferation. Our findings highlight new molecular pathways involved in melatonin's oncostatic activity, suggesting that it could act as an adjuvant agent in a potential therapy for cancer patients. We also found that high doses of melatonin, such as those used in this study for its cytotoxic impact on HNSCC cells, might lead to additional effects through melatonin receptors., Ministerio de Economia, Industria y Competitividad y por el Fondo de Desarrollo Regional FEDER, Spain SAF2013-49019 SAF2017-85903-P, Junta de Andalucia P07-CTS-03135 P10-CTS-5784 CTS-101, Ministerio de Educacion Cultura y Deporte, Spain, Plan Propio de Investigacion of the University of Granada

Tesis (Doctor en Ciencias Químicas) - - Universidad Nacional de Córdoba. Facultad de Ciencias Químicas, 2021 Resumen: La proteína transportadora de lípidos StarD7 pertenece a la superfamilia de proteínas con dominio START. La expresión evolutivamente conservada de estas proteínas en distintas especies resalta su importancia en el transporte, metabolismo y señalamiento intracelular lipídico. Está ampliamente demostrada la contribución de StarD7 en el mantenimiento de la homeostasis celular, participando en el transporte de fosfatidilcolina (PC) a la mitocondria, esencial para preservar la morfología y función mitocondrial. Además, el déficit de StarD7 se ha asociado a estrés de retículo endosplásmico y alteraciones en la proliferación y migración celular. En este trabajo de tesis se analizó la contribución de StarD7 sobre la expresión de conexina 43 (Cx43) y su participación en la migración celular polarizada usando como modelo de estudio la línea celular derivada de trofoblasto extravelloso HTR-8/SVneo, la cual exhibe un marcado fenotipo migratorio e invasivo. Se demostró que el silenciamiento de StarD7 conduce a una disminución del ARNm de Cx43 que se correlaciona con niveles disminuidos de su proteína. La disminución en los niveles de la proteína Cx43 se asoció a un aumento en los niveles de los transcriptos ITGA1, ITGA5, ITGB1 y NID1 así como en los niveles proteicos de integrina α1, integrina α5, integrina β1 madura, β-catenina y nidogen-1 y en la actividad de MMP9. Por otro lado, se demostró que el silenciamiento de StarD7 conduce a una disminución en la migración polarizada de las células HTR-8/SVneo con una reducción en los niveles proteicos de Twist1. Además, se detectó que las células con StarD7 silenciado tienen menor afinidad de adhesión sobre una superficie de fibronectina. Se estableció mediante ensayos de inmunofluorescencia que el silenciamiento de StarD7 conduce a una disrupción del aparato de Golgi y alteración en la localización del centro organizador de microtúbulos (MTOC) durante la migración polarizada. Mediante ensayos de recuperación se determinó que los niveles de Cx43 son dependientes de los niveles de expresión de StarD7. La recuperación de Cx43 en las células silenciadas contra StarD7 sugiere que los niveles de nidogen-1 son dependientes de la abundancia de la proteína Cx43 y que su recuperación permitiría restablecer la migración celular polarizada. En resumen, los hallazgos de este trabajo de tesis indican que la disminución en la expresión de Cx43 inducida por la disminución de la proteína transportadora de PC StarD7 tiene un impacto significativo en la expresión y/o función de otras proteínas. Además, se demostró que el silenciamiento de StarD7 conduce a alteración en la morfología del aparato de Golgi y en la localización del MTOC contribuyendo a una desregulación en la homeostasis celular. Finalmente, es importante destacar que las Cxs desempeñan un papel esencial en el desarrollo y la función del sistema reproductivo femenino de los mamíferos. Por lo tanto, los resultados de este trabajo de tesis resaltan la importancia de explorar el rol de StarD7 en patologías placentales. Summary: The StarD7 lipid transporter protein belongs to the START-domain protein superfamily. The evolutionarily preserved expression of these proteins in different species highlights their importance in lipid intracellular transport, metabolism and signaling. It is widely demonstrated the contribution of StarD7 to maintain cellular homeostasis, participating in the transport of phosphatidylcholine (PC) to the mitochondria, essential for mitochondrial function and morphogenesis. In addition, StarD7 deficiency has been associated with endoplasmic reticulum stress as well as with alterations in cell proliferation and migration. This thesis work analyzed StarD7's contribution to the connexin 43 (Cx43) expression and its participation in polarized cell migration using as a study model the cell line derived from HTR-8/SVneo extravillous trophoblast, which exhibits a marked migratory and invasive phenotype. It was shown that StarD7 silencing leads to a decrease in Cx43 mRNA that correlates with decreased levels of the Cx43 protein. The decrease in the Cx43 protein levels was associated with an increase in the levels of ITGA1, ITGA5, ITGB1 and NID1 transcripts as well as with the protein levels of integrin α1, integrin α5, mature integrin β1, β-catenin, and nidogen-1, and in MMP9 activity. On the other hand, it was shown that StarD7 silencing leads to a decrease in polarized cell migration of HTR-8/SVneo cells with a decrease Twist1 protein levels. In addition, cells with silenced StarD7 were found to have lower adhesion affinity on a fibronectin surface. It was established by immunofluorescence assays that StarD7 silencing leads to a disruption of the Golgi apparatus and alteration in the location of the microtubule organizing center (MTOC) during polarized migration. Recovery assays determined that Cx43 levels are dependent on StarD7 expression levels. Cx43 recovery experiments on StarD7-silenced cells suggest that nidogen-1 levels are dependent on the abundance of the Cx43 protein and that its recovery would restore the polarized cell migration. In summary, the findings of this thesis work indicate that the decrease in the expression of Cx43 induced by the decrease of the PC transfer protein StarD7 has a significant impact on the expression and/or function of other proteins. Additionally, StarD7 silencing leads to alteration on the morphology of Golgi apparatus and also to the location of MTOC contributing to deregulation in cellular homeostasis. Finally, it is important to note that Cxs play an essential role in the development and in the function of the female reproductive system of mammals. Therefore, the results of this thesis work highlight the importance of exploring StarD7's role in placental pathologies. 2023-03-31 Fil: Arzud Cruz del Puerto, Mariano Matías. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas; Argentina. Fil: Genti de Raimondi, Susana Del Valle. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina. Fil: Genti de Raimondi, Susana Del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Fil: Hallak, Marta Elena. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica; Argentina. Fil: Hallak, Marta Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina. Fil: Panzetta de Dutari, Graciela María Del Valle. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina. Fil: Panzetta de Dutari, Graciela María Del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Fil: Touz, María Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina. Fil: Damiano, Alicia E. Universidad de Buenos Aires; Argentina. Fil: Damiano, Alicia E. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina.

Cells respond to stress by activating a variety of defense signaling pathways, including cell survival and cell death pathways. Although cell survival signaling helps the cell to recover from acute insults, cell death or senescence pathways induced by chronic insults can lead to unresolved pathologies. Arterial hypertension results from chronic physiological maladaptation against various stressors represented by abnormal circulating or local neurohormonal factors, mechanical stress, intracellular accumulation of toxic molecules, and dysfunctional organelles. Hypertension and aging share common mechanisms that mediate or prolong chronic cell stress, such as endoplasmic reticulum stress and accumulation of protein aggregates, oxidative stress, metabolic mitochondrial stress, DNA damage, stress-induced senescence, and proinflammatory processes. This review discusses common adaptive signaling mechanisms against these stresses including unfolded protein responses, antioxidant response element signaling, autophagy, mitophagy, and mitochondrial fission/fusion, STING (signaling effector stimulator of interferon genes)-mediated responses, and activation of pattern recognition receptors. The main molecular mechanisms by which the vasculature copes with hypertensive and aging stressors are presented and recent advancements in stress-adaptive signaling mechanisms as well as potential therapeutic targets are discussed.

Currently, the drugs used for toxoplasmosis have severe toxicity to human cells, and the treatment still lacks effective and safer alternatives. The search for novel drug targets is timely., Toxoplasmosis, a protozoan infection caused by Toxoplasma gondii, is estimated to affect around 2.5 billion people worldwide. Nevertheless, the side effects of drugs combined with the long period of therapy usually result in discontinuation of the treatment. New therapies should be developed by exploring peculiarities of the parasite’s metabolic pathways, similarly to what has been well described in cancer cell metabolism. An example is the switch in the metabolism of cancer that blocks the conversion of pyruvate into acetyl coenzyme A in mitochondria. In this context, dichloroacetate (DCA) is an anticancer drug that reverts the tumor proliferation by inhibiting the enzymes responsible for this switch: the pyruvate dehydrogenase kinases (PDKs). DCA has also been used in the treatment of certain symptoms of malaria; however, there is no evidence of how this drug affects apicomplexan species. In this paper, we studied the metabolism of T. gondii and demonstrate that DCA also inhibits T. gondii’s in vitro infection with no toxic effects on host cells. DCA caused an increase in the activity of pyruvate dehydrogenase followed by an unbalanced mitochondrial activity. We also observed morphological alterations frequently in mitochondria and in a few apicoplasts, essential organelles for parasite survival. To date, the kinases that potentially regulate the activity of pyruvate metabolism in both organelles have never been described. Here, we confirmed the presence in the genome of two putative kinases (T. gondii PDK [TgPDK] and T. gondii branched-chain α-keto acid dehydrogenase kinase [TgBCKDK]), verified their cellular localization in the mitochondrion, and provided in silico data suggesting that they are potential targets of DCA. IMPORTANCE Currently, the drugs used for toxoplasmosis have severe toxicity to human cells, and the treatment still lacks effective and safer alternatives. The search for novel drug targets is timely. We report here that the treatment of T. gondii with an anticancer drug, dichloroacetate (DCA), was effective in decreasing in vitro infection without toxicity to human cells. It is known that PDK is the main target of DCA in mammals, and this inactivation increases the conversion of pyruvate into acetyl coenzyme A and reverts the proliferation of tumor cells. Moreover, we verified the mitochondrial localization of two kinases that possibly regulate the activity of pyruvate metabolism in T. gondii, which has never been studied. DCA increased pyruvate dehydrogenase (PDH) activity in T. gondii, followed by an unbalanced mitochondrial activity, in a manner similar to what was previously observed in cancer cells. Thus, we propose the conserved kinases as potential regulators of pyruvate metabolism and interesting targets for new therapies.

Recently, it was proposed that the thiophene ring is capable of promoting mitochondrial accumulation when linked to fluorescent markers. As a noncharged group, thiophene presents several advantages from a synthetic point of view, making it easier to incorporate such a side moiety into different molecules. Herein, we confirm the general applicability of the thiophene group as a mitochondrial carrier for drugs and fluorescent markers based on a new concept of nonprotonable, noncharged transporter. We implemented this concept in a medicinal chemistry application by developing an antitumor, metabolic chimeric drug based on the pyruvate dehydrogenase kinase (PDHK) inhibitor dichloroacetate (DCA). The promising features of the thiophene moiety as a noncharged carrier for targeting mitochondria may represent a starting point for the design of new metabolism-targeting drugs. ispartof: PHARMACEUTICS vol:13 issue:2 ispartof: location:Switzerland status: published

As epilepsias afetam aproximadamente 1% da população mundial, sendo que 80% dos pacientes habitam países subdesenvolvidos ou em desenvolvimento e de 20 a 30% ou não tem acesso ou não respondem aos tratamentos disponíveis. As epilepsias causam grande prejuízo à qualidade de vida dos pacientes pelos sintomas associados e os problemas acometem também familiares e cuidadores visto que essas doenças ainda são muito estigmatizadas. Embora as etiologias das epilepsias sejam conhecidas em sua maioria, algumas causas desconhecidas desencadeiam episódios de crises epilépticas. O metabolismo encefálico de forma geral é investigado para esclarecimento da fisiopatologia de várias doenças neurodegenerativas, mais especificamente a função e/ou disfunção mitocondrial vem tendo muito mais atenção da comunidade científica. A disfunção mitocondrial é apontada como um fator de risco em alguns tipos de epilepsias genéticas, mas em casos de epilepsias adquiridas o seu papel permanece elusivo. Técnicas sofisticadas como a respirometria de alta resolução são empregadas e aprimoradas constantemente para a avaliação da função mitocondrial. Nesse contexto, avaliamos por meio de diferentes estratégias investigativas o efeito de indutores de crises epilépticas em modelos animais (peixe-zebra e camundongos) sobre a função mitocondrial. As questões deste estudo são: i) Os indutores de crises promovem danos mitocôndrias durante e após as crises epilépticas? ii) O pentilenotetrazol possui algum outro efeito sobre mitocôndrias além do estresse oxidativo já demonstrado? Os resultados observados mostram que o efeito agudo de pentilenotetrazol in vitro quando adicionado ao meio contendo células intactas e dissociadas de tecido encefálico de peixe-zebra aumenta a eficiência de acoplamento (p/O2), o que pode indicar uma hidrólise aumentada de ATP neuronal. Também observamos aumento na capacidade de reserva no grupo status epilepticus induzido por ácido caínico in vivo, o que pode estar relacionado à capacidade mitocondrial de responder a um aumento na demanda de energia, especialmente em neurônios. Além disso, buscamos entender o papel das variações da concentração Ca2+ empregando concentrações de 500nm (repouso) e 5μM (ativação) em homogeneizado de tecido encefálico de peixe-zebra adulto. Nós observamos que a bioenergética mitocondrial é prejudicada de forma concentração-dependente, o estado 3, estado 4 e o desacoplamento são diminuídos pela presença de ambas concentrações de Ca2+, corroborando com a diminuição do controle respiratório, indicativo de integridade mitocondrial. No entanto, o entendimento do papel do Ca2+ no metabolismo encefálico e na bioenergética permanece como perspectiva deste trabalho. Após, realizamos o isolamento de mitocôndrias a partir de fígado e encéfalo de camundongos e observamos que as mesmas mitocôndrias são afetadas quando expostas diretamente ao pentilenotetrazol, especificamente o complexo I é inibido, independente de desidrogenases e transporte e por mecanismo diferente a inibição causada por rotenona. Inferimos que as mitocôndrias encefálicas são mais sensíveis ao pentilenotetrazol porque concentrações mais baixas foram capazes de diminuir a atividade do complexo I em comparação com as mitocôndrias hepáticas. Por fim, o presente trabalho traz evidências que sustentam o conceito de que crises epilépticas estão relacionadas com comprometimento de funções mitocondriais, o que de forma translacional pode indicar contribuição da disfunção mitocondrial para a condição clínica de pacientes diagnosticados com epilepsias adquiridas. Isto é, indica-se considerar o dano mitocondrial como parte da fisiopatologia de epilepsias adquiridas. Dessa forma, a prevenção e/ou o reparo do dano mitocondrial podem ser estratégias de prevenção e tratamento adicionados às diretrizes clínicas. Epilepsies affect approximately 1% of the world population, 80% of the patients live in underdeveloped or developing countries and 20% to 30% either do not have access to or do not respond to available treatments. Epilepsies impair the quality of life of patients due to associated symptoms; the problems also affect family members and caregivers as these diseases are still highly stigmatized. Although the etiologies of epilepsies are mostly known, some unknown causes trigger seizure episodes. Brain metabolism, in general, is investigated to clarify the pathophysiology of several neurodegenerative diseases. More specifically, mitochondrial function and/or dysfunction has been receiving much more attention from the scientific community. Mitochondrial dysfunction is identified as a risk factor in some types of genetic epilepsies, but its role remains elusive in cases of acquired epilepsies. Sophisticated techniques such as high-resolution respirometry are constantly applied and improved to assess mitochondrial function. In this context, we evaluated through different investigative strategies the effect of SE inducers in animal models (zebrafish and mice) on mitochondrial function. The questions of this study are: i) Do seizure inducers promote mitochondrial damage during and after epileptic seizures? ii) Does pentylenetetrazole have any other effects on mitochondria besides the oxidative stress already reported? We observed that the acute exposure to PTZ in vitro in the medium containing intact and dissociated cells from zebrafish brain tissue increases the coupling efficiency (p/O2), which may indicate an increased hydrolysis of neuronal ATP. We also observed an increase in reserve capacity in the SE group induced by kainic acid in vivo, which may be related to the mitochondrial ability to respond to an increase in energy demand, especially neurons. We also sought to understand the role of variations in Ca2+ concentration using concentrations of 500nm (resting) and 5μM (activation) in adult zebrafish brain tissue homogenate. We observed that mitochondrial bioenergetics is concentration-dependently impaired, state 3, state 4 and uncoupled are impaired by the presence of both concentrations of Ca2+, corroborating to the decrease in respiratory control, indicative of mitochondrial integrity. However, the understanding of the role of Ca2+ in brain metabolism and bioenergetics remains a perspective of this work. Afterwards, we performed the isolation of mitochondria from the liver and brain of mice and observed that the same mitochondria are affected when directly exposed to PTZ, specifically complex I is inhibited, independent of dehydrogenases and transport and by a different mechanism of the inhibition caused by rotenone. We imply that brain mitochondria are more sensitive to pentylenetetrazole because lower concentrations were able to impair complex I activity compared to liver mitochondria. Finally, the present work provides evidence that supports the concept that epileptic seizures are related to impairment of mitochondrial functions, which in a translational way may indicate the contribution of mitochondrial dysfunction to the clinical condition of patients diagnosed with acquired epilepsies. In other words, we should consider mitochondrial damage as part of the pathophysiology of acquired epilepsies. Thus, the prevention and/or repair of mitochondrial damage can be prevention and treatment strategies added to clinical guidelines.

The COVID-19 pandemic brought attention to studies about viral infections and their impact on the cell machinery. SARS-CoV-2, for example, invades the host cells by ACE2 interaction and possibly hijacks the mitochondria. To better understand the disease and to propose novel treatments, crucial aspects of SARS-CoV-2 enrolment with host mitochondria must be studied. The replicative process of the virus leads to consequences in mitochondrial function, and cell metabolism. The hijacking of mitochondria, on the other hand, can drive the extrusion of mitochondrial DNA (mtDNA) to the cytosol. Extracellular mtDNA evoke robust proinflammatory responses once detected, that may act in different pathways, eliciting important immune responses. However, few receptors are validated and are able to detect and respond to mtDNA. In this review, we propose that the mtDNA and its detection might be important in the immune process generated by SARS-CoV-2 and that this mechanism might be important in the lung pathogenesis seen in clinical symptoms. Therefore, investigating the mtDNA receptors and their signaling pathways might provide important clues for therapeutic interventions.

[eng] Brown and beige adipose tissues are mediators of adaptive energy expenditure in mammals, in contrast with the energy storage role of white fat. Mitochondrial activity, including uncoupled respiration, and the release of the so-called “batokines” in brown and beige adipocytes account for the majority of local and systemic adaptations to energy expenditure requirements mediated by these cells. One of these batokines is fibroblast growth factor 21 (FGF21), which has been recently proposed to be an antiaging hormone. Aging is associated with a decline in brown adipose tissue (BAT) activity and in browning of white adipose tissue (WAT). It has been proposed that the extent of BAT decline in aging may play a causative role in the enhanced propensity to age-associated metabolic conditions, and it has even been speculated that BAT reactivation may reverse them. On the other hand, aging is associated with increased amount of WAT. A loss of mitochondrial homeostasis by defective mitochondrial quality control as a result of decreased biogenesis but also to decreased degradation through mitophagy has been proposed as an underlying cause of aging in multiple tissues. Recently, Parkin has been identified as a key component of adipose tissue plasticity in response to thermogenic requirements, associated with the adaptive control of mitophagy. In the present study we investigated the impact of aging on adipose tissues in mice and humans with special focus on the status of the FGF21 system. We also tried to establish the role of Parkin in our mice model at distinct stages of aging. Parkin transcript and protein levels were up-regulated in relation to aging in mice adipose tissues, which may point to a compensatory homeostasis against diminished Parkin- independent mitophagy. Middle-aged wild-type mice presented an age-associated phenotype reminiscent of obesity and signs of BAT malfunction in middle-aged mice, which did not affect thermogenic response at this specific aging stage. Thus, our data would not support a massive effect of BAT in systemic derangements associated with aging. Parkin-KO mice were protected against age-associated obesity found in control animals. BAT activity does not seem to conduct the protection of Parkin-KO mice against aging-associated adiposity, as Parkin-KO mice do not present enhanced BAT- or browning-mediated energy expenditure. This phenomenon might rather be attributable to diminished food intake and risen energy expenditure-related processes in Parkin-KO mice. Our data denote that Parkin is possibly involved in the in the metabolic flexibility of lipid versus carbohydrate metabolism. FGF21 expression and its secretion in mice may be induced as stress hormone by mitochondrial dysfunctions in aged tissues. However, the chronic metabolic and stress-related disorders might elicit an FGF21 resistance, menacing healthy aging. Middle-aged mice lacking Parkin abolish increased levels of FGF21, which might be associated to the prevention of obesity occurring in those animals. In humans, serum FGF21 levels were increased in parallel with indicators of mildly deteriorated glucose homeostasis. FGF21- responsiveness machinery was not disrupted in subcutaneous adipose tissue from elderly individuals relative to those from young controls. Therefore, in contrast to what is observed in chronic metabolic pathologies or mice aging, high levels of FGF21 in healthy aging are not associated with repressed FGF21-responsiveness machinery in adipose tissue.

Lymphoma is the most common type of blood cancer today and, as its name suggests, it begins in the lymphatic system. The origin of this disease is related to mitochondrial defects, generated by mutations in the PNPase enzyme or polynucleotide phosphorylase, whose main functions are import and degradation mitochondrial RNA. For this reason, the main objective of the present work was to carry out a bibliographic review of scientific publications that described the important role of this enzyme in relation to mitochondria as the cause of lymphatic cancer. The methodology used consisted of a bibliometric analysis based on the use of different databases, in which search equations formed from keywords were introduced. Then, the selection of articles related to the study topic and published in the last 20 years was carried out. Subsequently, the journals were analyzed, based on the h-index, in order to determine which supported the hypothesis of the role of PNPase in lymphoma and which stated the opposite. The results showed that a total of 441,288 scientific publications were obtained, of which 133 were selected to carry out this work. As for the journals, those with the highest h-index were Nature and Cell . It can be concluded that PNPase plays a very important role in the transport of mitochondrial RNA and that the nuclear transcription factor (NF-Y) is involved in the control of cell growth, therefore, both have a crucial role in the development of this disease. Therefore, research on both PNPase and NF-Y is essential to establish the specific genetic characteristics that define the early lesions of lymphatic cancer and the consequent determination of their treatment. El linfoma es el tipo de cáncer de sangre más común en la actualidad y, como su nombre indica, comienza en el sistema linfático. El origen de esta enfermedad está relacionado con defectos mitocondriales, generados por mutaciones en la enzima PNPasa o polinucleótido fosforilasa, cuyas principales funciones son las de importación y degradación del ARN mitocondrial. Por este motivo, el objetivo principal del presente trabajo fue la realización de una revisión bibliográfica acerca de publicaciones científicas, que hicieran relevancia al papel de esta enzima, en relación con la mitocondria como causante del cáncer linfático. La metodología empleada consistió en un análisis bibliométrico basado en la utilización de diferentes bases de datos, en las que se introdujeron ecuaciones de búsqueda formadas a partir de palabras clave. Después, se llevó a cabo la selección de los artículos relacionados con el tema de estudio y publicados en los últimos 20 años. Posteriormente, se analizaron las revistas, en función del índice H, para poder observar cuáles apoyaban la hipótesis del papel de PNPasa en linfoma y cuáles decían lo contrario. Los resultados mostraron que se obtuvo un total de 441.288 publicaciones científicas, de las que fueron seleccionadas 147 para poder llevar a cabo este trabajo. En cuanto a las revistas, las que presentaron un índice H mayor fueron Natura y Cell. Se puede concluir que la PNPasa desempeña un papel muy importante en el transporte del ARN mitocondrial, y que el factor NF-Y está involucrado en el control del crecimiento de las células, por lo que, ambos presentan una función crucial en el desarrollo de esta enfermedad. Por ello, la investigación, tanto de PNPasa como de NF- Y, es fundamental para establecer las características genéticas concretas, que definan las lesiones tempranas del cáncer linfático y la consiguiente determinación de su tratamiento. Ciencias Experimentales

Oxidative distress and mitochondrial dysfunction, are key factors involved in the pathophysiology of Parkinson's disease (PD). The pleiotropic hormone insulin-like growth factor II (IGF-II) has shown neuroprotective and antioxidant effects in some neurodegenerative diseases. In this work, we demonstrate the protective effect of IGF-II against the damage induced by 1-methyl-4-phenylpyridinium (MPP+) in neuronal dopaminergic cell cultures and a mouse model of progressive PD. In the neuronal model, IGF-II counteracts the oxidative distress produced by MPP + protecting dopaminergic neurons. Improved mitochondrial function, increased nuclear factor (erythroid-derived 2)-like2 (NRF2) nuclear translocation along with NRF2-dependent upregulation of antioxidative enzymes, and modulation of mammalian target of rapamycin (mTOR) signalling pathway were identified as mechanisms leading to neuroprotection and the survival of dopaminergic cells. The neuroprotective effect of IGF-II against MPP + -neurotoxicity on dopaminergic neurons depends on the specific IGF-II receptor (IGF-IIr). In the mouse model, IGF-II prevents behavioural dysfunction and dopaminergic nigrostriatal pathway degeneration and mitigates neuroinflammation induced by MPP+. Our work demonstrates that hampering oxidative stress and normalising mitochondrial function through the interaction of IGF-II with its specific IGF-IIr are neuroprotective in both neuronal and mouse models. Thus, the modulation of the IGF-II/IGF-IIr signalling pathway may be a useful therapeutic approach for the prevention and treatment of PD., Graphical abstract Image 1, Highlights • IGF-II hampers oxidative damage and promotes survival in a cellular model of PD. • IGF-II avoids mitochondrial damage in dopaminergic cells in a model of PD. • IGF-II receptor mediates the neuroprotective effect of IGF-II in a cellular model of PD. • IGF-II prevents nigrostriatal degeneration and inflammation in a mice model of PD. • IGF-II prevents behavioural dysfunction in a mice model of PD.

Mitofusins (MFN1/2) have been demonstrated to play a role in several mitochondrial functions such as mitochondrial dynamics and bioenergetics, interorganelle communication, immune response, autophagy, cell cycle progression, cellular senescence and apoptosis. However, the role of MFN1/2 in the molecular mechanisms underlying these processes remains poorly understood. Here, we have studied MFN1/2 functions and regulation from an interactome perspective in order to gain insight into the accurate mechanisms by which they are involved in those cellular events. To this end, we have generated two HeLa cell lines expressing an HA-tag fused to the C-terminal domain of MFN1 and MFN2 applying CRISPR/Cas9 technology. This has facilitated MFN1/2 pulldown under endogenous expression levels in basal conditions and in conditions of nutrient deprivation. The fact that MFN1/2 expression levels remain endogenous is key since most of their binding partners known so far have been studied under overexpression conditions. Subsequently, we have performed mass spectrometry to identify interactors of MFN1 and MFN2 in basal or starvation conditions. We have observed that RNA-binding proteins are enriched in basal conditions among MFN1 and MFN2 binding candidates and that they are absent in starvation conditions. We have validated that FXR1, AGO2, RAB5C and NDFIP2 interact with MFN1 and MFN2, and that S100A8, S100A9 and SLC27A2 interact with MFN2. We have found that RAB5C is necessary for mitochondrial morphology maintenance. Our data have also shown that SLC27A2 is a protein important for the process of autophagy.

Producción Científica, The FASTK family of proteins have been recently reported to play a key role in the post-transcriptional regulation of mitochondrial gene expression, including mRNA stability and translation. Accumulated studies have provided evidence that the expression of some FASTK genes is altered in certain types of cancer, in agreement with the central role of mitochondria in cancer development. Here, we obtained a pan-cancer overview of the genomic and transcriptomic alterations of FASTK genes. FASTK, FASTKD1, FASTKD3 and FASTKD5 showed the highest rates of genetic alterations. FASTK and FASTKD3 alterations consisted mainly of amplifications that were seen in more than 8% of ovarian and lung cancers, respectively. FASTKD1 and FASTKD5 were the most frequently mutated FASTK genes, and the mutations were identified in 5–7% of uterine cancers, as well as in 4% of melanomas. Our results also showed that the mRNA levels of all FASTK members were strongly upregulated in esophageal, stomach, liver and lung cancers. Finally, the protein-protein interaction network for FASTK proteins uncovers the interaction of FASTK, FASTKD2, FASTKD4 and FASTKD5 with cancer signaling pathways. These results serve as a starting point for future research into the potential of the FASTK family members as diagnostic and therapeutic targets for certain types of cancer., Junta de Castilla y León (grants GRS 1971/A/19 and GRS 2201/A/2020), Junta de Castilla y León (grant VA172P20), Ministerio de Ciencia e Innovación (Severo Ochoa SVP-2014–068895)

Made available in DSpace on 2022-04-29T08:30:43Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01 The mitogenome of the South American parthenogenetic lizard Loxopholis percarinatum Müller, 1923 (Squamata: Gymnophthalmidae), a uni-bisexual species complex, was recovered for three individuals from Rio Negro region, Amazonas, Brazil. The content and order of genes are typical for vertebrate mitochondrial genomes, and we recovered 13 protein-coding genes, 22 tRNA, and two rRNA (12S and 16S), in addition to partial fragments of the Control Region. A maximum likelihood phylogenetic analysis with mitogenomes of selected lizard families recovered L. percarinatum with Iphisa elegans Gray, 1851, the only other Gymnophthalmidae species available in GenBank. Departamento de Zoologia Instituto de Biociências Universidade de São Paulo Departamento de Biodiversidade e Centro de Aquicultura Instituto de Biociências Universidade Estadual Paulista (UNESP) Amphibian and Reptile Diversity Research Center and Department of Biology University of Texas at Arlington Departamento de Ecologia e Biologia Evolutiva Universidade Federal de São Paulo Departamento de Biodiversidade e Centro de Aquicultura Instituto de Biociências Universidade Estadual Paulista (UNESP)

O acúmulo tecidual do ácido 3-metilglutárico (MGA) é um achado bioquímico característico de um grupo de doenças conhecidas como acidúria 3- metilglutacônica (MGTA), bem como da acidúria 3-hidróxi-3-metilglutárica (HMGA). Tanto as MGTA quanto a HMGA são doenças genéticas de herança autossômica recessiva cujos sintomas podem aparecer na infância ou na adolescência. Os pacientes geralmente apresentam anormalidades no cérebro, fígado e coração. Considerando que os mecanismos responsáveis pelo dano tecidual encontrado nessas doenças não estão totalmente estabelecidos, esta tese avaliou os efeitos da administração de MGA sobre o metabolismo energético, estresse oxidativo, biogênese mitocondrial e vias metabólicas relacionadas em córtex cerebral e fígado de ratos. Além disso, investigamos os efeitos do bezafibrato (BEZ), uma molécula potencialmente protetora, frente aos efeitos de MGA visto que o tratamento para as MGTA e a HMGA é bastante limitado. Para isso, ratos Wistar jovens receberam um pré-tratamento por gavagem com óleo de milho ou BEZ (30 ou 100mg/kg/dia) por sete dias, uma vez ao dia. No 30° dia de vida, os animais receberam, após 1 h da gavagem com óleo (veículo) ou BEZ, três injeções intraperitoneais de MGA ou NaCl (primeira injeção de 10μmol.g-1 seguida de duas de 5μmol.g-1) com intervalo de 90 minutos entre as injeções. Após eutanásia dos ratos, o córtex cerebral e o fígado foram usados para avaliar os parâmetros. O MGA aumentou os níveis de malondialdeído e o conteúdo de heme oxigenase-1 e ainda modulou a atividade de algumas enzimas antioxidantes, indicando um quadro de estresse oxidativo. O MGA também diminuiu o conteúdo de glutationa no fígado. Foi visto ainda que o MGA alterou a atividade das enzimas da cadeia transportadora de elétrons em ambos os tecidos, sugerindo alterações na bioenergética. O MGA diminuiu o conteúdo nuclear do PGC-1α, principal regulador da biogênese mitocondrial, no cérebro e mostrou forte tendência em causar a mesma alteração no fígado. Além disso, o MGA diminuiu o conteúdo de sirtuína 1 nos dois tecidos, sendo esta uma proteína chave para ativação de PGC-1α. O MGA ainda diminuiu o conteúdo de Akt nos tecidos, indicando que alterações nesta via também podem estar prejudicando a ativação de PGC-1α. No que concerne especificamente ao córtex cerebral, o MGA também diminuiu o conteúdo de sinaptofisina e aumentou a fosforilação da Tau. Este ácido ainda aumentou a fosforilação da ERK1/2 e p38, e aumentou o conteúdo de AMPKα1, sendo este último também aumentado no fígado. No que diz respeito ao fígado, o MGA reduziu o conteúdo de PPARα nuclear em relação ao citosólico, e também alterou o conteúdo de mitofusina 1 e proteína relacionada à dinamina 1, indicando um prejuízo à dinâmica mitocondrial. Nossos dados sugerem uma toxicidade relevante do MGA neste modelo, causando disfunção mitocondrial, estresse oxidativo e danos aos tecidos estudados. Além disso, nossos resultados mostraram que o BEZ exerceu efeitos benéficos na prevenção dos efeitos do MGA tanto no córtex cerebral quanto em fígado. Contudo, foi observado que o BEZ exerceu maior ação protetora no cérebro. Portanto, nossos dados sugerem que o MGA tem um papel importante na fisiopatologia dos danos cerebral e hepático verificados nos pacientes com MGTA e HMGA e que o BEZ pode ser considerado um fármaco promissor para o tratamento dessas patologias. Tissue accumulation of 3-methylglutaric acid (MGA) is an important biochemical finding observed in a group of disorders collectively called 3-methylglutaconic acidurias (MGTA), and also in 3-hydroxy-3-methylglutaric aciduria (HMGA). All types of MGTA and HMGA are autosomal recessive inherited diseases. Symptoms usually appear during childhood or adolescence, and affect especially the central nervous system, liver and heart. Since the mechanisms underlying the organ abnormalities commonly observed in patients affected by MGTA and HMGA are not fully elucidated, we investigated here the effects of MGA administration on redox homeostasis, mitochondrial bioenergetics, biogenesis and dynamics, and signaling pathways in brain and liver of young rats. As treatment for these disorders is limited, the effects of a pre-treament with bezafibrate (BEZ), a potential protective molecule, were also determined. In our model, young Wistar rats received three intraperitoneal injections of NaCl (control) or MGA (one injection of 10 μmol/g followed by two injections of 5 μmol/g with a 90-min interval) and were euthanized 1 h after the last injection. Pre-treatment with BEZ (30 or 100 mg/kg/day) was performed by gavage for 7 days before NaCl or MGA injection. After euthanasia of animals, liver and brain homogenates were prepared to evaluate the parameters. MGA administration induced marked toxicity in both tissues evaluated. We verified that MGA increased the levels of malondialdehyde and heme oxgenase-1, and altered the activity of several antioxidant enzymes, indicative of oxidative stress. MGA also decreased GSH levels in liver. Furthermore, MGA altered the activities of respiratory chain complexes in brain and liver, suggesting bioenergetic dysfunction. MGA also decreased the content of nuclear PGC-1α in brain, and showed a strong tendency to decrease this protein in liver. MGA further reduced the content of situin 1 in both tissues. Considering that situin 1 is an activator of PGC-1α, the master regulator of mitochondrial biogenesis, our data indicate mitochondrial biogenesis disruption. In addition, MGA decreased the content of Akt, which may be also involved in the mitochondrial biogenesis disturbances. Regarding only the brain, MGA resulted in decreased content of synaptophysin and increased Tau protein phosphorylation. MGA also increased the phosphorylation of ERK1/2, p38, and the content of AMPKα1 in the brain. In liver, MGA decreased the nuclear content of PPARγ compared to the cytosolic compartment, augmented AMPKα1 levels and altered the levels of mitofusin 1 and dynamin-related protein 1, reinforcing the data showing that MGA disturbs in mitochondrial biogenesis and also indicating mitochondrial dynamics alterations. We also verified that bezafibrate was able to prevent most deleterious effects caused by MGA. Of note, these protective effects elicited by bezafibrate were more pronounced in the cerebral cortex than in liver. These findings indicate that MGA plays an important role in the neurological symptoms and hepatic dysnfunction observed in MGTA and HMGA by inducing oxidative stress and causing mitochondrial dysfunction. Finally, our study suggests that bezafibrate may be a novel therapeutic strategy for these disorders.

The Papillon&ndash, Lef&egrave, vre syndrome (PLS) is a rare autosomal recessive disorder caused by mutations in the Cathepsin C (CTSC) gene, characterized by periodontitis and palmoplantar hyperkeratosis. The main inflammatory deficiencies include oxidative stress and autophagic dysfunction. Mitochondria are the main source of reactive oxygen species, their impaired function is related to skin diseases and periodontitis. The mitochondrial function has been evaluated in PLS and mitochondria have been targeted as a possible treatment for PLS. We show for the first time an important mitochondrial dysfunction associated with increased oxidative damage of mtDNA, reduced CoQ10 and mitochondrial mass and aberrant morphologies of the mitochondria in PLS patients. Mitochondrial dysfunction, determined by oxygen consumption rate (OCR) in PLS fibroblasts, was treated with CoQ10 supplementation, which determined an improvement in OCR and a remission of skin damage in a patient receiving a topical administration of a cream enriched with CoQ10 0.1%. We provide the first evidence of the role of mitochondrial dysfunction and CoQ10 deficiency in the pathophysiology of PLS and a future therapeutic option for PLS.

Ca2+ signaling has been involved in controling critical cellular functions such as activation of proteases, cell death, and cell cycle control. The endoplasmatic reticulum plays a significant role in Ca2+ storage inside the cell, but mitochondria have long been recognized as a fundamental Ca2+ pool. Protozoan parasites such as Plasmodium falciparum, Toxoplasma gondii, and Trypanosoma cruzi display a Ca2+ signaling toolkit with similarities to higher eukaryotes, including the participation of mitochondria in Ca2+-dependent signaling events. This review summarizes the most recent knowledge in mitochondrial Ca2+ signaling in protozoan parasites, focusing on the mechanism involved in mitochondrial Ca2+ uptake by pathogenic protists.

This study was supported by the grant #3650 managed by Fundacion General EmpresaUniversidad de Granada and by the investigation group CTS-454 "Impacto fisiologico del estres oxidativo, deporte, actividad fisica y salud"., We tested whether physiological doses of hydroxytyrosol (HT) may alter the mRNA transcription of key metabolic genes in exercised skeletal muscle. Two groups of exercise-trained Wistar rats, HTlow and HTmid, were supplemented with 0.31 and 4.61 mg/kg/d of HT, respectively, for 10 weeks. Another two groups of rats were not supplemented with HT; one remained sedentary and the other one was exercised. After the experimental period, the soleus muscle was removed for qRT-PCR and western blot analysis. The consumption of 4.61 mg/kg/d of HT during exercise increased the mRNA expression of important metabolic proteins. Specifically, 4.61 mg/kg/d of HT may upregulate long-chain fatty acid oxidation, lactate, and glucose oxidation as well as mitochondrial Krebs cycle in trained skeletal muscle. However, a 4.61 mg/kg/d of HT may alter protein translation, as in spite of the increment showed by CD36 and GLUT4 at the mRNA level this was not translated to higher protein content., 3650 CTS-454

O câncer é um problema de saúde pública que leva milhares de pessoas à óbito todo ano. O maior desafio para a terapia é a evolução tumoral, e embora a seleção de células resistentes tenha um importante papel nesse processo, está cada vez mais claro que outros mecanismos estão envolvidos. Entre esses mecanismos, características celulares que mudam ao longo do tempo (dinâmicas fenotípicas) devem contribuir para a tolerância de células tumorais, que eventualmente podem se tornar resistentes. Nesse sentido, esta tese tem como principal objetivo identificar características que contribuam para o desenvolvimento da tolerância através de flutuações que geram diferenças entre células clonais (geneticamente idênticas). Os resultados alcançados mostram que a capacidade de deixar descendentes (fitness) varia entre células clonais, em situação controle e após tratamento com temozolomida (TMZ). Esses achados são de extrema relevância para a biologia tumoral já que o fitness é a característica que vai determinar se a doença irá progredir ou não, e foram possíveis através da formulação de uma nova metodologia para avaliar dinâmica fenotípica chamada de Dynafit (análise da dinâmica de fitness – Dynamic Analysis of Fitness). Alguns fenótipos que contribuem para essa instabilidade foram identificados: atividade de ERK, dessincronização do ciclo celular, dano ao DNA e indução de senescência. Ainda, a modulação epigenética reduziu a instabilidade fenotípica e a morte fractional em colônias. Por fim, foi demonstrado a ocorrência de partição assimétrica mitocôndrial, que contribui para a geração de heterogenidade, que por sua vez é um dos principais obstáculos no tratamento de glioblastomas. Uma vez que a massa mitocôndrial tem um impacto sobre o fitness, como sugerido por outros e confirmado na presente tese, a ocorrência de partição assimétricas dessa organela gera células irmãs com diferentes níveis de sensibilidade. Também, foi demonstrado que a massa e a rede mitocôndrial são alteradas em resposta à TMZ. As conclusões deixam claro que para o aumento da eficácia do tratamento de tumores a instabilidade fenotípica deve ser levada em consideração, uma vez que a tolerância gerada por essa instabilidade contribui para o desenvolvimento da resistência e recidiva da doença. Cancer is a public health problem that kills thousands of people every year. The major challenge to therapy is the tumor evolution, and even thought the selection of resistant clones have an important role in this process, it is clear now that other process are involved. Among them, cellular characteristics that change over time (phenotypic dynamics) must contribute to the tolerance of tumoral cells, that eventually can become resistant. In that regard, this tesis have as a major aim identify characteristics that contribute to the development of tolerance through fluctuations that generate differences in clonal cells (genetically identical). The results show that the capacity to leave descendants (fitness) varies between clones, both in control and after treatment with temozolomide (TMZ). These findings are relevant to tumoral biology once the fitness is the characteristic that will determine if the disease progresses or not, and were achieved through the formulation of a new methodological approach to evaluate the phenotypic dynamic named Dynafit (Dynamic Analysis of Fitness). Same phenotypes that contribute to the instability of fitness were identify: ERK activity, cell cycle desynchronization, DNA damage and induction of senescence. Yet, the epigenetic modulation reduced the phenotypic instability and the fractional killing inside colonies. Finally, it was demonstrated the occurrence of asymmetric partition of mitochondria, that contributes to the generation of heterogeneity, that in turn is one of the main obstacles in the treatment of glioblastomas. Once the mitochondrial mass has an impact on fitness, as suggested by others and confirmed in this thesis, the occurrence of asymmetric partition of mitochondria generate sister cells with differences in sensitivity. Additionally, it was demonstrated that the mitochondrial mass and network are modified in response to TMZ. The conclusions clarify that to increase the effectiveness of tumor treatment, the phenotypic instability must be considered, once the tolerance generated through phenotypic instability contributes to the development of resistance and relapse of the disease.

A doença de Huntington (DH) é uma doença neurodegenerativa herdada de forma dominante e causada por uma expansão de repetição de bases de nucleotídeos CAG no gene huntingtin, resultando na síntese de uma proteína huntingtina mutante (mHTT). A tríade sintomatológica da DH inclui disfunções motoras, cognitivas e psiquiátricas. A primeira região cerebral a apresentar anormalidades é o estriado, sendo relatada uma degeneração de neurônios GABAérgicos em estágio avançando da doença. Outros achados, como perda de função de células gliais, bem como uma ativação de astrócitos, contribuem para um fenótipo neurotóxico na DH. Além disso, vários eventos celulares e moleculares desempenham um papel importante na patologia da DH. Em destaque estão as possíveis alterações mitocondriais, uma vez que a mitocôndria é uma organela essencial para diversos processos celulares, como a produção de ATP, metabolismo do cálcio e homeostase redox. Neste contexto, já é bem estabelecido que doenças neurodegenerativas apresentam disfunção mitocondrial, podendo alterar os processos envolvidos na dinâmica mitocondrial e nas interações dessa organela com outros constituintes celulares, em particular o retículo endoplasmático (RE). Assim, no presente estudo um modelo experimental animal para a DH induzido pela administração intraperitoneal do ácido 3-nitropropiônico (3-NP, 20 mg/kg) durante 3 dias consecutivos, investigamos parâmetros chave relacionados à mitocôndria no período de 7, 14, 21 e 28 dias após indução. Nossos resultados mostraram alterações na homeostase redox em estriado, com aumento da peroxidação lipídica 28 dias após a administração de 3-NP, e diminuição de defesas antioxidantes enzimáticas e não-enzimáticas em diferentes tempos. Também observamos disfunção do metabolismo bioenergético, com redução do estado 3, estado 4 e não-acoplado em 7 dias após a administração de 3-NP, aumento dos estados 4 e não acoplado e capacidade de reserva em 28 dias, e diminuição da atividade dos complexos da cadeia respiratória em 7 e 28 dias. Observamos alterações na dinâmica mitocondrial através do aumento da expressão proteica da mitofusina 1 (MFN1) em 7, 14 e 28 dias, uma importante proteína para a fusão mitocondrial, sugerindo uma resposta compensatória ao insulto oxidativo. Além disso, nossos resultados sugerem que em 28 dias após a administração 3-NP ocorreram alterações na interação mitocôndria-RE, com o aumento do conteúdo proteico de canal aniônico voltagem dependente 1 (VDAC1) e da proteína reguladora de glicose 75 kDa (GRP75). Também houve um aumento inicial observado no conteúdo da proteína sinaptofisina, seguido de diminuição, sugerindo uma resposta transitória da atividade sináptica, buscando a manutenção da neurotransmissão normal. Nossos achados mostram que um prejuízo grave na função mitocondrial, com alteração na dinâmica mitocondrial, homeostase do cálcio e redox, bem como um dano na interação mitocôndria-RE os quais estão envolvidos na fisiopatologia do dano estriatal observado na DH. Huntington's disease (HD) is a dominantly inherited neurodegenerative condition caused by a CAG nucleotide base repeat expansion in the huntingtin gene, resulting in the production of a mutant huntingtin protein (mHTT). The symptomatologic triad of HD confers motor, cognitive and psychiatric dysfunctions. In the initial stage, patients present with abnormalities in the striatum caused by mHTT, whereas degeneration of GABAergic neurons is observed in an advanced stage of the disease. Other findings, such as glial cells dysfunctions, particularly reactive astrocytes, contribute to a neurotoxic phenotype in HD. On the other hand, several cellular and molecular events have been shown to play a role in HD pathology. In this scenario, mitochondrial dysfunction has a key role since mitochondria are essential organelles for various cellular processes, such as ATP production, calcium metabolism and redox homeostasis. Furthermore, it is suggested that disrupted mitochondrial dynamics and alterations in the interactions of mitochondria with other cellular constituents, in particular the endoplasmic reticulum (ER), are common findings in neurodegenerative diseases. Thus, in the present study an experimental animal model for HD induced by intraperitoneal administration of 3- nitropropionic acid (3-NP, 20 mg/kg) for 3 consecutive days, we investigated key parameters related to mitochondria in the period of 7, 14, 21 and 28 days after induction. Our results showed changes in redox homeostasis in striatum, with an increase in lipid peroxidation 28 days after 3-NP treatment, and a decrease in defenses enzymatic and non-enzymatic antioxidant at different periods after 3-NP exposure. We also observed bioenergetic dysfunction, with reduced state 3, state 4 and non-coupled at 7 days, augmented state 4 and non-coupled state and reserve capacity at 28 days, and decreased activity of the respiratory chain complexes at 7 and 28 days. We observed changes in mitochondrial dynamics through increased protein expression of mitofusin 1 (MFN1) at 7, 14, and 28 days, an important protein for mitochondrial fusion, suggesting a compensatory response to 3-NP induced oxidative insult. Furthermore, our results demonstrated that, at 28 days after 3-NP administration, alterations in mitochondria-ER interactions occurred, with increased protein content of voltage-dependent anion channel 1 (VDAC1) and 75 kDa glucose regulated protein (GRP75). Additionally, an initial increase of synaptophysin protein content followed by a reduction of this content was seen, indicating a transient response aiming the maintenance of a normal neurotransmission. Our findings show that impaired mitochondrial function, with changes in mitochondrial dynamics, calcuim and redox homeostasis, as well as disturbances in mitochondria-endoplasmic reticulum interactions are involved in the pathophysiology of striatal damage seen in DH.

Tesis (Magíster en Biotecnología y Ciencias de la Vida) Financiada por el proyecto FONDECYT N°1180983: “Targeting anemia through enhancing mitochondrial dynamics and mitophagy: Investigation of two novel mitochondrial proteins”. Las mitocondrias son organelos eucariotas que cumplen roles desde la homeostasis del calcio, producción de ATP, hasta definir la sobrevivencia celular. Las mitocondrias son dinámicas y su morfología y biomasa se ajustan para satisfacer las demandas energéticas de las células. La biomasa mitocondrial está balanceada por los procesos de biogénesis, dinámica mitocondrial y autofagia selectiva de mitocondrias o mitofagia. Uno de los parámetros de biomasa mitocondrial es el número de copias de ADN mitocondrial (ADNmt) presente en las células, el cual es resultado del balance entre los procesos de biogénesis y mitofagia, por lo cual al haber una exacerbación de la mitofagia disminuirá la biomasa mitocondrial. ERYM3 es una nueva proteína putativa de las mitocondrias, monoexónica y exclusiva de los primates que fue encontrada mediante análisis in silico de microarreglos de la eritropoyesis. Su expresión a nivel de trascrito aumenta hacia el estadío final de la eritropoyesis. ERYM3 tiene un 86% de probabilidad de ser importada a la mitocondria, un patrón de expresión similar a las proteínas mitofágicas LC3 y NIX; y un dominio putativo LIR (Región de interacción con LC3) para la posible unión con LC3-II unido al fagóforo. Resultados preliminares de la sobreexpresión de la proteína de fusión ERYM3:GFP muestran su localización en el citosol, colocalización con mitocondrias y lisosomas, disminución de la marca mitocondrial y aumento en el número de lisosomas al inducirse la mitofagia, lo que sugiere la participación de ERYM3 en la mitofagia. Por lo tanto, se hipotetiza que ERYM3 estimula la disminución de la biomasa mitocondrial mediada por mitofagia. El objetivo general es determinar el efecto de la sobreexpresión de erym3 sobre la biomasa mitocondrial y la expresión de genes mitofágicos. Los resultados mostraron que a las 48 horas de sobreexpresión de erym3 disminuyó significativamente la biomasa mitocondrial según la cuantificación del ADNmt, mientras que los niveles de expresión de los genes mitofágicos medidos, correspondientes a pink1 y fundc1, no mostraron cambios significativos. La caracterización funcional de ERYM3 sugiere una posible nueva vía de mitofagia que permitirá el desarrollo de nuevas herramientas diagnósticas y terapéuticas para disfunciones mitocondriales.

Imbalance in the oxidative status in neurons, along with mitochondrial damage, are common characteristics in some neurodegenerative diseases. The maintenance in energy production is crucial to face and recover from oxidative damage, and the preservation of different sources of energy production is essential to preserve neuronal function. Fingolimod phosphate is a drug with neuroprotective and antioxidant actions, used in the treatment of multiple sclerosis. This work was performed in a model of oxidative damage on neuronal cell cultures exposed to menadione in the presence or absence of fingolimod phosphate. We studied the mitochondrial function, antioxidant enzymes, protein nitrosylation, and several pathways related with glucose metabolism and glycolytic and pentose phosphate in neuronal cells cultures. Our results showed that menadione produces a decrease in mitochondrial function, an imbalance in antioxidant enzymes, and an increase in nitrosylated proteins with a decrease in glycolysis and glucose-6-phosphate dehydrogenase. All these effects were counteracted when fingolimod phosphate was present in the incubation media. These effects were mediated, at least in part, by the interaction of this drug with its specific S1P receptors. These actions would make this drug a potential tool in the treatment of neurodegenerative processes, either to slow progression or alleviate symptoms.

[ES] La familia de proteínas Bcl-2 regula la apoptosis a través de una compleja red de interacciones. Las células tumorales suelen presentar mutaciones que afectan a su expresión o sus interacciones para mejorar la progresión tumoral. Además, alteraciones en su regulación también promueven la migración de células cancerígenas, la invasión y la metástasis. Para llevar a cabo sus funciones, las proteínas Bcl 2 interaccionan entre sí tanto en el citoplasma como en las membranas intracelulares. Los equilibrios de interacción de los dominios Bcl citosólicos se han investigado ampliamente y recientemente, se han propuesto como dianas terapéuticas. Sin embargo, el interactoma de los dominios transmembrana (TMD, del inglés transmembrane domains) sigue siendo poco conocido. Por ello, un conocimiento profundo de la biología de las proteínas Bcl-2 es necesario para explotar eficientemente sus superficies de unión en el tratamiento del cáncer. Para llevar a cabo este objetivo, nos hemos centrado en tres áreas: 1. La comprensión detallada de la contribución del TMD de Mcl-1 a su interactoma en membrana y su función. 2. El descubrimiento de nuevos inhibidores de Mcl-1 que actúen sobre su TMD y que permitan desarrollar una clase de drogas anticancerígenas aún por explorar. 3. La caracterización molecular de mutaciones relacionadas con el cáncer descritas en los TMD de Bcl-2 y Bcl-xL y sus implicaciones en la supervivencia de las células tumorales. La proteína antiapoptótica Mcl-1 inhibe a los miembros proapoptóticos Bak, Bax, Bok, Noxa, etc. Aunque se ha estudiado en detalle su actividad promoviendo la supervivencia celular, el mecanismo molecular por el cuál previene la apoptosis mediada por Bok aún no está claro. Además, el conocimiento de las actividades de Mcl-1, descritas hasta ahora, se basa exclusivamente en las estructuras resueltas de las regiones solubles en agua y en estudios centrados en los dominios citosólicos. Por primera vez, hemos demostrado la relevancia del TMD de Mcl-1 en su equilibrio de interacción. En este trabajo describimos su capacidad específica para homo- y hetero-oligomerizar con el TMD de Bok. También ponemos de manifiesto la influencia de estas interacciones en la modulación de apoptosis y resaltamos la relevancia clínica de los mutantes del TMD de Mcl-1 identificados en pacientes con cáncer. Muchos tumores hematológicos y sólidos sobre-expresan Mcl-1 como mecanismo para adquirir quimiorresistencia. Se han desarrollado miméticos de BH3 específicos para modular su actividad antiapoptótica en células cancerosas. Sin embargo, aún no disponemos de datos científicos que informen sobre su toxicidad y eficacia en humanos. En este trabajo, proponemos la novedosa interacción de los TMDs de Mcl-1 y Bok como un nuevo sitio de acción de fármacos quimioterapéuticos. Hemos identificado tres inhibidores de esta interacción con características que los hacen prometedores candidatos para el desarrollo farmacéutico, así como buenas herramientas moleculares para estudiar la interacción de los TMDs de Mcl-1 y Bok. Para modular la apoptosis, las células tumorales también presentan versiones mutadas de las proteínas antiapoptóticas Bcl-2 y Bcl-xL. En nuestro conocimiento, este es el primer estudio que analiza mutaciones somáticas de sus TMDs. Nuestro trabajo demuestra cómo estas mutaciones alteran el equilibrio en membrana de las proteínas. Además, nuestros resultados explican la influencia que algunos mutantes somáticos ejercen en la regulación de la apoptosis. En general, los resultados científicos que aparecen en esta tesis resaltan el papel de los Bcl TMDs en el interactoma de las proteínas Bcl-2. Estos hallazgos corroboran que las interacciones laterales entre los TMDs son específicas y contribuyen activamente a la funcionalidad de la proteína. Por lo tanto, comprender los Bcl TMDs puede proporcionar nuevos conocimientos sobre la biología de las proteínas Bcl., [CA] La família de proteïnes Bcl-2 regula l'apoptosi a través d'una complexa xarxa d'interaccions. Les cèl·lules tumorals solen presentar mutacions que afecten la seua expressió o les seues interaccions per a millorar la progressió tumoral. A més, alteracions en la seua regulació també promouen la migració de cèl·lules cancerígenes, la invasió i la metàstasi. Per a dur a terme les seues funcions, les proteïnes Bcl-2 interaccionen entre si tant en el citoplasma com en les membranes intracel·lulars. Els equilibris d'interacció dels dominis Bcl citosòlics s'han investigat àmpliament i recentment, s'han proposat com a dianes terapèutiques. No obstant això, l'interactoma dels dominis transmembrana (TMD, de l'anglés transmembrane domains) continua sent poc conegut. Per això, un coneixement profund de la biologia de les proteïnes Bcl-2 és necessari per a explotar eficientment les seues superfícies d'unió en el tractament del càncer. Per a dur a terme aquest objectiu, ens hem centrat en tres àrees: 1. La comprensió detallada de la contribució del TMD de Mcl-1 al seu interactoma en membrana i la seua funció. 2. El descobriment de nous inhibidors de Mcl-1 que actuen sobre el seu TMD i que permeten desenvolupar una classe de drogues anticanceroses encara per explorar. 3. La caracterització molecular de mutacions relacionades amb el càncer descrites en els TMD de Bcl-2 i Bcl-xL i les seues implicacions en la supervivència de les cèl·lules tumorals. La proteïna anti apoptòtica Mcl-1 inhibeix als membres pro apoptòtics Bak, Bax, Bok, Noxa, etc. Encara que s'ha estudiat detalladament la seua activitat promovent la supervivència cel·lular, el mecanisme molecular pel qual prevé l'apoptosi mediada per Bok encara no és clar. A més, el coneixement de les activitats de Mcl-1, descrites fins ara, es basa exclusivament en les estructures resoltes solubles en aigua i en estudis centrats en els dominis externs a la membrana. Per primera vegada, hem demostrat la rellevància del TMD de Mcl-1 el seu equilibri d'interacció. En aquest treball descrivim la seua capacitat específica per a unir-se amb si mateix i per a hetero-oligomeritzar amb el TMD de Bok. També expliquem la influència d'aquestes interaccions en l'apoptosi i ressaltem la rellevància clínica dels mutants del TMD de Mcl-1 identificats en pacients amb càncer. Molts tumors hematològics i sòlids sobre-expressen Mcl-1 com un mecanisme per a adquirir quimioresistència. S'han desenvolupat mimètics de BH3 específics per a modular la seua activitat anti apoptòtica en cèl·lules canceroses. No obstant això, encara no disposem de dades científiques que informen sobre la seua toxicitat i eficàcia en humans. Per això, proposem la nova interacció dels TMDs de Mcl-1 i Bok com un lloc d'actuació de fàrmacs quimioterapèutiques. Hem identificat tres inhibidors d'aquesta interacció amb característiques que els fan prometedors candidats per al desenvolupament farmacèutic, així com bones eines moleculars per a estudiar la interacció dels TMDs de Mcl-1 i Bok. Per a modular l'apoptosi, les cèl·lules tumorals també presenten versions mutades de les proteïnes anti apoptòtiques Bcl-2 i Bcl-xL. En el nostre coneixement, aquest és el primer estudi que analitza mutacions somàtiques de les seues TMDs. El nostre treball demostra com aquestes mutacions alteren l'equilibri en membrana de les proteïnes. A més, els nostres resultats expliquen la influència que alguns mutants somàtics exerceixen en la regulació de l'apoptosi. En general, els resultats científics que apareixen en aquesta tesi ressalten el paper dels Bcl TMDs en l'interactoma de les proteïnes Bcl-2. Aquestes troballes corroboren que les interaccions laterals entre els TMDs són específiques de la seqüència i contribueixen activament a la funcionalitat de la proteïna. Per tant, comprendre els Bcl TMDs pot proporcionar nous coneixements sobre la biologia de les proteïnes Bcl, [EN] The family of the Bcl-2 proteins modulates the apoptotic pathway by a complex network of interactions. Tumor cells frequently present mutations that affect Bcl-2 proteins expression or interactions to enhance cancer progression. Dysregulation of these proteins also promotes cancer cell migration, invasion, and metastasis. To execute their functions, Bcl-2 proteins interact in both the cytosol and intracellular membranes. Binding equilibria of Bcl extramembrane domains has been largely investigated and recently proposed as chemotherapeutic targets. However, the interactome of transmembrane domains (TMDs) remains poorly understood. In this scenario, a deep knowledge of the biology of Bcl-2 proteins is needed to exploit efficiently their binding surfaces for cancer treatment. To address this aim, our research focuses on three areas: 1. The detailed comprehension of the TMD contribution to both the Mcl-1 membrane interactome and protein functionality. 2. The discovery of new Mcl-1 inhibitors that target the transmembrane surface to develop a class of anticancer drugs currently unexplored. 3. The molecular characterization of cancer-related mutations within the Bcl-2 and Bcl-xL TMDs and their implications for the survival of cancer cells. Antiapoptotic Mcl-1 protein inhibits the proapoptotic members Bak, Bax, Bok, and Noxa, among others. Although its prosurvival activity has been well studied, the molecular mechanism to prevent Bok-mediated apoptosis remains unclear. Furthermore, understanding of Mcl-1 activities described to date is only based on water-soluble structures and studies focused on extramembrane domains. For the first time, we uncover the relevance of the Mcl-1 TMD in the interaction equilibria of the protein. In the present work, we describe its specific capacity to self-associate and hetero-oligomerize with the Bok TMD. We also explain the influence of these interactions in the apoptotic pathway and highlight the clinical relevance of Mcl-1 TMD mutants identified in tumor patients. Many hematological and solid malignancies overexpress Mcl-1 as an acquired chemoresistance mechanism. To modulate its antiapoptotic activity in cancer cells, specific BH3 mimetics have been developed; however, there is no scientific data yet regarding human toxicity and efficacy. In this work, we propose the novel Mcl-1 and Bok TMDs interaction interface as a drugging site in the development of chemotherapeutics. We identify three potential inhibitors of such molecular interface with promising features to become both drug candidates for pharmaceutical development and research toosl for the molecular study of the Mcl-1 and Bok TMDs interaction. To take advantage of apoptosis modulation, tumor cells also present mutated versions of the antiapoptotic members Bcl-2 and Bcl-xL. To our knowledge, this is the first study that analyzes patient-derived mutations within Bcl-2 and Bcl-xL TMDs and demonstrates how said mutations alter the membrane equilibria of these proteins. The results presented here also explain the functional influence of some somatic mutants in apoptosis regulation. Overall, the scientific results exhibited in this Thesis highlight the role of Bcl TMDs in the interactome of Bcl-2 proteins. These findings corroborate that lateral interactions between TMDs are sequence-specific and actively contribute to protein functionality. Therefore, understanding of Bcl transmembrane segments may provide new insights into the biology of Bcl 2 proteins for their pharmaceutical modulation in antitumoral therapy., The student has been granted with a PhD fellowship and a short-term fellowship from the Generalitat Valenciana (Subvenciones para la contratación de personal investigador de carácter predoctoral, 2016-2019, and Grant for predoctoral stays out of the Comunitat Valenciana, 2019). This work has been supported by the Spanish Ministry of Economy and Competitiveness (projects SAF2014-52614-R and SAF2017-84689-R

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) As células necessitam de energia para a manutenção de vários processos metabólicos. Adenosina trifosfato (ATP) é um metabólito central no metabolismo celular, participando de inúmeras transações energéticas, e assim considerado uma fonte de energia biológica universal em todos organismos vivos. Em tecidos heterotróficos a mitocôndria é a organela responsável pela maior parte do suprimento do ATP necessário para os processos metabólicos. Para sua utilização, os adenilatos (adenosina monofosfato, AMP; adenosina difosfofato, ADP; e ATP) são distribuídos entre os compartimentos celulares via proteínas carreadoras especializadas em transporte. Dentre essas proteínas se encontram os carreadores do tipo antiporte ADP/ATP (AACs), que exportam o ATP da matriz mitocondrial para o citosol e em troca importam o ADP do citosol. Em Arabidopsis thaliana são encontradas três isoformas de AACs (AAC1, AAC2 e AAC3). Entre essas isoformas, o AAC1 é a mais expressa em diferentes tecidos e estágios fenológicos. Contudo, a sua função fisiológica ainda não foi elucidada em plantas. Neste trabalho, avaliou-se o papel do transportador mitocondrial AAC1 em A. thaliana. Para tal, foram utilizadas uma linha mutante com inserção de T-DNA (KO) e quatro linhas antisenso sob o controle do promotor 35S (AS2, AS4, AS5 e AS7) com reduzida expressão de AAC1. Em folhas expandidas de plantas deficientes na expressão de AAC1, observou-se redução nastaxas de respiração noturna sem alterações em parâmetros de fotossíntese em relação a plantas selvagens. Adicionalmente, foram observados diminuição no número de folhas e na área foliar específica. Verificou-se também acúmulo de açucares (glicose, frutose, sacarose) e malato em folhas das plantas com baixa expressão de AAC1. Observou-se maiores teores de aminoácidos em paralelo com menores conteúdos de proteínas em folhas. Nas raízes, a deficiência na expressão de AAC1 resultou em menor crescimento, o qual foi associado a menores taxas respiratórios nos tecidos desse órgão. Em conjunto, os resultados apresentados sugerem que AAC1 desempenha um papel importante na respiração mitocondrial impactando o crescimento da planta como um todo, sem alterações marcantes na eficiência fotossintética. Palavras-chave: Translocador 1 do Nucleotídeo Adenina. Metabolismo. Respiração celular. Mitocôndrias. Células - Cells need energy to maintain various metabolic processes. Adenosine triphosfate (ATP) is a central metabolite in cellular metabolism, participating in numerous energy transactions, and thus considered a source of universal biological energy in all living organisms. In heterotrophic tissues, mitochondria are the organelle responsible for most of the ATP supply necessary for metabolic processes. For their use, adenylates (AMP, ADP and ATP) are distributed among the cell compartments via transport-specialized carries proteins. Among these proteins are carriers of the Type Antiport ADP/ATP (AACs), which export the ATP from the mitochondrial matrix to the cytosol and in return import the ADP of the cytosol. In Arabidopsis thaliana, three isoforms of AACs are found: AAC1, AAC2 and AAC3. Among these isoforms, AAC1 is the most expressed in different tissues and phenological stages. However, its physiological function has not yet been fully elucidated in plants. In this work, the role of mitochondrial transporter AAC1 in A. thaliana was evaluated. For this, we used a mutant line with T-DNA (KO) insertion and four antisense lines under the control of promoter 35S (AS2, AS4, AS5 and AS7) with reduced expression of AAC1. In expanded leaves of plants deficient in the expression of AAC1, there was a reduction in dark respiration rates without changes in photosynthesis parameters in relation to wild plants. Additionally, was observed a decrease in the number of leaves and in the specific leaf area. There was also accumulation of sugars (glucose, fructose, sucrose), and malate in leaves of plants with low expression of AAC1. A decrease in the number of leaves and in the specific leaf area was observed. There was also accumulation of sugars (glucose, fructose, sucrose) and malate in leaves of plants with low expression of AAC1. A decrease in the number of leaves and in the specific leaf area was observed. There was also accumulation of sugars (glucose, fructose, sucrose), and malate in leaves of plants with low expression of AAC1. Higher amino acid contents were observed in parallel with lower protein contents in leaves. In the roots, the deficiency in the expression of AAC1 resulted in lower growth, which was associated with lower respiratory rates in the tissues of this organ. Together, the results presented suggest that AAC1 plays an important role in mitochondrial respiration impacting plant growth as a whole, without marked changes in photosynthetic efficiency.

Purpose To evaluate the effects of treatment with Indigo Carmine (IC) on rat livers subjected to ischemia-reperfusion injury. Methods The animals were subdivided into 4 groups: 1.SHAM group(SH) - saline; 2.SHAM group with IC-2mg/Kg(SHIC); 3.IR group - rats submitted to ischemia and reperfusion with saline(IR); 4.IR group with IC-2mg/Kg(IRIC). The IR protocol consists of liver exposure and administration of drug or saline intravenously, followed by 60 minutes of ischemia and 15 of reperfusion. Liver samples were collected for biochemical analysis. Results State 3 of mitochondrial respiration showed a significant worsening of the IRIC group in relation to all others. State 4 showed a difference between IRIC and SHIC. The Respiratory Control Ratio showed statistical decrease in IR and IRIC versus Sham. The osmotic swelling showed significant difference between SHxIR; SHICxIRIC and SHxIRIC. There was a significant increase in ALT in the IRIC group in relation to all the others. Concerning the nitrate dosage, there was a decrease in the group treated with IC(IRxIRIC). There was no difference regarding the dosage of Malondialdehyde. Conclusion IC was not able to protect mitochondria from IR injury and proved to be a potentiating agent, acting in synergy with the IR injury promoting damage to the hepatocyte membranes.

Cancer stem cells (CSC) are associated with the mechanisms of chemoresistance to different cytotoxic drugs or radiotherapy, as well as with tumor relapse and a poor prognosis. Various studies have shown that mitochondria play a central role in these processes because of the ability of this organelle to modify cell metabolism, allowing survival and avoiding apoptosis clearance of cancer cells. Thus, the whole mitochondrial cycle, from its biogenesis to its death, either by mitophagy or by apoptosis, can be targeted by different drugs to reduce mitochondrial fitness, allowing for a restored or increased sensitivity to chemotherapeutic drugs. Once mitochondrial misbalance is induced by a specific drug in any of the processes of mitochondrial metabolism, two elements are commonly boosted: an increment in reactive nitrogen/oxygen species and, subsequently, activation of the intrinsic apoptotic pathway. Yes

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 30-06-2020, Esta tesis tiene embargado el acceso al texto completo hasta el 30-12-2021, La regulación de la respiración neuronal se ha atribuido tradicionalmente al transportador de Ca2+ mitocondrial (MCU), ya que el Ca2+ mitocondrial (Ca2+-mit) activa las deshidrogenasas del ciclo de Krebs. Sin embargo, los transportadores mitocondriales regulados por Ca2+ (CaMCs) tienen un papel muy relevante regulando la respiración en respuesta a estímulos que producen pequeñas señales de Ca2+ citosólicas. Aralar/AGC1/Slc25a12 es el transportador de aspartato-glutamato neuronal, y el punto de regulación de la lanzadera de NADH malato-aspartato (MAS). En este estudio se demuestra que la activación por Ca2+ de Aralar, y no MCU/Ca2+-mit, es esencial para la estimulación de la respiración en respuesta a la estimulación por NMDA. La activación por Ca2+ de Aralar-MAS en neuronas que usan glucosa es necesaria para estimular la síntesis de piruvato, activando su transporte a la mitocondria y la respiración y la glucolisis, algo que no ocurre en ausencia de Ca2+. Además, se demuestra que las neuronas aumentan la captura de glucosa de forma Ca2+-dependiente e independiente de Aralar tras la estimulación por NMDA. Sorprendentemente, el silenciamiento de Mcu no sólo no disminuye la respiración estimulada por NMDA, sino que tiene un efecto potenciador en parte atribuible a los efectos tóxicos del Ca2+ mitocondrial. La deficiencia en Aralar/AGC1 causa una enfermedad rara del neurodesarrollo caracterizada por retraso en el crecimiento, epilepsia e hipomielinización. Los ratones aralar-KO tienen una corta esperanza de vida y alteraciones motoras asociadas a una grave afección en el estriado. En humanos, la dieta cetogénica (KD) revierte la epilepsia y mejora parcialmente la hipomielinización. Este estudio revela que el principal cuerpo cetónico sintetizado tras el tratamiento con KD, β-Hidroxibutirato (βOHB) es beneficioso en la deficiencia en Aralar. βOHB recupera la respiración mitocondrial en glucosa en neuronas aralar-KO y las protege de la excitotoxicidad inducida por glutamato. In vivo, βOHB recupera parcialmente marcadores dopaminérgicos estriatales, como el contenido en dopamina, el transportador vesicular VMAT2 y el mediador postsináptico DARPP-32. βOHB también aumenta la síntesis de proteínas de mielina. Aunque no se recuperan los niveles de aspartato y NAA cerebrales, βOHB los aumenta en neuronas aralar-KO, sugiriendo que su uso está aumentado en el ratón, reforzando el papel de NAA como precursor en la síntesis de mielina en oligodendrocitos. A pesar de las limitaciones impuestas por el fenotipo tan severo de los ratones aralar-KO, este estudio demuestra que el βOHB es un sustrato alternativo efectivo e independiente de aralar que revierte las principales alteraciones de esta enfermedad. Un estudio más profundo de sus funciones neuroprotectoras, también a través de vías de señalización, sería muy relevante para identificar terapias sustitutivas de la dieta cetogénica en humanos, Regulation of neuronal respiration has been traditionally attributed to mitochondrial Ca2+ uniporter (MCU), as it allows Ca2+ activation of matrix dehydrogenases and TCA cycle. However, Ca2+-regulated mitochondrial carriers (CaMCs) have revealed an important role in stimulating neuronal respiration in response to agents evoking of small cytosolic Ca2+ signals. Aralar/AGC1/Slc25a12 is the aspartate-glutamate carrier expressed in neurons, and the regulatory step in the malate-aspartate NADH shuttle (MAS). This study demonstrates that Ca2+ activation of Aralar, rather than MCU/mitochondrial Ca2+ (Ca2+- mit), controls the upregulation of respiration in response to NMDA in neurons using glucose. Ca2+ activation of Aralar-MAS is necessary to increase endogenous pyruvate synthesis, activating its transport into mitochondria and upregulating respiration and glycolysis, neither of which occurring in the absence of Ca2+. In addition, this study shows that following NMDA stimulation, neurons upregulate glucose uptake in a Ca2+-dependent and Aralar-independent way. Surprisingly, Mcu silencing not only fails to decrease NMDAstimulated respiration but in fact has a potentiator effect, probably due to suppression of the toxic effect of mitochondrial Ca2+. Aralar/AGC1 deficiency causes a neurodevelopmental rare disease characterized by growth retardation, epilepsy and secondary postnatal hypomyelination. Aralar-KO mice have reduced life expectancy and altered motor coordination with severe affectation of striatum. In human patients, ketogenic diet (KD) alleviates epilepsy and partially improves myelination. This study reveals that the main ketone body synthesized during treatment with KD, β-hydroxybutyrate (βOHB), reports beneficial effects in Aralar/AGC1 deficiency. βOHB rescues impaired mitochondrial respiration on glucose in aralar-KO primary cortical neurons, and also protects them from glutamate-induced excitotoxicity. In vivo, βOHB administration partially rescues striatal dopaminergic system in aralar-KO mice, increasing the levels of neuronal markers as dopamine, VMAT2 and DARPP-32. βOHB administration also increases the synthesis of myelin proteins, but not aspartate and NAA levels. However βOHB supply to aralar-KO neurons resulted in increased synthesis of aspartate and NAA, suggesting their increased use in the aralar-KO brain and reinforcing neuronal NAA as an essential precursor for myelin lipid synthesis in oligodendrocytes. In spite of the limitations imposed by the strong phenotype of aralar- KO mice, this study demonstrates that βOHB is an effective alternative substrate that bypasses Aralar rescuing the main hallmarks of Aralar/AGC1 deficiency. A deeper study of the neuroprotective roles of βOHB, also acting through signaling pathways, would be very interesting with the aim of finding an effective therapy that substitutes KD in human patients

Em grande parte das infecções parasitárias, existe uma relação dinâmica entre o agente etiológico e o sistema imunológico do hospedeiro, cujo equilíbrio, se alterado, pode influenciar o prognóstico da doença. Na infecção malárica, o Plasmodium spp. desencadeia a produção de altos níveis de citocinas inflamatórias que são essenciais para o controle do parasito, mas também são responsáveis pelos sintomas da doença. Nosso grupo demonstrou que os monócitos desempenham um papel importante na proteção contra o P. vivax, constituindo uma importante fonte de citocinas inflamatórias e de espécies reativas de oxigênio. A fim de modular a inflamação, citocinas anti-inflamatórias também são produzidas. Estudos sugerem que as diferentes apresentações clínicas dos pacientes com malária causada pelo P. vivax estão fortemente associadas a um desequilíbrio no balanço entre citocinas inflamatórias e anti-inflamatórias. Apesas dos avanços no entendimento acerca dos mecanismos protetores e imunopatológicos, pouco é sabido em relação a manutenção desse equilíbrio durante a malária. O presente estudo demonstrou que durante a infecção pelo P. vivax, há uma reprogramação no metabolismo dos monócitos e a mitocôndria desempenha um papel central nesse fenômeno. Essa transição consiste em um aumento da captação de glicose, bem como na produção de ATP via glicólise. Essas alterações estão associadas a aumento no potencial de membrana mitocondrial, favorecendo a produção de mROS. Esse mecanismo pode estar envolvido no controle do parasito, visto que a mitocôndria é capaz de migrar para o fagolisossomo contendo o Pv-RET e produzir mROS. Por outro lado, os monócitos também atuam na modulação dessa resposta inflamatória. Essas células expressam níveis mais altos de ectonucleotidases em relação aos demais leucócitos, indicando um importante papel na modulação do ATP extracelular e consequentemente, na produção de adenosina. Apesar dos níveis plasmáticos de adenosina terem diminuído em pacientes com malária aguda causada pelo P. vivax, os mesmos pacientes exibem uma expressão aumentada de receptores purinérgicos P1 nas subpopulações de monócitos. Ademais, a adenosina aumenta a produção de IL-10 e diminui a de TNF pelos monócitos, sendo esse efeito completamente abolido com o bloqueio do receptor A2A. Em conjunto, nossos dados demonstram que os monócitos têm um papel duplo durante a malária causada pelo P. vivax, tentando simultaneamente controlar a infecção e a potencial resposta inflamatória deletéria. In most parasitic infections, there is a relationship between the etiologic agent and the host's immune system, whose balance, if altered, can influence the host’s susceptibility and, therefore, the prognosis of the disease. During malaria, Plasmodium spp. triggers the production of high levels of inflammatory cytokines that are both essential for the control of the parasite and responsible for the symptoms observed during the disease. Our group demonstrated that monocytes play an important role in host defense against Plasmodium vivax infection and represent the main source of inflammatory cytokines and reactive oxygen species. In order to modulate inflammation, anti-inflammatory cytokines, such as IL-10, are also produced. Studies suggested that different clinical presentations of malaria are strongly associated with an imbalance in inflammatory and anti-inflammatory cytokines production. Based on that, despite advances in our understanding of the involvement of protective and immunopathological immunity, little is known about maintaining this balance during malaria. The present study demonstrates that during P. vivax infection monocyte metabolism is altered with mitochondria playing a major function in this switch. This transition involves a reprograming in monocyte metabolism in which the cells increase glucose uptake and produce ATP via glycolysis instead of via oxidative phosphorylation (OXPHOS). Morevover, the above changes are associated with altered mitochondrial membrane potential leading to an increase in mROS instead of ATP production, which can be involved in parasite control since these organells are able to migrate to the phagolysosome containing the Pv-RET and produced mROS. On the other hand, monocytes also participate in the modulation of this inflammatory response during malaria. They express higher levels of ectonucleotidases than other leukocytes, indicating their important role in extracellular ATP modulation and consequently in adenosine production. Despite plasmatic levels of adenosine were decreased in patients experiencing acute malaria caused by P. vivax, the same patients display an increased expression of P1 purinergic receptors on monocyte subsets. In addition, adenosine increases IL-10 and decreases TNF production by monocytes. Importantly, this effect was completely abolished with the blockage of A2a receptor. All together our data show that monocytes have a dual role during malaria caused by P. vivax, simultaneously attempting to control the infection and the potential deleterious inflammatory response.

Objective. Describe the influence of Fetal bovine serum (FBS) on the survival, growth and expression of cellular organelles in rat dental epithelial cells. Material and methods. Cell cultures of rat dental epithelial cells were carried out at 37°C in a humid atmosphere, in the absence and at a concentration of 10% FBS. Morphological evaluation was performed during the proliferation and confluence of cell in culture. Double immunofluorescence labels were made using anti-Actin, anti-TOMM20A, and anti-LAMP1 antibodies. Results. Circular or ovoid dental epithelial cells with bulky nuclei were evidenced during proliferation and confluences in a similar manner in culturing cells in the presence and absence of FBS. The lack of FBS negatively impacts the proliferation of epithelial cells. No alterations were observed in the localization of the anti-actin, anti-TOMM20 and anti-LAMP1 immunomarkers in both conditions of experimental cultures. Conclusions. FBS suppression in rat dental epithelial cells decreased survival, proliferation and suggests not having an impact on the organelles evaluated., Objetivo. Describir la influencia del Suero Fetal Bovino (SFB) en la supervivencia, crecimiento y expresión de organelas celulares en las células epiteliales dentales de rata. Materiales y métodos. Cultivos de células epiteliales dentales de rata fueron llevados a cabo a 37°C en una atmosfera húmeda, en ausencia y a una concentración de 10% de SFB. Una evaluación morfológica fue realizada durante la proliferación y confluencia de las células en cultivo. Dobles marcajes por inmunofluorencia fueron efectuados haciendo uso de anticuerpos anti-actina, anti-TOMM20 y anti-LAMP1. Resultados. Se evidenciaron células epiteliales dentales circulares u ovoides con núcleos voluminosos durante la proliferación y confluencias de manera similar en las células cultivas en presencia y ausencia de SFB. La carencia de SFB impactó negativamente la proliferación de las células epiteliales. No fueron observadas alteraciones en la localización de los inmunomarcajes anti-actina, anti-TOMM20 y anti-LAMP1 en las dos condiciones de cultivos experimentales. Conclusiones. La supresión del SFB en el cultivo de células epiteliales dentales de rata disminuyó la supervivencia, proliferación y sugiere no tener un impacto sobre las organelas evaluadas.

Background Patients with peripheral artery disease (PAD) undergo frequent episodes of ischemia-reperfusion in lower extremity muscles that may negatively affect mitochondrial health and are associated with impaired mobility. We hypothesized that skeletal muscle from PAD patients will show high mitochondrial DNA heteroplasmy, especially in regions more susceptible to oxidative damage, such as the displacement loop, and that the degree of heteroplasmy will be correlated with the severity of ischemia and mobility impairment. Methods and Results Mitochondrial mutations and deletions and their relative abundance were identified by targeted mitochondrial DNA sequencing in biopsy specimens of gastrocnemius muscle from 33 PAD (ankle brachial index 0.9) subjects aged >= 60 years. The probability of heteroplasmy per DNA base was significantly higher for PAD subjects than non-PAD within each region. In adjusted models, PAD was associated with higher heteroplasmy than non-PAD (P=0.003), but the association was limited to microheteroplasmy, that is heteroplasmy found in 1% to 5% of all mitochondrial genomes (P=0.004). Heteroplasmy in the displacement loop and coding regions were significantly higher for PAD than non-PAD subjects after adjustment for age, sex, race, and diabetes mellitus (P=0.037 and 0.004, respectively). Low mitochondrial damage, defined by both low mitochondrial DNA copy number and low microheteroplasmy, was associated with better walking performance. Conclusions People with PAD have higher low frequency heteroplasmy in gastrocnemius muscle compared with people without PAD. Among people with PAD, those who had evidence of least mitochondrial damage, had better walking performance than those with more mitochondrial damage. Registration URL: http://www.clinicaltrials.gov. Unique identifier: NCT02246660., This work was supported by the National Institute on Aging (R21-AG047510), by the National Heart, Lung, and Blood Institute (R01-HL107510, R01HL109244, R01-HL088589, R01HL122846), the Office of Dietary Supplements, and the Nathan Shock Center for Aging.

In Trypanosoma cruzi, the etiological agent of Chagas disease, the amino acid proline participates in processes related to T. cruzi survival and infection, such as ATP production, cell differentiation, host-cell invasion, and in protection against osmotic, nutritional, and thermal stresses and oxidative imbalance. However, little is known about proline biosynthesis in this parasite. Δ1-Pyrroline-5-carboxylate reductase (P5CR, EC 1.5.1.2) catalyzes the biosynthesis of proline from Δ1-pyrroline-5-carboxylate (P5C) with concomitant NADPH oxidation. Herein, we show that unlike other eukaryotes, T. cruzi biosynthesizes proline from P5C, which is produced exclusively from glutamate. We found that TcP5CR is an NADPH-dependent cytosolic enzyme with a Kmapp for P5C of 27.7 μM and with a higher expression in the insect-resident form of the parasite. High concentrations of the co-substrate NADPH partially inhibited TcP5CR activity, prompting us to analyze multiple kinetic inhibition models. The model that best explained the obtained data included a non-competitive substrate inhibition mechanism (Kiapp=45±0.7μM). Therefore, TcP5CR is a candidate as a regulatory factor of this pathway. Finally, we show that P5C can exit trypanosomatid mitochondria in conditions that do not compromise organelle integrity. These observations, together with previously reported results, lead us to propose that in T. cruzi TcP5CR participates in a redox shuttle between the mitochondria and the cytoplasm. In this model, cytoplasmic redox equivalents from NADPH pools are transferred to the mitochondria using proline as a reduced metabolite, and shuttling to fuel electrons to the respiratory chain through proline oxidation by its cognate dehydrogenase.

[eng] OPA1 is a dynamin-related protein that is responsible for the fusion of the internal mitochondrial membrane and essential to control the morphology of the mitochondrial cristae. Thus, it directly impacts the efficiency of OxPhos and the stability of mitochondrial DNA. In this study, we have explored the effects of hepatic deletion of OPA1 on mitochondrial function and metabolism. We have shown that the ablation of OPA1 in the liver produces a mitochondrial dysfunction characterized by alterations in mitochondrial cristae structure, concomitant with a reduced respiratory capacity and mtDNA copy number, and perturbed mitochondrial proteostasis. The mitochondrial dysfunction caused by the ablation of OPA1 in liver triggers the activation of a mitochondrial stress response, including mitochondrial unfolded stress response (UPRmt) that is probably mediated by the transcription factor ATF5. Interestingly, we have observed that OPA1 deficiency in the liver causes better glucose tolerance and protects against diet-induced obesity and insulin resistance concomitant to a high circulating levels of the metabolic modulator FGF21. Here we suggest that the systemic protective effects associated to OPA1 ablation are due to the action of FGF21, probably mediated by the mitochondrial stress response associated to OPA1 loss-of-function via ATF5 activation., [spa] OPA1 es una proteína relacionada con la dinamina que es responsable de la fusión de la membrana mitocondrial interna y esencial para controlar la morfología de las crestas mitocondriales, afectando directamente la eficiencia de OxPhos y la estabilidad del DNA mitocondrial. En este estudio, hemos explorado los efectos de la eliminación hepática de OPA1 sobre la función mitocondrial y el metabolismo. Hemos demostrado que la ablación de OPA1 en el hígado produce una disfunción mitocondrial caracterizada por alteraciones en la estructura de las crestas mitocondriales, concomitante con una capacidad respiratoria reducida y menor número de copias de DNA miocondrial, y perturbación en la proteostasis mitocondrial. La disfunción mitocondrial causada por la ablación de OPA1 en el hígado desencadena la activación de una respuesta al estrés mitocondrial, incluyendo la respuesta a las proteínas mal plegadas de la mitocondria, que probablemente es mediada por el factor de transcripción ATF5. Curiosamente, hemos observado que la deficiencia de OPA1 en el hígado causa una mejor tolerancia a la glucosa y protege contra la obesidad y resistencia a la insulina inducida por la dieta, en paralelo con un aumento de los niveles de FGF21 circulante, un factor involucrado en la modulación metabólica. Con este estudio proponemos que los efectos sistémicos protectores asociados a la ablación de OPA1 se deben a la acción de FGF21 que probablemente es mediada por la respuesta al estrés mitocondrial asociada a la pérdida de función de OPA1 a través de la activación de ATF5.

Glutaminase (GA) catalyzes the first step in mitochondrial glutaminolysis playing a key role in cancer metabolic reprogramming. Humans express two types of GA isoforms: GLS and GLS2. GLS isozymes have been consistently related to cell proliferation, but the role of GLS2 in cancer remains poorly understood. GLS2 is repressed in many tumor cells and a better understanding of its function in tumorigenesis may further the development of new therapeutic approaches. We analyzed GLS2 expression in HCC, GBM and neuroblastoma cells, as well as in monkey COS-7 cells. We studied GLS2 expression after induction of differentiation with phorbol ester (PMA) and transduction with the full-length cDNA of GLS2. In parallel, we investigated cell cycle progression and levels of p53, p21 and c-Myc proteins. Using the baculovirus system, human GLS2 protein was overexpressed, purified and analyzed for posttranslational modifications employing a proteomics LC-MS/MS platform. We have demonstrated a dual targeting of GLS2 in human cancer cells. Immunocytochemistry and subcellular fractionation gave consistent results demonstrating nuclear and mitochondrial locations, with the latter being predominant. Nuclear targeting was confirmed in cancer cells overexpressing c-Myc- and GFP-tagged GLS2 proteins. We assessed the subnuclear location finding a widespread distribution of GLS2 in the nucleoplasm without clear overlapping with specific nuclear substructures. GLS2 expression and nuclear accrual notably increased by treatment of SH-SY5Y cells with PMA and it correlated with cell cycle arrest at G2/M, upregulation of tumor suppressor p53 and p21 protein. A similar response was obtained by overexpression of GLS2 in T98G glioma cells, including downregulation of oncogene c-Myc. Furthermore, human GLS2 was identified as being hypusinated by MS analysis, a posttranslational modification which may be relevant for its nuclear targeting and/or function. Our studies provide evidence for a tumor suppressor role of GLS2 in certain types of cancer. The data imply that GLS2 can be regarded as a highly mobile and multilocalizing protein translocated to both mitochondria and nuclei. Upregulation of GLS2 in cancer cells induced an antiproliferative response with cell cycle arrest at the G2/M phase.