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Heart and blood vessel disorders, such as coronary heart disease, brain vessel disease, rheumatic heart disease, and others, are together referred to as cardiovascular disease (CVD). In this study, we sought to determine how mitochondrial Leucine Transfer RNA genes and CVDs are related (MT-L1 and MT-L2). From CVD patients in Peshawar, a total of 27 saliva samples were taken. Leu-tRNA genes expressed by mitochondria were amplified using polymerase chain reaction after DNA was removed. Ten samples were sent for sequencing after PCR and gene cleaning. We obtained all of the sequenced results, which were subsequently aligned and evaluated against the mitochondrial revised Cambridge Reference Sequence (rCRS). However, in our sequenced samples, Leu-tRNA MT-L1 and MT-L2 genes were determined to be unaltered. Thus, it is suggested that a large population be taken into account while screening for mutations in the mitochondrial encoded Leu-tRNA MT-L1 and MT-L2 genes of cardiac patients in areas of Pakistan. Additionally, it is recommended that patients with cardiac problems should also have other mitochondrial encoded genes checked for potential mutations. This could result in the identification of genetic markers that could be used for early CVD screening in Pakistan. Resumo Distúrbios do coração e dos vasos sanguíneos, como doença cardíaca coronária, doença dos vasos cerebrais, doença cardíaca reumática entre outros, são referidos juntos como doença cardiovascular (DCV). Neste estudo, procuramos determinar como os genes mitocondriais do RNA de transferência de leucina e as DCVs estão relacionados (MT-L1 e MT-L2). Foi coletado um total de 27 amostras de saliva de pacientes com DCV em Peshawar. Genes de Leu-tRNA expressos por mitocôndrias foram amplificados usando reação em cadeia da polimerase (PCR) após a remoção do DNA. Dez amostras foram enviadas para sequenciamento após PCR e limpeza gênica. Obtivemos todos os resultados sequenciados, que foram posteriormente alinhados e avaliados em comparação com a Sequência de Referência de Cambridge revisada (rCRS). No entanto, em nossas amostras sequenciadas, os genes Leu-tRNA MT-L1 e MT-L2 foram determinados como inalterados. Assim, sugere-se que uma grande população seja levada em consideração durante a triagem de mutações nos genes Leu-tRNA MT-L1 e MT-L2 mitocondriais codificados de pacientes cardíacos em áreas do Paquistão. Além disso, recomenda-se que outros genes mitocondriais codificados de pacientes com problemas cardíacos também sejam verificados quanto a possíveis mutações. Isso pode resultar na identificação de marcadores genéticos que podem ser usados para triagem precoce de DCV no Paquistão.

Background: At present, the etiology and pathogenesis of Moyamoya disease (MMD) are not completely clear. Patients are usually diagnosed after cerebrovascular events. Therefore, it is of great clinical significance to explore the predictive factors of MMD. Objective: This study aimed to investigate the serum level of CoQ10B, the amount of endothelial progenitor cells (EPCs), and mitochondrial function of EPCs in MMD patients. Methods: Forty-one MMD patients and 20 healthy controls were recruited in this study. Patients with MMD were divided into two groups: Ischemic type (n=23) and hemorrhagic type (n=18). Blood samples were collected from the antecubital vein and analyzed by CoQ10B ELISA and flow cytometry. Measures of mitochondrial function of EPCs include oxygen consumption rate (OCR), mitochondrial membrane potential, Ca2+ concentration, adenosine triphosphatases activity and ROS level. Results: The serum CoQ10B level in MMD patients was significantly lower than that in healthy controls (p

Microglial activation involves both fragmentation of the mitochondrial network and changes in cellular Ca2+ homeostasis, but possible modifications in mitochondrial calcium uptake have never been described in this context. Here we report that activated microglial BV-2 cells have impaired mitochondrial calcium uptake, including lower calcium retention capacity and calcium uptake rates. These changes were not dependent on altered expression of the mitochondrial calcium uniporter. Respiratory capacity and the inner membrane potential, key determinants of mitochondrial calcium uptake, are both decreased in activated microglial BV-2 cells. Modified mitochondrial calcium uptake correlates with impaired cellular calcium signaling, including reduced ER calcium stores, and decreased replenishment by store operated calcium entry (SOCE). Induction of mitochondrial fragmentation through Mfn2 knockdown in control cells mimicked this effect, while inhibiting LPS-induced mitochondrial fragmentation by a dominant negative form of Drp1 prevented it. Overall, our results show that mitochondrial fragmentation induced by LPS promotes altered Ca2+ homeostasis in microglial cells, a new aspect of microglial activation that could be a key feature in the inflammatory role of these cells.

Tesis (Doctora en Ciencias Químicas) - - Universidad Nacional de Córdoba. Facultad de Ciencias Químicas, 2022 Resumen: Las mitocondrias son organelas esenciales para la supervivencia de las células eucariotas, ya que ejercen múltiples funciones bioquímicas, incluida la producción de ATP a través de la fosforilación oxidativa (OXPHOS), la síntesis de aminoácidos, grupo hemo, lípidos y la biogénesis de los grupos de hierro y azufre. Experimentan cambios dinámicos en tamaño, forma, número y distribución dependiendo de estímulos fisiológicos y patológicos. Estos cambios adaptativos ocurren a través de ciclos coordinados de fisión y fusión de mitocondrias. Las proteínas mitofusina 1 (Mfn1) y 2 (Mfn2) localizadas en la membrana mitocondrial externa (MME) y la proteína Optic atrophy 1 (OPA1) que se encuentra en la membrana mitocondrial interna (MMI) forman parte de la maquinaria de fusión. Por otro lado, la proteína citosólica Dynamin-related 1 (Drp1) media los eventos de fisión mitocondrial. La composición lipídica de las membranas mitocondriales es fundamental para preservar la actividad y estabilidad de los complejos proteicos implicados en la función, distribución y biogénesis de esta organela. Las mitocondrias sintetizan varios lípidos como fosfatidilglicerol, cardiolipina, fosfatidiletanolamina y ácido fosfatídico; mientras que otros como fosfatidilcolina (PC), fosfatidilserina, fosfatidilinositol, esteroles y esfingolípidos, deben importarse de otras organelas, principalmente desde el retículo endoplásmico (RE). StarD7, implicada en el transporte de PC a la mitocondria, se sintetiza como una proteína precursora (StarD7.I) que contiene una señal de localización mitocondrial la cual se escinde en la mitocondria originando la isoforma madura StarD7.II, que se libera al citoplasma o permanece en el espacio intermembrana mitocondrial (EIM). En estudios previos se demostró que el knockdown de StarD7 induce alteraciones en las mitocondrias y el RE con una reducción en el contenido de PC; sin embargo, se desconoce si StarD7 modula la dinámica y función mitocondrial. En este trabajo de tesis se analizó la participación de StarD7 en la morfología, dinámica y función mitocondrial utilizando dos modelos celulares: la línea HTR-8/SVneo derivada de trofoblasto humano de primer trimestre y la línea de mioblastos C2C12 derivada de tejido muscular de ratón. Se generaron células HTR-8/SVneo y C2C12 que sobre-expresan establemente las isoformas StarD7.I (D7.I) y StarD7.II (D7.II). Además ambas líneas celulares se silenciaron en forma transiente (siD7) y estable (shD7). Se demostró en la línea HTR-8/SVneo que la sobre-expresión de D7.I altera la morfología mitocondrial aumentando su fragmentación, mientras que no se observaron cambios en las células que sobre-expresan D7.II en comparación con las células estables control (Ct). Las células D7.I estables transportan mayor cantidad de PC a las mitocondrias que las células Ct, producen fusiones mitocondriales, mantienen el potencial de membrana y generan niveles más bajos de especies reactivas de oxígeno (ERO). Además, expresan mayor nivel de las proteínas Drp1 y Mfn2, con menor cantidad de Mfn1. Por otro lado, las células D7.I transfectadas con plásmidos que codifican a las mutantes Drp1-K38A, Drp1-S637D o Drp1-S637A indican que el fenotipo mitocondrial observado es dependiente de la actividad GTPasa de Drp1 y del grado de fosforilación de la serina 637. En contraste, el silenciamiento de StarD7 en la línea celular HTR-8/SVneo disminuye la expresión de las proteínas de fusión Mfn1 y Mfn2 sin modificar la cantidad de Drp1. Además, se observó que estas células aumentan los niveles de ERO y presentan mitocondrias fragmentadas en forma de “donut”, indicativas de estrés metabólico (Rojas et al., 2021) En la línea C2C12, se demostró que cambios en los niveles de StarD7 no modifican la morfología mitocondrial, evidenciada mediante tinción con MitoTracker Deep Red. Tampoco alteran la expresión de las proteínas de fisión/fusión Mfn1, Mfn2, Drp1, ni la expresión de PCG1α implicada en la biogénesis mitocondrial. Sin embargo, las observaciones realizadas por microscopía electrónica muestran, en las células shD7, una significativa desorganización en sus crestas mitocondriales con menor expresión de L-OPA1, proteína involucrada en su remodelación y mantenimiento. Finalmente, se demostró que el silenciamiento de StarD7 en las células C2C12, disminuye el potencial de membrana, impide la diferenciación celular a miotubos, mientras que incrementa el ECAR (extracellular acidification rate), la captación de glucosa y la acumulación de lípidos neutros. En conjunto, estos hallazgos proporcionan evidencia novedosa que indica que las alteraciones en la expresión de StarD7 producen cambios significativos en la morfología, dinámica y función mitocondrial dependiente del contexto celular. 2024-03-31 Fil: Rojas, María Laura. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas; Argentina.

La principal característica de los tumores malignos de crecimiento rápido es el metabolismo de tipo Warburg, que está directamente relacionado con una cantidad extremadamente alta de hexocinasa (HK) unida a los canales de aniones dependientes de voltaje (VDAC) en la membrana externa mitocondrial. Anteriormente, explicamos el metabolismo de tipo Warburg como resultado de la supresión eléctrica de las mitocondrias debido al cierre de VDAC libres que no están unidos a HK. Aquí, proponemos un posible nuevo mecanismo de actividad anticancerígena de vitamina C (ascorbato) en dosis altas estimado con un modelo computacional simplificado. De acuerdo con la hipótesis propuesta y el modelo, la oxidación del ascorbato en las mitocondrias conduce a la generación del potencial de membrana externa (PMO) negativo de signo opuesto al PMO positivo generado por los complejos VDAC-HK en las células cancerosas. la reapertura de los VDAC eléctricamente cerrados, reprogramando así el metabolismo energético de las células. Según la hipótesis, los mediadores redox, que aumentan la tasa de oxidación del ascorbato en las mitocondrias, deberían aumentar sinérgicamente los efectos anticancerígenos del ascorbato en dosis altas de acuerdo con los datos experimentales registrados en el literatura. El modelo muestra que incluso pequeños cambios en la sensibilidad al voltaje VDAC y/o la cantidad de complejos VDAC-HK, que se sabe que son causados ​​por varios factores fisiológicos, podrían influir fuertemente en el mecanismo mitocondrial propuesto de la actividad anticancerígena del ascorbato. The main feature of fast-growing malignant tumors is the Warburg-type metabolism, which is directly related to an extremely high quantity of hexokinase (HK) bound to the voltage-dependent anion channels (VDACs) in the mitochondrial outer membrane. Previously, we explained the Warburg type metabolism as a result of the electrical suppression of mitochondria due to the closure of free VDACs that are not bound to HK. Here, we propose a possible new mechanism of high-dose vitamin C (ascorbate) anticancer activity estimated with a simplified computational model. According to the proposed hypothesis and the model, ascorbate oxidation in mitochondria leads to the generation of the negative outer membrane potential (OMP) of opposite sign to the positive OMP generated by the VDAC-HK complexes in cancer cells. The model demonstrates that negative OMP generated by any mechanism, even of relatively low magnitudes, leads to the reopening of the electrically closed VDACs, thus reprogramming cell energy metabolism. According to the hypothesis, redox mediators, which increase the rate of ascorbate oxidation in mitochondria, should synergistically increase anticancer effects of high-dose ascorbate in accordance with experimental data recorded in the literature. The model shows that even small changes in the VDAC-voltage sensitivity and/or quantity of the VDAC-HK complexes, known to be caused by various physiological factors, might strongly influence the proposed mitochondrial mechanism of ascorbate anticancer activity

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de Lectura: 11-04-2022, Metabolic pathologies and their complications are one of the main causes of death in western high-income countries. Mitochondria are key controllers of metabolism and the skeletal muscle (Skm) structure. However, whether Skm mitochondrial dysfunction is a cause or consequence of metabolic disorders is still controversial. In order to unveil the role of mitochondria in the onset and progression of these pathologies, in this thesis we have developed and characterized the first mouse model of conditional and Skm-specific inhibition of the mitochondrial H+-ATP synthase by the overexpression of its constitutively active inhibitor, ATPIF1|H49K. 6-month-old mice presenting the inhibition of the mitochondrial oxidative phosphorylation (OXPHOS) show a higher body weight compared to control littermates, due to deep alterations in glucose, lipid and amino acid metabolism in both muscle and white adipose tissue. Upon mitochondrial inhibition, Skm increases lactate production, reduces fatty acid β-oxidation, and relies on branched-chain amino acids catabolism as major source of acetyl-CoA for the de novo lipid synthesis. Moreover, acetyl-CoA accumulates in muscle and feedbacks the mitochondrial dysfunction by increasing the production of reactive oxygen species. As a result, mice with restrained OXPHOS become more prone to develop insulin resistance when fed a high-fat diet. Furthermore, Skm mitochondrial dysfunction alters motor behaviour, an effect that increases with ageing. 18-month-old mice presenting a chronic inhibition of the H+-ATP synthase show deep alterations in Skm organization, developing a myopathy characterized by the presence of tubular aggregates (TA), honeycomb-like arrays of sarcoplasmic-reticulum (SR) tubules that disorganize the sarcomere structure. The long-term OXPHOS inhibition reprograms soleus oxidative fibers to glycolytic ones, and increases mitochondrial fission and mitophagy, presumably to recycle defective mitochondria. TA appeared as the result of SR tubulation aimed at increasing mitochondria/SR contact sites to buffer calcium overloads due to prolonged OXPHOS dysfunction. Hence, enhancing OXPHOS activity appears as a promising strategy for therapeutic intervention in metabolic and muscle disorders. With this aim, we screened 702 drugs and identified edaravone as a new mitochondrial antioxidant and enhancer. Remarkably, in vivo edaravone treatment restores a healthy phenotype both in 6-month and 18-month old mice. Altogether, these results suggest that Skm mitochondrial alterations contribute to the setting of metabolic and muscular disorders, proposing edaravone as a potential treatment for these pathologies, This work has been supported by a predoctoral fellowship from the Spanish Ministerio de Economía y Competitividad (BES-2017-079909). The project was funded by the Spanish Ministerio de Economía y Competitividad (Grants SAF2016-76028- R and PID2019- 104241RB-I00)

Fil: Pandi Perumal, Seithikurippu R. Somnogen Canada Inc; Canadá Fil: Cardinali, Daniel Pedro. Pontificia Universidad Católica Argentina. Facultad de Ciencias Médicas; Argentina Fil: Reiter, R. UT Health San Antonio. Department of Cell Systems and Anatomy; Estados Unidos Fil: Brown, Gregory M. Univeridad de Toronto. Centro para la Adicción y la Salud Mental. Ciencias Moleculares del Cerebro; Canadá Abstract: That the pineal gland is a source of melatonin is widely known; however, by comparison, few know of the much larger pool of extrapineal melatonin. That pool is widely distributed in all animals, including those that do not have a pineal gland, e.g., insects. Extrapineal melatonin is not released into the blood but is used locally to function as an antioxidant, anti-inflammatory agent, etc. A major site of action of peripherally-produced melatonin is the mitochondria where it neutralizes reactive oxygen species (ROS) that are generated during oxidative phosphorylation. Its role also includes major actions as an immune modulator reducing overreactions to foreign agents while simultaneously boosting immune processes. During a pandemic such as coronavirus disease 2019 (COVID-19), caused by the virus SARS-CoV-2, melatonin is capable of suppressing the damage inflicted by the cytokine storm. The implications of melatonin in susceptibility and treatment of COVID-19 disease are discussed.

Resumen Introducción. Los fibroblastos gingivales (FGs) son células del tejido conjuntivo gingival que han tomado en los últimos años una relevancia promisoria por su probable utilización en la terapia celular, dadas sus capacidades de multipotencialidad y de autorrenovación. Objetivo. Conocer y describir el impacto de la ausencia en la suplementación de Suero Fetal Bovino (SFB) en la supervivencia de fibroblastos gingivales en cultivos. Materiales y métodos. Fibroblastos gingivales fueron aislados de tejido gingival de pacientes sanos y cultivados en medios de cultivos DMEM (Dulbecco's Modified of Eagle Medium) en ausencia y suplementados con 0.2% de SFB a 37°C en una atmósfera húmeda con 5% de CO2. Se llevó a cabo una evaluación morfológica, de supervivencia y proliferación de los FGs, así como la identificación mediante la técnica de inmunofluorescencia de marcadores del citoesqueleto celular como la actina y mitocondrias. Resultados. Los FGs cultivados en ausencia y con suplementación de 0.2% de SFB evidenciaron una forma fusiforme, con núcleos ovalados y numerosas prolongaciones citoplasmáticas durante el tiempo de cultivo. Un leve aumento en la proliferación de FGs fue observado en aquellas células en contacto con el medio DMEM+0.2% de SFB comparadas con el medio donde estuvo ausente la suplementación. El inmunomarcaje de la actina y las mitocondrias dejó en evidencia que la ausencia y suplementación a 0.2% de SFB no afectó su localización en los FGs evaluados. Conclusión. Los fibroblastos gingivales sobreviven y proliferan en ausencia de SFB, conservando sus características morfológicas celulares. Abstract Introduction. Gingival fibroblasts (GF) are cells of gingival connective tissue that have taken promising relevance in recent years due to their probable use in cell therapy, given their multipotencial and self-renewal capabilities. Objective. To know and to describe the impact of the absence of Fetal Bovine Serum (FBS supplementation on the survival of gingival fibroblasts in cultures. Materials and methods. Gingival fibroblasts were isolated from gingival tissue of healthy patients and cultured in DMEM (Dulbecco's Modified of Eagle Medium) culture media in absence and supplemented with 0.2% FBS at 37 ° C in a humid atmosphere with 5% CO2. A morphological evaluation, survival and proliferation of GF were carried out, as well as the identification by the immunofluorescence technique of cellular cytoskeleton markers such as actin and mitochondria. Results. The GF grown in the absence and with supplementation of 0.2% FBS showed a fusiform shape, with oval nuclei and numerous cytoplasmic extensions during the culture time. A slight increase in the proliferation of GF was observed in those cells in contact with the DMEM medium +0.2% FBS compared to the medium where the supplementation was absent. Immunostaining of actin and mitochondria showed that the absence and supplementation to 0.2% of FBS did not affect its location in the evaluated. Conclusion. Gingival fibroblasts survive and proliferate in the absence of FBS, preserving their cellular morphological characteristics.

O câncer é uma das causas mais comuns de morte mundialmente. Os tratamentos disponíveis são associados a numerosos efeitos adversos, aliado à pequena porcentagem de pacientes que atingem a completa remissão, instigando a necessidade de novas estratégias terapêuticas. Tendo isto em vista, a utilização de ímãs estáticos no sistema nervoso central surge como uma técnica de neuromodulação não invasiva que oferece novas possibilidades de tratamento, sendo necessária, porém, uma compreensão completa dos mecanismos de ação subjacentes à resposta celular. O principal objetivo desta tese foi investigar os efeitos da Estimulação Magnética Estática (EME) sobre a viabilidade, morte, ciclo e proliferação celular, e autofagia e quantidade de mitocôndrias. Inicialmente foram testados diferentes tempos de estimulação (6, 12, 24, 36h, 72 horas e 6 dias) com intensidade de 0,3T na linhagem de neuroblastoma SH-SY5Y em cultura. Para verificar a resposta celular a EME, os parâmetros de viabilidade celular (MTT), morte e ciclo celular (coloração de anexina-V/PI e análise de iodeto de propídio, respectivamente), proliferação celular (CFSE), autofagia (laranja de acridina) e quantidade total mitocondrial (MitoTracker™ Verde) foram analisados. Verificou-se que a exposição a EME 0,3T por 24h e 6 dias, reduziu a viabilidade celular das células SH-SY5Y (P0,05). Adicionalmente, houve redução da autofagia e proliferação celular após 6 dias de exposição e diminuição do número de mitocôndrias em ambos os tempos (P0.05). Additionally, there was a reduction in autophagy and cell proliferation after 6 days of exposure and a decrease in the number of mitochondria at both times (P

La obesidad es una enfermedad multifactorial e incapacitante que se caracteriza por una sobrecarga metabólica del tejido adiposo (AT), la inflamación crónica de bajo grado, el estrés oxidativo y la disfunción endotelial. En la actualidad, la prevalencia mundial de la obesidad está aumentando, donde el estilo de vida influye de manera significativa en la resolución de esta enfermedad. En este contexto, ha aumentado la demanda de estrategias para revertir las comorbilidades y frenar su aumento. El procedimiento de bypass gástrico en Y de Roux (RYGB) es una herramienta eficaz para la pérdida de peso y la remisión de las comorbilidades metabólicas de la obesidad, aunque se desconocen los mecanismos moleculares que implican estas mejoras. Por lo tanto, el objetivo principal de la presente tesis doctoral fue determinar cómo la cirugía RYGB modula la inflamación crónica de bajo grado, el estrés oxidativo y la disfunción endotelial, explorando su efecto sobre el complejo del inflamasoma, la autofagia, la dinámica mitocondrial y el estrés del retículo endoplásmico (ER). También se abordó el papel de la metformina, un enfoque terapéutico alternativo para la obesidad y la diabetes de tipo 2 (T2D). Por lo tanto, otro objetivo de este estudio fue analizar los mecanismos moleculares relacionados con la inflamación y la autofagia en la AT y el efecto protector de la metformina contra las alteraciones metabólicas. Nuestra hipótesis fue que la pérdida de peso inducida por la RYGB modula la respuesta de los leucocitos, componentes primordiales del sistema inmunitario. Para comprobar esta hipótesis, analizamos las distintas vías metabólicas que se ven alteradas por la obesidad y exploramos si el AT responde de forma diferencial a la metformina. Observamos que la pérdida de peso producida tras 1 año de RYGB se acompañaba de una mejora del perfil lipídico proaterogénico y de la sensibilidad a la insulina. Esto se acompañó de una disminución de las citocinas proinflamatorias y de la producción de especies reactivas de oxígeno de los leucocitos y de una mayor respuesta antioxidante, lo que dio lugar a una menor interacción entre los leucocitos y el endotelio y a marcadores de disfunción endotelial. Además, un análisis más detallado de los mecanismos moleculares en los leucocitos tras la pérdida de peso reveló una mejora de la respuesta homeostática como resultado de un aumento de la AMPK, de los marcadores de autofagia, de la biogénesis y de la dinámica mitocondrial, junto con una disminución de las citoquinas proinflamatorias, de los marcadores de inflamación y del estrés del ER. En conjunto, estos resultados ayudan a arrojar luz sobre los mecanismos que subyacen al efecto protector de la pérdida de peso en el control metabólico y la homeostasis celular en la obesidad. Además, mediante un estudio transversal, observamos que los pacientes con obesidad y T2D tratados con metformina presentan una mejora del perfil inflamatorio y oxidativo con respecto a los sujetos con obesidad metabólicamente sana (MHO). Esta mejora parece estar modulada por cambios en la activación del complejo del inflamasoma y la autofagia en el AT visceral, lo que sugiere -a pesar de la creencia común- que los sujetos MHO no están tan protegidos cardiometabólicamente como se espera. Por lo tanto, se sugiere que estudios futuros presenten como objetivo determinar las dianas directas de la metformina responsables de mediar en estas respuestas. Obesity is a multifactorial and disabling disease characterized by metabolic overload in adipose tissue (AT), chronic low-grade inflammation, oxidative stress and endothelial dysfunction. Currently, the worldwide prevalence of obesity is increasing, with lifestyle having a significant influence on the resolution of this disease. In this context, there has been an increase in the demand for strategies to reverse comorbidities and to stop its increase. The Roux-en-Y gastric bypass (RYGB) procedure is an effective tool for weight loss and remission of the metabolic comorbidities of obesity, although the molecular mechanisms involving these improvements are unknown. Therefore, the main objective of the present PhD thesis was to determine how RYGB surgery modulates chronic low-grade inflammation, oxidative stress and endothelial dysfunction, by exploring its effect on the inflammasome complex, autophagy, mitochondrial dynamics and endoplasmic reticulum (ER) stress. The role of metformin, an alternative therapeutic approach to obesity and type 2 diabetes (T2D), was also addressed. Therefore, another aim of this study was to analyse the molecular mechanisms related to inflammation and autophagy in AT and the protective effect of metformin against metabolic alterations. Our hypothesis was that RYGB-induced weight loss modulates the response of leukocytes, primordial components of the immune system. To test this hypothesis, we analysed the various metabolic pathways that are altered by obesity and explored whether AT responds differentially to metformin. We observed that the weight loss produced after 1 year of RYGB was accompanied by an improvement in proatherogenic lipid profile and insulin sensitivity. This was accompanied by a decrease in proinflammatory cytokines and leukocyte reactive oxygen species production and an enhanced antioxidant response, resulting in less interaction between leukocytes and both the endothelium and markers of endothelial dysfunction. In addition, further analysis of molecular mechanisms in leukocytes after weight loss revealed an improvement in the homeostatic response as a result of an increase in AMPK, markers of autophagy, biogenesis and mitochondrial dynamics, along with a decrease in proinflammatory cytokines, markers of inflammation and ER stress. Considered together, these results help to shed light on the mechanisms underlying the protective effect of weight loss on metabolic control and cellular homeostasis in obesity. Moreover, through a cross-sectional study, we reported that obese T2D patients treated with metformin exhibit improved inflammatory and oxidative profile with respect to metabolically healthy obesity (MHO) subjects. This improvement seems to be modulated by changes in the activation of the inflammasome complex and autophagy in VAT, suggesting – despite common belief – that MHO subjects are not as cardiometabolically protected as expected. Future mechanistic studies should aim to determine the direct targets of metformin responsible for mediating these responses.

Fil: Balparda, Manuel. University of Bonn. Molecular Plant Physiology; Alemania Fil: Elsasse, Marlene. University of Bonn. Institute for Cellular and Molecular Botany. Molecular Evolution; Alemania Fil: Elsasse, Marlene. University of Munster. Institute of Plant Biology and Biotechnology. Plant Energy Biology; Alemania Fil: Badía, Mariana B. Heinrich Heine University. Institute of Developmental and Molecular Biology of Plants. Plant Molecular Physiology and Biotechnology. Cluster of Excellence on Plant Sciences; Alemania Fil: Badía, Mariana B. Pontificia Universidad Católica Argentina. Facultad de Química e Ingeniería del Rosario; Argentina Fil: Giese, Jonas. University of Munster. Institute of Plant Biology and Biotechnology. Plant Physiology; Alemania Fil: Bovdilova, Anastasiia. Heinrich Heine University. Institute of Developmental and Molecular Biology of Plants. Plant Molecular Physiology and Biotechnology. Cluster of Excellence on Plant Sciences; Alemania Fil: Hüdig, Meike. University of Bonn. Molecular Plant Physiology; Alemania Fil: Hüdig, Meike. Heinrich Heine University. Institute of Developmental and Molecular Biology of Plants. Plant Molecular Physiology and Biotechnology. Cluster of Excellence on Plant Sciences; Alemania Fil: Reinmuth, Lisa. University of Bonn. Institute for Cellular and Molecular Botany. Molecular Evolution; Alemania Fil: Eirich, Jürgen. University of Munster. Institute of Plant Biology and Biotechnology. Plant Physiology; Alemania Fil: Schwarzlander, Markus. University of Munster. Institute of Plant Biology and Biotechnology. Plant Energy Biology; Alemania Fil: Finkemeier, Iris. University of Munster. Institute of Plant Biology and Biotechnology. Plant Physiology; Alemania Fil: Schallenberg Rüdinger, Mareike. University of Bonn. Institute for Cellular and Molecular Botany. Molecular Evolution; Alemania Fil: Maurino, Verónica G. University of Bonn. Molecular Plant Physiology; Alemania Fil: Maurino, Verónica G. Heinrich Heine University. Institute of Developmental and Molecular Biology of Plants. Plant Molecular Physiology and Biotechnology. Cluster of Excellence on Plant Sciences; Alemania Summary: Plants need to rapidly and flexibly adjust their metabolism to changes of their immediate environment. Since this necessity results from the sessile lifestyle of land plants, key mechanisms for orchestrating central metabolic acclimation are likely to have evolved early. Here, we explore the role of lysine acetylation as a post-translational modification to directly modulate metabolic function. We generated a lysine acetylome of the moss Physcomitrium patens and identified 638 lysine acetylation sites, mostly found in mitochondrial and plastidial proteins. A comparison with available angiosperm data pinpointed lysine acetylation as a conserved regulatory strategy in land plants. Focusing on mitochondrial central metabolism, we functionally analyzed acetylation of mitochondrial malate dehydrogenase (mMDH), which acts as a hub of plant metabolic flexibility. In P. patens mMDH1, we detected a single acetylated lysine located next to one of the four acetylation sites detected in Arabidopsis thaliana mMDH1. We assessed the kinetic behavior of recombinant A. thaliana and P. patens mMDH1 with site-specifically incorporated acetyl-lysines. Acetylation of A. thaliana mMDH1 at K169, K170, and K334 decreases its oxaloacetate reduction activity, while acetylation of P. patens mMDH1 at K172 increases this activity. We found modulation of the malate oxidation activity only in A. thaliana mMDH1, where acetylation of K334 strongly activated it. Comparative homology modeling of MDH proteins revealed that evolutionarily conserved lysines serve as hotspots of acetylation. Our combined analyses indicate lysine acetylation as a common strategy to fine-tune the activity of central metabolic enzymes with likely impact on plant acclimation capacity.

El contenido de ácido desoxirribonucleico (ADN) mitocondrial (ADNmt) por célula como marcador de viabilidad embrionaria se propuso como una estrategia muy innovadora para la priorización en la selección de embriones humanos en los tratamientos reproducción asistida, siendo los embriones con menor contenido mitocondrial por célula los candidatos a ser transferidos. La hipótesis de partida se basaba en que dichos embriones presentarían un estado metabólico más apropiado y un menor estrés celular, los cuales se traducirían en una mayor probabilidad de implantación. La elevada expectación inicialmente generada alrededor de este marcador, vino acompañada de publicaciones con resultados contradictorios en cuanto a su relación con distintos parámetros de éxito clínico, lo que ha desembocado en una falta de consenso sobre la relevancia clínica de su uso. Una hipótesis posible, que explique esta falta de consenso, podría ser que el abordaje del uso del contenido de ADNmt se ha realizado sin conocimientos suficientes sobre aspectos clave como son el significado del contenido del ADNmt durante el desarrollo embrionario o la asociación entre el contenido de ADNmt y el contenido y estado de actividad mitocondrial. El presente trabajo de tesis doctoral trata de profundizar, mediante el análisis del contenido y del estado mitocondrial en el blastocisto, sobre los aspectos moleculares en los que se sustenta esta hipótesis. Este estudio se realizará para determinar si existen factores que expliquen la disparidad de resultados difundidos sobre este tema. Para ello, se establecieron tres objetivos específicos con el fin de abordar el análisis mitocondrial mediante distintas aproximaciones: Objetivo específico 1. Caracterizar el contenido y el patrón de distribución del ADN mitocondrial durante el desarrollo embrionario humano preimplantacional. Objetivo específico 2. Describir el contenido de ADN mitocondrial y el estado mitocondrial en embriones humanos bajo condiciones de estrés. Objetivo específico 3. Identificar la relación existente entre el contenido de ADN mitocondrial por célula y la masa y la actividad mitocondrial en el blastocisto humano. Gracias a la consecución de estos objetivos se ha llegado a la conclusión de que el contenido de ADNmt por célula es capaz de verse modulado en cuestión de pocas horas en situaciones de estrés respecto al cultivo in-vitro normal, y no es representativo del contenido de mitocondrias y de la actividad mitocondrial en un momento que sería cercano a la transferencia embrionaria. No obstante, se ha demostrado una importante relación entre los valores altos de contenido de ADNmt y el estrés celular. Estos hallazgos pueden explicar, en parte, la falta de consenso que existe en cuanto al uso del contenido de ADNmt por célula como marcador de competencia embrionaria, y pueden ser útiles para establecer la base molecular necesaria que respalde, en un futuro, su uso como un indicador adicional para la priorización de la transferencia embrionaria.

The Carnitine Palmitoyltranferase I (CPT1) catalyzes the rate-limiting step of long-chain fatty acid (LCFA) mitochondrial β-oxidation. The enzyme promotes the conjugation of LCFA with l-carnitine, which allows LCFA to enter the mitochondria matrix. The structural features involved in CPT1 and LCFA-CoA interactions have not been fully elucidated, mainly due to the absence of CPT1 crystallographic data. Previous studies reported important residues (Lys556, Lys560, and Lys561) crucial to the CPT1 mechanism. Nonetheless, these studies have not explored the LCFA bindings. Using molecular modeling strategies, we aimed to understand the conformational changes in CPT1 structure induced by LCFA-CoA. For this purpose, a tridimensional CPT1A model was built by homology modeling using CRAT protein (PBD:1t7q, resolution 1.8 Å) as a template. We simulated the CPT1 structure in the presence and absence of LCFA-CoA by molecular dynamics (MD). By applying a principal component analysis (PCA), two states of apostructure CPT1 based on CoA-Loop (688-711) were observed. In contrast, just one state was evidenced along with smaller conformational subspaces in ligand-complexed simulations using LCFA-CoA. The CoA moiety of ligands interacts with charged residues, namely Lys560, Lys556, Arg563, and Arg645. The frequency of interactions observed for each of these residues is60% of simulation time, suggesting a dynamic profile of interactions in synergy with long-chain carbon interactions over α-I (478-492). Collectively, these features may be associated with the catalytic conformation of LCFA-CoA to CPT1a. Further calculations of free-energy for different fatty acids, such as alpha-linolenic (ALA), gamma-linolenic (GLA), and arachidonic (ARA) acids, yielded energy values ranging from -76.9 ± 15.9 to -68.5 ± 10.0 kcal mol

Mitochondrial complex I is a central metabolic enzyme that uses the reducing potential of NADH to reduce ubiquinone-10 (Q10) and drive four protons across the inner mitochondrial membrane, powering oxidative phosphorylation. Although many complex I structures are now available, the mechanisms of Q10 reduction and energy transduction remain controversial. Here, we reconstitute mammalian complex I into phospholipid nanodiscs with exogenous Q10. Using cryo-EM, we reveal a Q10 molecule occupying the full length of the Q-binding site in the ‘active’ (ready-to-go) resting state together with a matching substrate-free structure, and apply molecular dynamics simulations to propose how the charge states of key residues influence the Q10 binding pose. By comparing ligand-bound and ligand-free forms of the ‘deactive’ resting state (that require reactivating to catalyse), we begin to define how substrate binding restructures the deactive Q-binding site, providing insights into its physiological and mechanistic relevance.

Astaxantina (AST), uma xantofila, tem sido vista como uma molécula citoprotetora em diferentes modelos experimentais. AST é uma potente indutora da enzima hemeoxigenase-1 (HO-1), a qual está envolvida na degradação do heme, gerando monóxido de carbono (CO), íons de ferro livres e biliverdina (BV). Biliverdina redutase (BVR) converte BV em bilirrubina (BR). CO, BV e BR podem induzir efeitos antioxidantes e anti-inflamatórios em determinados tipos celulares. Metilglioxal (MG), um dicarbonil reativo, é um subproduto da glicólise e da degradação de certos aminoácidos, dentre outras moléculas. Ainda, MG é um contaminante na água, no solo e no ar. MG induz a produção de espécies reativas direta e indiretamente. Além disso, MG causa disfunção mitocondrial. As mitocôndrias são as principais fontes de adenosina trifosfato (ATP) em células animais. Por outro lado, as mitocôndrias são um importante sítio de produção de espécies reativas. Assim, dano mitocondrial pode resultar em colapso bioenergético e desequilíbrio redox. De fato, MG induz estresse oxidativo, falência bioenergética e inflamação em células humanas. No presente trabalho, investigamos se um pré-tratamento com AST atenuaria os efeitos causados por MG em células de neuroblastoma humano SH-SY5Y. AST a 5 – 40 μM foi administrada às células por 24 h antes da exposição ao MG por 24 h adicionais. Analisamos ambos os parâmetros celulares e mitocondriais nesse modelo experimental. Encontramos que AST a 20 μM preveniu o desequilíbrio redox e o colapso bioenergético nas células expostas ao MG. AST estimulou a atividade de HO-1 e a inibição desta enzima por protoporfirina-IX de zinco (ZnPP-IX) suprimiu os efeitos promovidos por AST nas mitocôndrias das células expostas ao MG. Além disso, AST preveniu o aumento induzido por MG nos níveis de interleucina-1β (IL-1β) e de fator de necrose tumoral-α (TNF-α), marcadores de inflamação, nas células expostas ao ZnPP-IX. Um co-tratamento com molécula-2 liberadora de monóxido de carbono (CORM-2) ou BR reduziu o impacto de MG na viabilidade das células SH-SY5Y. Assim, AST preveniu o dano celular, o colapso mitocondrial e a inflamação por um mecanismo associado com HO-1 em células expostas ao MG. Pesquisas adicionais são necessárias para analisar o mecanismo exato pelo qual AST promoveu citoproteção contra MG. Astaxanthin (AST), a xanthophyll, has been viewed as a cytoprotective molecule in different experimental models. AST is a potent inducer of the enzyme heme oxygenase-1 (HO-1), which is involved in the degradation of heme, generating carbon monoxide (CO), free iron ions, and biliverdin (BV). Biliverdin reductase (BVR) converts BV into bilirubin (BR). CO, BV, and BR can induce antioxidant and anti-inflammatory effects in certain cell types. Methylglyoxal (MG), a reactive dicarbonyl, is a by-product of glycolysis of the degradation of certain amino acids, among other molecules. Otherwise, MG is a contaminant in water, soil, and air. MG induce the production of reactive species by direct and indirect routes. Moreover, MG causes mitochondrial dysfunction. Mitochondria are a major source of adenosine triphosphate (ATP) in animal cells. On the other hand, mitochondria are an important site of reactive species production. Thus, mitochondrial damage may result in bioenergetics collapse and redox impairment. In fact, MG induces oxidative stress, bioenergetics failure, and inflammation in human cells. In the present work, we investigated whether a pre-treatment with AST would attenuate the effects caused by MG in the human neuroblastoma SH-SY5Y cells. AST at 5 – 40 μM was administrated to the cells for 24 h prior to an exposure to MG for additional 24 h. We analyzed both cellular and mitochondrial parameters in this experimental model. We found that AST at 20 μM prevented the redox impairment and bioenergetic collapse in the cells exposed to MG. AST stimulated HO-1 activity and inhibition of this enzyme by zinc protoporphyrin-IX (ZnPP-IX) suppressed the effects promoted by AST on the mitochondria of the cells exposed to MG. Furthermore, AST prevent the MG-induced increase in the levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), markers of inflammation, in the cells exposed to ZnPP-IX. A co-treatment with carbon monoxide releasing molecule-2 (CORM-2) or BR reduced the impact of MG on the viability of the SH-SY5Y cells. Thus, AST prevented the cellular impairment, mitochondrial collapse, and inflammation by a mechanism associated with HO-1 in the cells exposed to MG. Further research is necessary to analyze the exact mechanism by which AST promoted cytoprotection against MG.

A acidemia glutárica tipo I (AG I) é uma acidemia orgânica de herança autossômica recessiva caracterizada pela deficiência da enzima glutaril-CoA desidrogenase (GCDH), que participa do catabolismo dos aminoácidos lisina, hidroxilisina e triptofano. Os pacientes acometidos pela AG I apresentam sintomas iniciais inespecíficos até o aparecimento das crises encefalopáticas. Durante essas crises, que ocorrem após situações de catabolismo elevado, uma característica degeneração do estriado é observada nos pacientes, seguida de sintomas como distonia e discinesia, hipotonia, convulsões, rigidez muscular e espasticidade. Atualmente, o tratamento para a AG I possui pouca eficácia, sendo realizada restrição dietética de proteínas e suplementação de L-carnitina. Portanto, no presente estudo, foram avaliados a homeostase redox, o metabolismo energético e o controle de qualidade mitocondrial em cérebro de camundongos selvagens (Gcdh+/+) e nocautes para a enzima GCDH (Gcdh-/-) submetidos à dieta rica em lisina. Dessa forma, camundongos Gcdh+/+ e Gcdh-/- de 30 dias de idade receberam uma dieta de lisina (4,7%) por 72 h. Alguns animais ainda receberam três injeções intraperitoneais do antioxidante direcionado à mitocôndria JP4-039 (5 g/g), uma para cada dia da dieta. Quando a dieta completou 72 horas, os animais foram eutanasiados e tiveram seu estriado e córtex cerebral homogeneizados para a avaliação dos parâmetros. Os resultados mostraram um aumento nos níveis de malondialdeído (MDA) e na oxidação de 2’,7’-diclorofluoresceína (DCFH) no estriado e córtex de camundongos Gcdh-/-, comparados aos animais Gcdh+/+. Também foi verificado um aumento na atividade da superóxido dismutase (SOD), catalase (CAT) e glutationa transferase (GST) nas duas estruturas avaliadas dos camundongos nocautes. Ainda, foi observada uma diminuição na atividade da glutationa peroxidase (GPx) no estriado de camundongos Gcdh-/-. Os achados também demonstraram uma diminuição no metabolismo da glicose no córtex cerebral através da técnica MicroPET. A fosforilação da p38 e o conteúdo das proteínas canal de ânions dependente de voltagem (VDAC) e proteína cinase ativada por adenosina monofosfato (AMPK) também estavam alterados nas duas estruturas cerebrais. Já o tratamento com JP4-039 preveniu as alterações nos níveis de MDA, na oxidação de DCFH, na atividade das enzimas antioxidantes e no metabolismo de glicose. Estes achados mostram que estresse oxidativo, hipometabolismo da glicose e alterações na biogênese mitocondrial contribuem para a fisiopatologia da AG I, e que o JP4-039 é um candidato promissor para o tratamento dessa doença. Glutaric aciduria type I (GA I) is a neurometabolic disorder caused by the deficient activity of the mitochondrial enzyme glutaryl-CoA dehydrogenase (GCDH), which participates in the catabolism of the amino acids lysine, hydroxylysine and tryptophan. Patients affected by AG I have nonspecific symptoms until the onset of encephalopathic crises. These crises occur due to situations of high catabolism, such as fever, infections or prolonged fasting. After these crises, a characteristic bilateral striatal degeneration occurs in patients, followed by dystonia and dyskinesia, seizures, muscle stiffness and spasticity. Currently, the treatment for GA I is ineffective and consists only of dietary protein restriction and L-carnitine supplementation. In the present study, we evaluated redox homeostasis, energy metabolism and mitochondrial quality control in brain of wild type (Gcdh+/+) and knockout (Gcdh-/-) mice fed a high lysine diet. Thirty-day-old Gcdh+/+ and Gcdh-/- mice received a high lysine chow (4.7%) for 72 h. Some animals also received three intraperitoneal injections of JP4-039 (5 g/g) for each day of the diet. When the diet completed 72 h, mice were euthanized, and their striatum and cerebral cortex were homogenized for the evaluation of the parameters. Our results showed an augment in malondialdehyde (MDA) levels and 2’,7’-dichlorofluorescein (DCFH) oxidation in striatum and cerebral cortex of Gcdh-/- mice, compared to Gcdh+/+ animals. We also observed an increase in the activity of superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST) in both structures of the knockout mice. Moreover, the activity of GPx was decreased in striatum of Gcdh-/- mice. Furthermore, a decrease in glucose metabolism was seen in cerebral cortex of Gcdh-/- mice, assessed by MicroPET scan. Alterations in the phosphorylation of p38 and the content of voltage-dependent anion-selective channel (VDAC) and adenosine monophosphate-activated protein kinase (AMPK) were further verified in brain of Gcdh-/- animals. The treatment with JP4-039 prevented the alterations on MDA levels, DCFH oxidation, antioxidant enzyme activities and glucose metabolism. Our data show that oxidative stress, glucose metabolism and mitochondrial biogenesis alterations contribute to the pathophysiology of GA I, and that JP4-039 is a promising candidate for the treatment of this disorder.

Introdução: Sendo uma doença caracterizada pelo aumento da resistência vascular pulmonar e disfunção cardíaca direita, a hipertensão arterial pulmonar (HAP) também é marcada pela perturbação da homeostase redox e mitocondrial. Objetivo: Este trabalho teve como objetivo a investigação destas alterações no ventrículo direito (VD), no cérebro e nas vesículas extracelulares (EVs) circulantes de ratos com HAP induzida por monocrotalina (MCT). Além disso, em camundongos com HAP induzida por MCT, foram investigados os possíveis efeitos protetores de dois tratamentos: EVs derivadas de células-tronco mesenquimais (MSCs) e superexpressão da superóxido dismutase mitocondrial (MnSOD). Métodos: Na primeira etapa deste trabalho, ratos machos foram divididos em grupo Controle e MCT, e a doença foi induzida pela injeção única intraperitoneal de MCT (60 mg/kg). As avaliações hemodinâmicas e morfométricas e a coleta dos órgãos foi realizada após 21 dias. Após isto, as EVs foram isoladas a partir do sangue destes animais e foram preparadas, juntamente com o VD e o cérebro, para as análises dos parâmetros redox e mitocondriais. Na segunda etapa, camundongos de ambos os sexos foram divididos em grupo Controle, MCT, Controle + EVs e MCT + EVs, a doença foi induzida pela injeção semanal subcutânea de MCT (60 mg/kg) e o tratamento foi feito por injeção intravenosa semanal de MSC-EVs (de medula óssea humana) (3 x 106 partículas/ml) 24 após cada injeção de MCT/veículo, durante 4 semanas. Por fim, na terceira etapa, camundongos de ambos os sexos transgênicos foram divididos em grupo MnSODVE-NE (expressão normal da MnSOD) e MnSODVE-OE (superexpressão da MnSOD), e a doença foi induzida pela injeção semanal subcutânea de MCT (60 mg/kg) durante 4 semanas. Ao final deste período, em ambas as etapas, foi realizada a ecocardiografia e os órgãos foram coletados para posteriores análises morfométricas, histológicas e bioquímicas. Resultados e discussão: Na primeira etapa, foi observado um estado redox oxidativo no VD dos animais doentes, bem como o prejuízo na função de moléculas relacionadas à homeostase mitocondrial. Este mesmo perfil foi observado nas EVs circulantes destes animais. Por outro lado, no cérebro, as análises dos parâmetros redox indicaram um ambiente celular pouco oxidativo. Além disso, houve uma preservação da homeostase mitocondrial e do metabolismo energético celular neste tecido. Na segunda e terceira etapas, ambos os tratamentos foram capazes de impedir o desenvolvimento da HAP, uma vez que os animais do grupo MCT + EVs (na segunda etapa) e do grupo MnSODVE-OE (na terceira etapa) não apresentaram aumento significativo da resistência vascular pulmonar e da hipertrofia do VD, preservando o fluxo pela artéria pulmonar e a função cardíaca direita. Em ambas as etapas, este efeito benéfico foi relacionado com as diferenças na expressão de proteínas envolvidas com o metabolismo energético das células, como a GSK-3β, a FOXO1 e a CPT1A, avaliadas no tecido pulmonar, que parecem indicar a preservação do metabolismo energético normal celular pelo tratamento com as MSC-EVs e a superexpressão da MnSOD, ao invés da mudança metabólica característica da HAP. Conclusão: A função cardiopulmonar está severamente afetada pela HAP, com impacto sobre o metabolismo redox e mitocondrial celular no VD. Este estímulo parece alterar o conteúdo das EVs circulantes e estas, por sua vez, atuam como comunicadoras interórgãos para sinalizar o aumento de proteínas envolvidas na manutenção da homeostase redox e mitocondrial. Com base nisto, ambos os tratamentos com MSC-EVs e com superexpressão da MnSOD foram eficazes em proteger a função cardiopulmonar e impedir o desenvolvimento da HAP, através da preservação do metabolismo energético em favor da fosforilação oxidativa. Introduction: Being a disease characterized by increased pulmonary vascular resistance and right heart dysfunction, pulmonary arterial hypertension (PAH) is also marked by disturbance of redox and mitochondrial homeostasis. Objective: This study aimed to investigate these changes in the right ventricle (RV), brain and circulating extracellular vesicles (EVs) of rats with monocrotaline-induced PAH (MCT). Furthermore, in mice with MCT-induced PAH, the possible protective effects of two treatments were investigated: EVs derived from mesenchymal stem cells (MSCs) and overexpression of mitochondrial superoxide dismutase (MnSOD). Methods: In the first stage of this work, male rats were divided between Control and MCT groups, and the disease was induced by a single intraperitoneal injection of MCT (60 mg/kg). Hemodynamic and morphometric evaluations and organ collection were performed after 21 days. After that, the EVs were isolated from the blood of these animals and were prepared, together with the RV and the brain, for the analysis of redox and mitochondrial parameters. In the second stage, mice of both sexes were divided into Control, MCT, Control + EVs and MCT + EVs, the disease was induced by the weekly subcutaneous injection of MCT (60 mg/kg), and the treatment was performed by weekly intravenous injection of MSC-EVs (from human bone marrow) (3 x 106 particles/ml), 24h after each MCT/vehicle injection, for 4 weeks. Finally, in the third stage, transgenic mice of both sexes were divided into groups MnSODVE-NE (normal expression of MnSOD) and MnSODVE-OE (overexpression of MnSOD), and the disease was induced by the weekly subcutaneous injection of MCT (60 mg/kg) for 4 weeks. At the end of this period, in both stages, echocardiography was performed, and the organs were collected for further morphometric, histological, and biochemical analyses. Results and discussion: In the first stage, an oxidative redox state was observed in the RV of the sick animals, as well as the impairment in the function of molecules related to mitochondrial homeostasis. This same profile was observed in the circulating EVs of these animals. On the other hand, in the brain, analyzes of redox parameters indicated a low oxidative cellular environment. In addition, there was a preservation of mitochondrial homeostasis and cellular energy metabolism in this tissue. In the second and third stages, both treatments were able to prevent the development of PAH, since the animals in the MCT + EVs group (second stage) and the MnSODVE-OE group (third stage) prevented the increase in pulmonary vascular resistance and hypertrophy of the RV, preserving the flow through the pulmonary artery and the right heart function. In both stages, this beneficial effect was related to differences in the expression of proteins involved in the energy metabolism of cells such as GSK-3β, FOXO1 and CPT1A, evaluated in lung tissue, which seem to indicate the preservation of normal cellular energy metabolism performed by treatment with MSC-EVs and MnSOD overexpression, rather than of the characteristic metabolic change in PAH. Conclusion: Cardiopulmonary function is severely affected by PAH, with an impact on cellular redox and mitochondrial metabolism in the RV. This stimulus seems to alter the content of circulating EVs and these, in turn, act as interorgan communicators to signal the increase of proteins involved in the maintenance of redox and mitochondrial homeostasis. Based on this, both treatments with MSC-EVs and MnSOD overexpression were effective in protecting cardiopulmonary function and preventing the development of PAH, by preserving energy metabolism in favor of oxidative phosphorylation.

A quantitative model to expose the adrenal gland sub-cellular alterations produced by crotamine-like (C-L) from rattlesnake venom during 3, 6 and 24 hours (h), and also qualitative changes on mice neuromuscular structures in vivo were observed and calculated by transmission electron microscopy. A pure crotamine-like (C-L) isoform was obtained using a cationic exchange chromatography column from the rattlesnake Crotalus durissus cumanensis venom. The C-L SDS-PAGE (15.5 %) under non-reduced conditions exhibited a molecular mass of ~3 kDa single band. The C-L in vivo qualitative experiments induced ultrastructural changes in mouse neuromuscular structures at 3, 6 and 24 h, such as reduction in the number of acetylcholine vesicles, disorganisation of the secondary synaptic clefts, enlargement of the sub-sarcolemma space and alteration of the mitochondria morphology, number and cristae. Regarding neurotoxic actions in vivo, the animals injected with C-L presented spastic paralysis of the hind limbs. The quantitative alterations studied on the capillaries, the nucleus, the mitochondria the lipid inclusions, and the smooth endoplasmic reticulum were observed from 3 to 24 h after C-L injection. As far as it is known from the literature review, there are no quantitative records of similar sub-cellular alterations caused by crotamine. Se observó y calculó mediante microscopía electrónica de transmisión, un estudio cuantitativo para exponer las alteraciones subcelulares de la glándula suprarrenal producidas por crotamina-similar (C-L) del veneno de serpiente de cascabel, durante 3; 6 y 24 horas (h), y también los cambios cualitativos in vivo en las estructuras neuromusculares de ratones. Se obtuvo una isoforma pura similar a la crotamina (C-L) usando una columna de cromatografía de intercambio catiónico del veneno de Crotalus durissus cumanensis. La SDS-PAGE (15,5 %) de la C-L en condiciones no reducidas exhibió una masa molecular de ∼3 kDa de banda única. Los experimentos cualitativos in vivo de C-L indujeron cambios ultraestructurales, bajo microscopía electrónica en constituyentes neuromusculares de ratón a las 3; 6 y 24 h, tales como reducción en el número de vesículas de acetilcolina, desorganización de las hendiduras sinápticas secundarias, agrandamiento del espacio sub-sarcolema y alteración de las mitocondrias. En cuanto a las acciones neurotóxicas in vivo, los animales inyectados con C-L presentaron parálisis espástica de las extremidades posteriores. Las alteraciones cuantitativas estudiadas en los capilares, el núcleo, las mitocondrias, las inclusiones lipídicas y el retículo endoplásmico liso se observaron de 3 a 24 h después de la inyección de C-L. Hasta donde se conoce por la revisión de la literatura, no existen registros cuantitativos de alteraciones sub-celulares similares provocadas por la crotamina. rodriguezacosta1946@yahoo.es

Mitochondria have pivotal roles in cellular physiology including energy metabolism, reactive oxygen species production, Ca2+ homeostasis, and apoptosis. Altered mitochondrial morphology and function is a common feature of cancer cells and the regulation of mitochondrial homeostasis has been identified as a key to the response to chemotherapeutic agents in human leukemias. Here, we explore the mechanistic aspects of cytotoxicity produced by thioridazine (TR), an antipsychotic drug that has been investigated for its anticancer potential in human leukemia cellular models. TR exerts selective cytotoxicity against human leukemia cells in vitro. A PCR array provided a general view of the expression of genes involved in cell death pathways. TR immediately produced a pulse of cytosolic Ca2+, followed by mitochondrial uptake, resulting in mitochondrial permeabilization, caspase 9/3 activation, endoplasmic reticulum stress, and apoptosis. Ca2+ chelators, thiol reducer dithiothreitol, or CHOP knockdown prevented TR-induced cell death. TR also exhibited potent cytotoxicity against BCL-2/BCL-xL-overexpressing leukemia cells. Additionally, previous studies have shown that TR exhibits potent antitumor activity in vivo in different solid tumor models. These findings show that TR induces a Ca2+-mediated apoptosis with involvement of mitochondrial permeabilization and ER stress in leukemia and it emphasizes the pharmacological potential of TR as an adjuvant in antitumor chemotherapy.

[eng] SLIMP (Seryl-tRNA Synthetase-Like Insect Mitochondrial Protein) was identified during the generation of a mitochondrial human disease fly model. SLIMP was described as a previously uncharacterized paralog of the Drosophila mitochondrial seryl-tRNA synthetase (SerRS2), which became an essential protein, universally distributed in insects, echinoderms, and molluscs. Notably, SLIMP constitutes an aaRS-like protein that can bind to tRNAs, but it has lost the aminoacylation activity (Guitart, Bernardo, Sagalés, et al., 2010). Interestingly, we recently reported that SLIMP plays an essential role in the mitochondria by simultaneously regulating the mitochondrial protein synthesis and mtDNA copy number through interacting with SerRS2 and LON, respectively (Picchioni, Antolin-Fontes, et al., 2019). On the other hand, two reported studies pointed to SLIMP as an essential cell cycle regulator (Ambrus et al., 2009; Liang et al., 2014). Previous experiments in our group showed a G2 accumulation phenotype in SLIMP-KD cells, which can be rescued by overexpressing SLIMP without the mitochondria-targeting signal. Additionally, transcriptional upregulation of a core set of E2F1-target genes was detected upon SLIMP depletion. All these data point to a cell cycle-related role of SLIMP, potentially carried out from outside the organelle. Interestingly, here we first experimentally characterized the SLIMP MSP and proved that it is essential for driving the protein into the organelle. Moreover, we discovered an extra-mitochondrial population of SLIMP potentially shuttling between the cytosol and the nucleus, presumably in charge of the cell cycle role of SLIMP. Furthermore, our results suggest a role of SLIMP repressing the transition from the G1 to S phase during the cell cycle progression in an E2F1-independent pathway. Finally, interestingly, we demonstrate that the CDK1-P levels are increased upon SLIMP depletion, suggesting an activation of the G2/M checkpoint. In balance, the data presented in this thesis reinforces the idea of SLIMP playing a non-canonical function outside the mitochondria regulating the cell cycle progression near the R point. Thus, SLIMP would represent a single molecule at the crossroad between the mitochondrial homeostasis and cell cycle regulation., [cat] SLIMP (Seryl-tRNA Synthetase-Like Insect Mitochondrial Protein) es va identificar com un paraleg prèviament desconegut de la seril-tRNA sintetasa mitocondrial de Drosòfila (SerRS2), durant la generació d’un model de malaltia mitocondrial humana. Segurament SLIMP és fruit d’una duplicació gènica a la base dels animals i s’ha convertit en una proteïna essencial i universalment distribuïda en artròpodes, mol·luscs i equinoderms. A més, SLIMP ha perdut l’activitat aminoaciladora, tot i que encara conserva la capacitat d’unir-se a ARNs de transferència (Guitart, Bernardo, Sagalés, et al., 2010). El nostre laboratori ha demostrat recentment que SLIMP desenvolupa una funció clau a la mitocòndria, regulant simultàniament la traducció gènica a l’orgànul i el número de copies de ADN mitocondrial, a traves de la interacció amb SerRS2 i LON respectivament (Picchioni, Antolin-Fontes, et al., 2019). D’altre banda, dos estudis publicats en els últims anys vinculen SLIMP amb la regulació del cicle cel·lular (Ambrus et al., 2009; Liang et al., 2014). En aquest sentit, experiments prèviament realitzats al grup demostren que quan els nivells d’SLIMP baixen, les cèl·lules s’acumulen en la fase G2 del cicle. Curiosament, aquest fenotip es pot revertir sobre-expressant un forma truncada d’SLIIMP que no pot entrar a la mitocòndria. A més, en aquestes condicions, també s’observa una pujada a nivell transcripcional d’alguns gens regulats per E2F1. Conjuntament aquestes dades apunten a una possible funció d’SLIMP relacionada amb la regulació del cicle cel·lular potencialment portada a terme des de fora de la mitocòndria. En aquesta tesi, nosaltres, primer determinem la seqüència del pèptid senyal mitocondrial d’SLIMP experimentalment, i demostrem que es essencial i suficient per dirigir la proteïna cap a l’orgànul. A més, detectem una petita població d’SLIMP fora de la mitocòndria, present tant en el nucli com en el citoplasma. És important remarcar, que varis experiments presentats en aquesta tesi suggereixen que SLIMP estaria reprimint la progressió del cicle cel·lular a nivell de la transició G1-S, mitjançant un mecanisme alternatiu a E2F1. Finalment, demostrem que els nivells de CDK1-P son mes elevats en les cèl·lules que els hi manca SLIMP, cosa que indica una activació del punt de control de G2/M i que podria explicar la acumulació en G2 observada en aquestes cèl·lules. En conjunt les dades presentades en aquesta tesi, reforcen la idea que SLIMP desenvolupa una funció no canònica des de fora de la mitocòndria, reprimint l’entrada en la fase S del cicle cel·lular. Per tant, SLIMP representaria una molècula que vincula directament la homeòstasi mitocondrial i la regulació del cicle cel·lular.

Protein kinase Cε (PKCε) is highly expressed in nociceptor neurons and its activation has been reported as pro-nociceptive. Intriguingly, we previously demonstrated that activation of the mitochondrial PKCε substrate aldehyde dehydrogenase-2 (ALDH2) results in anti-nociceptive effects. ALDH2 is a major enzyme responsible for the clearance of 4-hydroxy-2-nonenal (4-HNE), an oxidative stress byproduct accumulated in inflammatory conditions and sufficient to induce pain hypersensitivity in rodents. Here we determined the contribution of the PKCε-ALDH2 axis during 4-HNE-induced mechanical hypersensitivity. Using knockout mice, we demonstrated that PKCε is essential for the nociception recovery during 4-HNE-induced hypersensitivity. We also found that ALDH2 deficient knockin mice display increased 4-HNE-induced nociceptive behavior. As proof of concept, the use of a selective peptide activator of PKCε (ΨεHSP90), which favors PKCε translocation to mitochondria and activation of PKCε-ALDH2 axis, was sufficient to block 4-HNE-induced hypersensitivity in WT, but not in ALDH2-deficient mice. Similarly, ΨεHSP90 administration prevented mechanical hypersensitivity induced by endogenous production of 4-HNE after carrageenan injection. These findings provide evidence that selective activation of mitochondrial PKCε-ALDH2 axis is important to mitigate aldehyde-mediated pain in rodents, suggesting that ΨεHSP90 and small molecules that mimic it may be a potential treatment for patients with pain.

[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.