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[ES] La regulación de calcio intracelular en cardiomiocitos tiene un rol esencial en la fisiología del corazón, debido a su relación directa con la capacidad contráctil de las fibras cardíacas y con su participación en mecanismos de arritmogénesis. Las mitocondrias presentes en los cardiomiocitos tienen una función crucial tanto en la producción de ATP como en la regulación de calcio en el interior de estas células. Sin embargo, actualmente solo unos pocos modelos computacionales de potencial de acción incluyen las mitocondrias. El presente trabajo de fin de grado se centra en el desarrollo de un modelo de potencial de acción que incluya a estos orgánulos, así como de una herramienta informática que permita la simulación computacional en Matlab®. El modelo implementado se adaptó del modelo de mitocondria de linfocitos B de Kim y otros (2012) a miocitos ventriculares de conejo, utilizando los datos experimentales obtenidos en la investigación de Lu y otros (2013). El modelado de las mitocondrias se introdujo en el modelo de potencial de acción de miocito ventricular de conejo de Shannon y otros (2004). Mediante el modelo computacional implementado se realizaron simulaciones en Matlab® en las que se compararon los resultados obtenidos mediante ambos modelos (el modelo inicial que no incluye las mitocondrias y el nuevo modelo que sí incluye las mitocondrias), así como simulaciones en las que se alteraron determinadas corrientes iónicas presentes en la célula con el objetivo de simular alteraciones debidas a intervenciones farmacológicas y/o mutaciones en la célula. Por último, se realizó un análisis de sensibilidad multifactorial de los resultados obtenidos en las diversas simulaciones para determinados biomarcadores de calcio con el objetivo de estudiar la influencia de las mitocondrias en la regulación intracelular de calcio., [EN] Intracellular calcium regulation in cardiomyocytes has an essential role in the heart physiology, due to its direct relationship with the contractile capacity of the cardiac fibers and its participation in arrithmogenesis mechanism. Cardiomyocyte mitochondria have a crucial function in ATP production and intracellular calcium regulation. However, actually only a few computational models include mitochondria. This thesis is focused on the development of an action potential model that includes these organelles, as well as a computational tool to simulate in Matlab®. The implemented model was adapted from the mitochondria model by Kim y otros (2012) to ventricular rabbit cardiomyocytes, using experimental data by Lu y otros (2013). The mitochondrial formulation was introduced in the action potential model of ventricular rabbit cardiomyocytes by Shannon et al. (2004). We conducted simulations in Matlab® using the implemented computational model. The results obtained using both models (the initial model without mitochondria and the new model with mitochondria) where compared, as well as the results of simulations where some ionic currents where altered to simulate pharmacological interventions and/or effect of mutations. Finally, we did a multifactorial analysis of the results obtained in the simulations for calcium biomarkers in order to study the influence of mitochondria in the intracellular calcium regulation.

Os esteroides anabolizantes androgênicos (EAA) como o decanoato de nandrolona (ND) são hormônios sintéticos derivados da testosterona. Sabe-se que um dos efeitos mais marcantes da administração abusiva destes esteroides é o aumento do comportamento agressivo. Evidências indicam que altas doses de EAA alteram a morfologia e causam hiperativação de sinapses glutamatérgicas, o que se correlaciona com um fenótipo agressivo exacerbado. Fisiologicamente o glutamato é considerado o principal neurotransmissor excitatório no cérebro de mamíferos, entretanto, em níveis elevados, pode causar hiperexcitabilidade neuronal mediada pelos receptores glutamatérgicos ionotrópicos do tipo N-metil-d-aspartato (NMDAr) e, consequentemente alterações no metabolismo mitocondrial. A terminação da sinalização excitatória glutamatérgica é realizada majoritariamente por transportadores existentes em astrócitos. Neste sentido, o transportador astrocitário de glutamato GLT-1 é responsável por mais de 90% da remoção do glutamato da fenda sináptica, contribuindo significativamente, para a manutenção da homeostasis da sinalizacão glutamatérgica. A administração do antibiótico β-lactâmico ceftriaxona (CEF) aumenta a expressão de GLT-1 e diminui a hiperexcitabilidade glutamatérgica, o que poderia potencialmente contrapor mecanismos cerebrais associados ao aumento do fenótipo agressivo induzidos pelo decanoato de nandrolona (ND). Entretanto, estas possíveis interacões moleculares e comportamentais ainda não foram exploradas. Assim, o objetivo primário deste trabalho foi investigar se o aumento da expressão do transportador astrocitário GLT-1 modula mecanismos glutamatérgicos envolvidos na agressividade induzida pelo ND, e a atividade mitocondrial. Para tanto, camundongos CF-1 machos de 60 dias de idade foram divididos em 4 grupos: veículo oleoso (VEH), nandrolona (ND), ceftriaxona (CEF) e nandrolona+ceftriaxona (ND/CEF). A nandrolona foi injetada por via subcutânea (15mg/Kg) por 19 dias. A ceftriaxona (200mg/Kg) ou solução salina foram administradas intraperitonealmente por 5 dias. Após a última injeção foi avaliada a latência para o primeiro ataque e o número de ataques no teste do intruso. Os animais foram sacrificados logo após o teste, e homogeneizados de córtex foram utilizados para imunoquantificação do GLT-1 e da fosforilação da subunidade pNR2Bser1232 do NMDAr. A atividade mitocondrial foi avaliada em sinaptossoma de cérebro total. Os níveis de glutamato foram medidos no líquido cefalorraquidiano. Comparado com o veículo, o tratamento com ND diminuiu significativamente a expressão do GLT-1, aumentou os níveis de glutamato e a expressão da subnidade pNR2Bser1232 o que foi mecanisticamente associado ao aumento do fenótipo agressivo; diminuicão da latência e aumento do número de ataques ao intruso. Ainda, a ND diminuiu o controle respiratório mitocondrial. A administração de CEF aumentou significativamente a expressão do GLT-1 e diminuiu os níveis de glutamato em relação ao grupo ND, enquanto que os níveis de pNR2Bser1232 e a agressividade foram similar ao grupo controle. No grupo ND/CEF o immunoconteúdo de GLT-1 e de pNR2Bser1232, os níveis de glutamato e o fenótipo agressivo, foram significativamente menores que no grupo ND, e similares ao grupo controle. Ainda, a CEF foi capaz de atenuar o prejuízo no controle respiratório mitocondrial causado pela ND. Nossos resultados demonstram que a interação bidirecional entre o transportador astrocitário GLT-1 e a subunidade pNR2Bser1232 neuronal mediada pelo glutamato, exercem um impacto regulatório no fenótipo agressivo induzido pela ND, e no controle respiratório mitocondrial. Desta maneira, este modelo reforça a importância da homeostasia funcional da sinapse tripartide glutamatérgica no fenótipo agressivo. Anabolic androgenic steroids (AAS) such as nandrolone decanoate (ND) are synthetic hormones derived from testosterone. It is known that one of the most important adverse effects of abusive administration of these steroids is the increase in aggressive behavior. Evidence indicates that high doses of AAS alter morphology and cause hyperactivation of glutamatergic synapses which correlates with an aggressive exacerbated phenotype. Physiologically, glutamate is considered the main excitatory neurotransmitter in the mammalian brain. At high glutamate levels, occurs neuronal hyperexcitability mainly trhough the ionotropic N-methyl-d-aspartate (NMDAr) type of glutamatergic receptors and, consequently, changes in mitochondrial metabolism. Existing transporters in astrocytes predominantly perform the termination of glutamatergic excitatory signaling. In this sense, the GLT-1 glutamate astrocytic transporter is responsible for more than 90% of glutamate removal from the synaptic cleft, contributing significantly to the maintenance of glutamatergic signaling homeostasis. Administration of the β-lactam antibiotic ceftriaxone (CEF) increases GLT-1 expression and decreases glutamatergic hyperexcitability, which could potentially counteract brain mechanisms associated to increased aggressive phenotype mediated by nandrolone decanoate (ND). However, a possible molecular and behavioral interaction has not yet been explored in context. Thus, the primary objective of this work was to investigate whether increased expression of the GLT-1 astrocyte transporter modulates the glutamatergic mechanisms involved in ND-induced aggressive phenotype, and mitochondrial activity. Sixty-day-old male CF-1 mice were divided into 4 groups: oil vehicle (VEH), nandrolone (ND), ceftriaxone (CEF) and nandrolone + ceftriaxone (ND / CEF). Nandrolone was injected subcutaneously (15mg / kg) for 19 days. Ceftriaxone (200mg / kg) or saline solution were administered intraperitoneally for 5 days. After the last injection, the latency for the first attack and the number of attacks on the intruder test were evaluated. The animals were sacrificed after the test, and homogeinized cortex were used for immunoquantification of GLT-1 and phosphorylation of the NMDAr pNR2Bser1232 subunit. Mitochondrial activity was evaluated in total brain sinaptossomes. Glutamate levels were measured in the cerebrospinal fluid. Compared to the vehicle group, treatment with ND significantly decreased the expression of GLT-1, increased glutamate levels and expression of the pNR2Bser1232 which was mechanistically associated with an increase in the aggressive phenotype; decrease in the latency and increase in the number of attacks. Also, ND decreased mitochondrial respiratory control. Administration of CEF significantly increased GLT-1 expression and decreased glutamate levels relative to the ND group, whereas pNR2Bser1232 levels and aggressive phenotype were similar to the control group. In the ND / CEF group the expression of GLT-1 and pNR2Bser1232, glutamate levels and aggressive phenotype were significantly lower than in the ND group, and similar to the control group. Furthermore, CEF was able to attenuate the alteration in the mitochondrial respiratory control caused by ND. Our results demonstrated that the levels of glutamate astrocytic transporter GLT-1 and pNR2Bser1232 are important mechanism behind the increased aggressive phenotype induced by ND, and decreased mitochondrial respiratory control. Also, this model reinforces the importance of glutamate levels and astrocytic molecular targets in the regulation of the aggressive phenotype.

[Background]:Leishmaniasis is the world's second deadliest parasitic disease after malaria, and current treatment of the different forms of this disease is far from satisfactory. Alkylphospholipid analogs (APLs) are a family of anticancer drugs that show antileishmanial activity, including the first oral drug (miltefosine) for leishmaniasis and drugs in preclinical/clinical oncology trials, but their precise mechanism of action remains to be elucidated., [Methodology/Principal Findings]:Here we show that the tumor cell apoptosis-inducer edelfosine was the most effective APL, as compared to miltefosine, perifosine and erucylphosphocholine, in killing Leishmania spp. promastigotes and amastigotes as well as tumor cells, as assessed by DNA breakdown determined by flow cytometry. In studies using animal models, we found that orally-administered edelfosine showed a potent in vivo antileishmanial activity and diminished macrophage pro-inflammatory responses. Edelfosine was also able to kill Leishmania axenic amastigotes. Edelfosine was taken up by host macrophages and killed intracellular Leishmania amastigotes in infected macrophages. Edelfosine accumulated in tumor cell mitochondria and Leishmania kinetoplast-mitochondrion, and led to mitochondrial transmembrane potential disruption, and to the successive breakdown of parasite mitochondrial and nuclear DNA. Ectopic expression of Bcl-XL inhibited edelfosine-induced cell death in both Leishmania parasites and tumor cells. We found that the cytotoxic activity of edelfosine against Leishmania parasites and tumor cells was associated with a dramatic recruitment of FOF1-ATP synthase into lipid rafts following edelfosine treatment in both parasites and cancer cells. Raft disruption and specific FOF1-ATP synthase inhibition hindered edelfosine-induced cell death in both Leishmania parasites and tumor cells. Genetic deletion of FOF1-ATP synthase led to edelfosine drug resistance in Saccharomyces cerevisiae yeast., [Conclusions/Significance]:The present study shows that the antileishmanial and anticancer actions of edelfosine share some common signaling processes, with mitochondria and raft-located FOF1-ATP synthase being critical in the killing process, thus identifying novel druggable targets for the treatment of leishmaniasis., This work was supported by grants from the Spanish Ministerio de Economia y Competitividad (SAF2014-59716-R and BIO2014-56930-P), Instituto de Salud Carlos III (RD12/0036/0065 from Red TemaÂtica de Investigación Cooperativa en Cáncer, cofunded by the EU's European Regional Development Fund ± FEDER),European Community's Seventh Framework Programme FP7-2007-2013 (grant HEALTH-F2-2011-256986, PANACREAS), and Spain-UK International Joint Project grant from The Royal Society-CSIC (2004GB0032). CG was supported by the RamoÂn y Cajal Program from the Spanish Ministerio de Ciencia e Innovación.AÂCM was recipient of Formación de Profesorado Universitario predoctoral fellowship from the Spanish Ministerio de Ciencia e Innovación.

[spa] TP53INP2 es una proteína nuclear que activa la autofagia en moscas y células eucariotas. El propósito de esta tesis doctoral fue investigar si TP53INP2 modula la autofagia mitocondrial dependiente de Parkin. Aquí, se demuestra que en un modelo de sobreexpresión de TP53INP2 se reduce la actividad mitofágica en respuesta al agente despolarizante CCCP o a los inhibidores de la cadena respiratoria Oligomicina/Antimicina A, en células que expresan Parkin. Además, este modelo causa modificaciones en el metabolismo mitocondrial. Así por ejemplo, la sobreexpresión de TP53INP2 produjo un incremento de la masa mitocondrial y una mayor susceptibilidad frente al estrés oxidativo mientras que la pérdida de función de TP53INP2 causó una reducción de la masa mitocondrial y del potencial de membrana mitocondrial. Asimismo, se pudo determinar que no hay cambios en la expresión de reguladores de la biogénesis mitocondrial (PGC-1α), esto sin duda refuerza la hipótesis de que el efecto de TP53INP2 sobre el metabolismo mitocondrial es debido a cambios en la mitofagia. Posteriormente, con el propósito de analizar los mecanismos por los cuales TP53INP2 disminuye la mitofagia dependiente de Parkin, se analizó la localización celular de la proteína. Sorprendentemente, se observó que ciertos estresores mitocondriales causan el reclutamiento de TP53INP2 a la mitocondria y este efecto es acelerado por Parkin. Prosiguiendo estos resultados en células que expresan Parkin, se descubrió que TP53INP2 inhibe la acumulación de PINK1 en condiciones en las cuales la membrana mitocondrial se encuentra despolarizada. Al mismo tiempo, se encontró una reducción en la estimulación de la fosforilación de la ubiquitina en el residuo S65. Por otra parte, se determinó que TP53INP2 colocaliza con Parkin en el núcleo en condiciones basales o en condiciones de daño mitocondrial. Esta aparente interacción no requiere PINK1 dado que células deficientes en esta proteína también mostraron una colocalización positiva en ambas situaciones. De igual forma, TP53INP2 interactúa físicamente con Parkin, esta interacción es mantenida con una forma corta de Parkin que carece del dominio UBL, la cual a diferencia de la forma larga no colocaliza con la mitocondria en condiciones de despolarización de la membrana mitocondrial. Sin embargo, la interacción fue abolida por una forma mutante de Parkin que carece de la actividad ubiquitina ligasa. En suma, en esta tesis se demuestra que TP53INP2 participa en el mantenimiento de la función e integridad mitocondrial a través de un mecanismo que implica la inhibición de PINK1 en la mitocondria dañada y por consiguiente la disminución de la mitofagia a través de la vía PINK1/Parkin. Los resultados de esta tesis indican que TP53INP2 representa una especie de interruptor que activa la autofagia general o no selectiva pero previene un tipo de autofagia selectiva, la mitofagia dependiente de PINK1/Parkin, en células de mamífero., [eng] TP53INP2/DOR is a nuclear protein that shuttles to the cytosol and stimulates autophagosomal formation and autophagy activity in mammalian cells and in flies. In this study we explored whether TP53INP2 also modulates mitochondrial autophagy. To this end, we analyzed the effects of TP53INP2 on Parkin-dependent mitophagy. TP53INP2 gain-of-function markedly reduced mitophagic activity in response to different stresses in Parkin-expressing cells. In keeping with these observations, genetic manipulation of TP53INP2 caused modifications of mitochondrial metabolism. Thus, TP53INP2 gain-of-function caused enhanced mitochondrial mass and high susceptibility to oxidative stress whereas TP53INP2 loss-of-function reduced mitochondrial mass and mitochondrial membrane potential. Again, in support of an effect of TP53INP2 mediated by changes in mitophagy, TP53INP2-dependent changes in mitochondrial metabolism were not a consequence of alterations in regulators of mitochondrial biogenesis (PGC-1α). In order to analyze the mechanisms by which TP53INP2 attenuates Parkin-dependent mitophagy, we analyzed the cellular localization of the protein. Surprisingly, mitochondrial stressors caused the recruitment of TP53INP2 to the mitochondria and this was accelerated by Parkin. In keeping with the mitochondrial localization of the protein, TP53INP2 inhibited PINK1 accumulation following mitochondrial depolarization, and in parallel it reduced the stimulation of ubiquitin Ser65 phosphorylation, and protein ubiquitination upon mitochondrial damage in Parkin-expressing cells. TP53INP2 showed a surprising property that is to colocalize with Parkin in the nucleus under basal conditions or in mitochondria upon mitochondrial damage. The colocalization of Parkin and TP53INP2 does not require PINK1 (PINK1-deficient cells also show the colocalization under basal or upon mitochondrial damage). Furthermore, TP53INP2 physically interacts with Parkin. This interaction is maintained under conditions in which mitochondrial clustering does not form by mutant forms of Parkin lacking the UBL domain. Furthermore, the interaction was prevented by Parkin mutant forms lacking ubiquitin ligase activity. In all, we document that TP53INP2 participates in the maintenance of mitochondrial integrity and function through a mechanism that entails the inhibition of PINK1 in damaged mitochondria, and in consequence the attenuation of the PINK1/Parkin pathway of mitophagy. Thus, TP53INP2 represents a switch that activates general macroautophagy but prevents PINK1/Parkin-dependent mitophagy in mammalian cells.

La silimarina es un flavonoglicano extraído de las semillas y el fruto de Silybum marianum (L.) Gaertn (Cardo mariano o Cardo asnal). Es una mezcla de tres compuestos diferentes: silibina, silidianina y silicristina. Ha sido utilizada en el tratamiento de las intoxicaciones y en padecimientos hepáticos por su efecto regenerador celular, inhibidor de leucotrienos y efecto antioxidante, ya que actúa como antioxidante en las células de este órgano, protegiéndolas de los daños causados por radicales libres, e incrementa la capacidad de regeneración mediante la producción de nuevas células, así como la eliminación de toxinas del organismo. El objetivo de este trabajo fue analizar la capacidad antioxidante de diferentes dosis de silimarina sobre el daño oxidativo en membranas de microsomas y mitocondrias obtenidos de cerebro de ratas Wistar. Se sometió a las membranas a un sistema in vitro ascorbato - Fe+2 dependiente, durante 180 min a 37 ºC en presencia de cantidades crecientes de silimarina: 6.25, 12.5, y 25 μg/mg de proteína microsomal o mitocondrial. La peroxidación fue cuantificada en un contador de centelleo líquido Hewlett Packard con programa de quimioluminiscencia (cuentas totales por minuto/mg de proteína)., Silymarin is a flavonoglycan extracted from seeds and fruit of Silybum marianum (L.) Gaertn (Cardo asnal or Cardo mariano). Is a mixture of three different compounds: silybin, silidianin and silicristin. Silymarin has been used in poisoning treatment and liver disease due to its cellular regenerative effect, inhibitor of leukotrienes and antioxidant effect, since it acts as anantioxidant in liver cells, protecting them from the free radicals damage and increases the regeneration capacity through the production of new cells, as well as the elimination of toxins from the organism. The present investigation was carried out to analyze the antioxidant capacity of different doses of Silymarin on the oxidative damage in microsomal and mitochondrial membranes from brain of Wistar rats. Membranes were subjected to an in vitro ascorbate - Fe+2 dependent system, during 180 min at 37 °C in the presence of increasing amounts of Silymarin: 6.25, 12.5, and 25 μg/mg of microsomal and mitochondrial protein. Peroxidation was quantified with a liquid scintillation analyzer Packard 1900 TR with chemiluminescence program (determined total counts per minute /mg ofprotein)., Facultad de Ciencias Veterinarias

[eng] Sporadic inclusion body myositis (sIBM) is the most common myopathy in elderly. This disease causes muscle wasting with both distal and proximal affectation. Quadriceps and finger flexors are muscle typically affected. At pathological level, three different features are present in muscle biopsies: inflammation, mitochondrial abnormalities and degeneration. The presence of T-cell infiltrate, ragged-red fibers and rimmed vacuoles are some features linked to these pathological processes. Protein misfolding and aggregation lead to the formation of the mentioned rimmed vacuoles, composed by many different misfolded proteins: β-amyloid, caveolin, phosphor-Tau among others. The accumulation of β-amyloid is also a hallmark of Alzheimer’s disease (AD), which presents some parallelisms with sIBM. In this work, we aimed to evaluate the mitochondrial state in muscle from sIBM patients but also in peripheral blood mononuclear cells (PBMC) to describe the molecular abnormalities that mitochondria could present in this disease. In addition, we aimed to measure plasmatic molecules related to inflammation, mitochondria and degeneration, in search for plasmatic biomarkers in sIBM patients but also in dermatomyositis and polymyositis, both diseases from the inflammatory myopathy group, like sIBM. Regarding mitochondrial analysis, muscle biopsies from 23 sIBM patients were analyzed, as well as 18 controls free of muscle disease. In addition, PBMC from 14 sIBM patients and from 20 controls were assessed as well. MtDNA levels and also mitochondrial respiratory chain complex IV (COX) activity were significantly decreased in muscle from sIBM patients compared to controls. Interestingly, when analyzing PBMC, dysfunction in COX activity was also found. In this tissue, a deregulation in mitochondrial protein synthesis was also found. As 57% of the sIBM patients presented mtDNA deletions, we aimed to evaluate if the presence of mtDNA deletions correlate with impaired mitochondrial parameters. sIBM patients with mtDNA deletions presented the lowest amount of mtDNA, and those patients without deletions showed values more similar to controls. A similar pattern was found when correlating the presence of MFN-2, a protein involved in mitochondrial dynamics. Again, patients with mtDNA deletions presented the lowest amount of this protein, and patients without deletions showed an intermediate values between patients with deletions and controls. Regarding the analysis of plasmatic molecules related to sIBM pathological features, inflammatory, mitochondrial and amyloidogenic molecules were analyzed in plasma samples from 21 sIBM, 20 controls and also in 14 plasma samples from dermatomyositis (DM) and polymyositis (PM) patients, which constitute an inflammatory myopathy different from sIBM group. Inflammatory (IL-6 and TNF-α) and mitochondrial-related (circulating mtDNA, FGF-21 and CoQ) molecules did not show significant differences between groups. However, amyloidogenic molecules (BACE-1, PS-1 and sAPPβ) were increased in sIBM patients respect to controls but also respect to the DM and PM group confirming its implication in sIBM pathogenesis. Sensitivity and specificity test showed that BACE-1 would be the most suitable biomarker for sIBM diagnosis. This thesis describes at molecular level the mitochondrial implication in the disease, and also reinforces the amyloidogenic component in sIBM. In addition, it proposes a plasmatic and non-invasive biomarker that could help in the sIBM diagnosis, especially in discriminating between other inflammatory myopathies, like polymyositis., [cat] La miositis per cossos d’inclusió en la seva forma esporàdica (MCI) és la miopatia més comú en individus de més de 50 anys tot i ser una malaltia rara. Cursa amb atròfia muscular progressiva distal i proximal i actualment no es coneix cura. A nivell histopatològic presenta un component inflamatori, un component mitocondrial i un component degeneratiu. Degut al seu component degeneratiu i a la similitud de les proteïnes que formen aquests cossos d’inclusió, s’ha establert un possible paral·lelisme amb la malaltia d’Alzheimer. Els objectius d’aquesta tesi doctoral són explorar a nivell molecular les alteracions mitocondriales en la MCI en múscul, però també en cèl·lules mononuclears de sang perifèrica (CMSP), ja que és un teixit menys invasiu. A més, com a segon objectiu principal pretén d’estudiar mol·lècules relacionades amb la inflamació, amb el mitocondri i amb la degeneració en plasma d’aquests pacients per tal de demostrar la seva implicación amb la etiopatogènia i a més per establir nous marcadors menys invasius que permetin diagnosticar la malaltia i diferenciarla d’altres malalties similars com la dermatomiositis i la polimiositis. Fent referència a l’estudi mitocondrial, tant la quantitat de DNA mitocondrial com l’activitat del complex IV de la cadena mitocondrial (COX) es van trobar disminuïts en músculs dels pacients amb MCI. D’altra banda, amb l’estudi de les CMSP, també vam trobar disminuïda l’activitat de la COX, i a més una desregulación de la síntesis de proteïnes mitocondrials. Donat que un 57% dels pacients va presentar delecions múltiples al DNA mitocondrial, la presència d’aquestes delecions correlacionava amb una menor quantitat de DNA mitocondrial i a més amb un decrement de proteïna MFN-2, implicada en la dinàmica mitocondrial. Amb l’estudi de les molècul·les plasmàtiques, es van analitzar en plasma de pacients amb MCI, en controls però també en pacients amb dermatomiositis i polimiositis mol·lècules relacionades amb la inflamació (IL-6 i TNF-α), amb el mitocondri (DNA mitocondrial circulant, FGF-21 i enzim CoQ) i amb la amiloidogènesi (BACE-1, PS-1 i sAPPβ). Les mol.lècules amiloidogèniques es trobaven incrementades en els pacients amb MCI respecte controls i altres miopatíes inflamatòries, demostrant la seva impliació en la etiopatogènia i obtenint un cert valor diagnòstic. Amb aquesta tesi, s’ha demostrat la implicació mitocondrial en la etiopatogènia de la MCI, i s’han trobat alteracions en plasma de mol·lècules amiloidogèniques que, a més, tenen potencial diagnòstic per diferenciar aquesta malaltia d’altres miopatíes inflamatòries com la polimiositis.

[eng] Homeostasis of the hematopoietic stem/progenitor cell pool relies on a finely tuned balance between self-renewal, differentiation and proliferation. Recent work has revealed the importance of mitochondria during stem cell differentiation; however, it remains unclear whether mitochondrial content/function affects human hematopoietic stem versus progenitor function. We sorted cord blood-derived CD34+ cells on the basis of mitochondrial mass and examined their homeostasis and clonogenic potential in vitro and hematopoietic repopulation potential in vivo. CD34+ cells with high mitochondrial mass contained and expressed 2-fold high ATP levels and mitochondrial-specific genes than cells with low mitochondrial mass, however, HIF-1α and MEIS1 were high in the CD34+ cells with low mitochondria. We found that CD34+ cells with low mitochondrial content were enriched for hematopoietic stem cell function as demonstrated by significantly higher hematopoietic reconstitution potential in immunodeficient mice. By contrast, CD34+ cells with high mitochondrial content were enriched for hematopoietic progenitor function with high in vitro clonogenic capacity. Coenzyme Q10 (CoQ10) plays a critical role in mitochondria as an electron carrier within the electron transport chain (ETC) and is an essential antioxidant. Mutations in genes responsible for CoQ10 biosynthesis (COQ genes) cause primary CoQ10 deficiency, a rare and heterogeneous mitochondrial disorder with no clear genotype-phenotype association, mainly affecting tissues with high energy demand including brain and skeletal muscle (SkM). A four-year-old girl was identified with a heterozygous mutation (c.483G>C; E161D) in COQ4, associated with a reduction in [CoQ10], CoQ10 biosynthesis and ETC activity affecting complexes I/II+III. Bona fide induced pluripotent stem cell (iPSC) lines carrying the COQ4 mutation (CQ4-iPSCs) were generated, characterized and genetically corrected using CRISPR/Cas9 genome-editing (CQ4ed-iPSCs). Comprehensive differentiation and metabolic analysis of control-iPSCs, CQ4-iPSCs and CQ4ed-iPSCs faithfully reproduced the disease phenotype. Accordingly, the COQ4 mutation in iPSCs was associated with CoQ10 deficiency, metabolic dysfunction and impaired differentiation into SkM. Remarkably, differentiation of CQ4-iPSCs into dopaminergic or motor neurons was unaffected. This study offers an unprecedented iPSC model recapitulating CoQ10 deficiency-associated functional and metabolic phenotypes caused by COQ4 mutation.

Producción Científica, Tumor cells undergo a critical remodeling of intracellular Ca2+ homeostasis that contribute to important cancer hallmarks. Store-operated Ca2+ entry (SOCE), a Ca2+ entry pathway modulated by mitochondria, is dramatically enhanced in colon cancer cells. In addition, most cancer cells display the Warburg effect, a metabolic switch from mitochondrial metabolism to glycolysis that provides survival advantages. Accordingly, we investigated mitochondria control of store-operated currents (SOCs) in two cell lines previously selected for representing human normal colonic cells and colon cancer cells. We found that, in normal cells, mitochondria are important for SOCs activity but they are unable to prevent current inactivation. In contrast, in colon cancer cells, mitochondria are dispensable for SOCs activation but are able to prevent the slow, Ca2+-dependent inactivation of SOCs. This effect is associated to increased ability of tumor cell mitochondria to take up Ca2+ due to increased mitochondrial potential (ΔΨ) linked to the Warburg effect. Consistently with this view, selected non-steroidal anti-inflammatory drugs (NSAIDs) depolarize mitochondria, inhibit mitochondrial Ca2+ uptake and promote SOC inactivation, leading to inhibition of both SOCE and cancer cell proliferation. Thus, mitochondria sustain store-operated currents in colon cancer cells but not in normal colonic cells and this effect is counteracted by selected NSAIDs providing a mechanism for cancer chemoprevention., Ministerio de Economía, Industria y Competitividad (grant BFU2015-70131R), Junta de Castilla y León (grant VA145U13)

O estresse é uma resposta não específica do organismo a ameaças à sua homeostase e o estresse crônico parece contribuir para o desenvolvimento de diversas patologias. Em situações estressantes são liberados glicocorticóides (GCs) pelo córtex adrenal, os quais se ligam aos receptores de glicocorticóides (RGs) para exercer suas ações. As mitocôndrias são organelas com diversas funções, dentre elas, a geração de energia na célula. Nos neurônios elas são essenciais para a função sináptica e sobrevivência neuronal, e podem ser encontradas tanto no corpo quanto na região sináptica. Uma vez que os GCs estão envolvidos na expressão de proteínas mitocondriais, alterações nos níveis de GCs ou RGs podem afetar as mitocôndrias e, consequentemente, a célula como um todo. Nessa tese, o foco de estudo foram as alterações que os GCs podem provocar na mitocôndria do sistema nervoso. No Capítulo I desta tese foi investigado os efeitos do estresse crônico por contenção sobre a cadeia respiratória mitocondrial e o conteúdo de RGs, de estriado, hipocampo e córtex de ratos machos e fêmeas, bem como a massa mitocondrial (MM) e o potencial de membrana mitocondrial (PMM) em dissociado de células e sinaptossomas destas mesmas estruturas cerebrais. Foi verificado que a resposta ao estresse envolveu alterações mitocondriais de forma sexo-específica, porém, não foi possível esclarecer se houve alteração na quantidade de mitocôndrias nestas respostas ao estresse. No Capítulo II estudamos se a ativação direta dos RGs com doses não tóxicas de dexametasona (DEX) provocaria alteração na quantidade de mitocôndrias em curto prazo. Para isso, células SH-SY5Y diferenciadas com ácido retinóico, que são corriqueiramente utilizadas como modelo neuronal, foram tratadas por 24 ou 48 horas com diferentes doses não tóxicas de DEX e foram avaliados MM e PMM. A DEX per se não afetou, em nenhuma dose ou tempo de tratamento, nem a MM, nem o PMM das células SHSY5Y diferenciadas, portanto, nos tempos e doses testadas, não altera a quantidade de mitocôndrias. No Capítulo III testamos os efeitos de altas doses de corticosterona (CORT) por um período mais longo, in vivo permitindo assim adaptações do animal. As estruturas analisadas foram hipocampo e estriado de camundongos machos, e as análises foram realizadas em células totais e sinaptossomas. Também testamos o efeito da diminuição dos GCs no cérebro utilizando um modelo hipocorticóide com DEX. O tratamento com DEX aumentou a MM e PMM de células totais, MM de neurônios e PMM de sinaptossomas de hipocampo; em estriado, reduziu a MM. O tratamento com CORT aumentou MM e PMM de sinaptossomas de estriado. Portanto, neste capítulo verificamos que a depleção dos GCs com DEX gerou aumento na quantidade de mitocôndrias no hipocampo demonstrada pelos aumentos na MM e PMM. Esse aumento na quantidade de mitocôndrias no corpo celular se refletiu também em um aumento na quantidade de mitocôndrias nos sinaptossomas. Com esse estudo concluímos que os principais efeitos do estresse sobre as mitocôndrias neuronais parecem ocorrer por diminuição da sensibilidade aos GCs e não pela presença de altas doses do hormônio. Essas alterações parecem ser adaptativas, favorecendo o manejo do estresse pelo animal, porém são necessários mais estudos para avaliar se, a mais longo prazo, essas adaptações continuam ocorrendo e como essas células adaptadas irão responder a insultos. Stress is a non-specific response to factors that threatens homeostasis and chronic stress seems to contribute to the development of several pathologies. In stressful situations, adrenal cortex releases glucocorticoids (GCs) which bind to glucocorticoid receptors (RGs) to exert their actions. Mitochondria are organelles with several functions, among them, the energy generation in the cell. In neurons they are essential for synaptic function and neuronal survival, and can be found in both the body and the synaptic region. Since GCs are involved in mitochondrial protein expression, changes in GCs or RGs may affect mitochondria and, consequently, the cell as a whole. In this thesis our focus was the changes that GCs can cause in the mitochondria present in the nervous system. In Chapter I we investigated the effects of chronic restraint stress on the mitochondrial respiratory chain and the RG content in striatum, hippocampus and cortex of male and female rats, as well as mitochondrial mass (MM) and mitochondrial membrane potential (PMM) in dissociated cells and synaptosomes in these same brain structures. It was verified that the stress response involved mitochondrial alterations in a sex-specific way; however, it was not possible to clarify if there was alteration in mitochondria quantity in these stress responses. In Chapter II we studied whether the direct RG activation with non-toxic doses of dexamethasone (DEX) would cause in short term changes in the mitochondrial amount. For this, retinoic acid-differentiated SH-SY5Y cells, which are commonly used as neuronal model, were treated for 24 or 48 hours with different non-toxic doses of DEX and were evaluated MM and PMM. DEX per se did not affect, at any dose or time of treatment, either MM or PMM of differentiated SH-SY5Y cells, therefore at the times and doses tested it does not alter the mitochondrial amount. In Chapter III we tested the effects of high doses of corticosterone (CORT) for a longer period, in vivo thus allowing animal adaptations to GC. We analyzed male mice’s hippocampus and striatum, and analyzes were performed on total cells and synaptosomes. We also tested the effect of GC decrease in the brain using a hipocorticoid model with DEX. Treatment with DEX increased the MM and PMM of total cells, MM of neurons and in PMM of hippocampal synaptosomes; in striatum, MM was reduced. Treatment with CORT increased synaptosomal MM and PMM in striatum. Therefore, in this chapter we verified that GCs depletion with DEX generated an increase in the mitochondrial amount in hippocampus demonstrated by increases in MM and PMM. This increase in the amount of mitochondria in the cell body was also reflected in an increase in the synaptosomal amount of mitochondria. With this study we conclude that the main effects of stress on neuronal mitochondria seem to occur due to decreased sensitivity to GCs and not to high doses of the hormone. These changes appear to be adaptive, favoring animal stress management, but further studies are needed to assess whether these adaptations continue to occur in the longer term and how these adapted cells will respond to insults.

Mitochondria supply cellular energy and also perform a role in the adaptation to metabolic stress. In mammals, the ataxia-telangiectasia mutated (ATM) kinase acts as a redox sensor controlling mitochondrial function. Subsequently, transcriptomic and genetic studies were utilized to elucidate the role played by a fungal ATM homolog during carbon starvation. In Aspergillus nidulans, AtmA was shown to control mitochondrial function and glucose uptake. Carbon starvation responses that are regulated by target of rapamycin (TOR) were shown to be AtmA-dependent, including autophagy and hydrolytic enzyme secretion. AtmA also regulated a p53-like transcription factor, XprG, inhibiting starvation-induced XprG-dependent protease secretion and cell death. Thus, AtmA possibly represents a direct or indirect link between mitochondrial stress, metabolism, and growth through the influence of TOR and XprG function. The coordination of cell growth and division with nutrient availability is crucial for all microorganisms to successfully proliferate in a heterogeneous environment. Mitochondria supply cellular energy but also perform a role in the adaptation to metabolic stress and the cross-talk between prosurvival and prodeath pathways. The present study of Aspergillus nidulans demonstrated that AtmA also controlled mitochondrial mass, function, and oxidative phosphorylation, which directly or indirectly influenced glucose uptake. Carbon starvation responses, including autophagy, shifting metabolism to the glyoxylate cycle, and the secretion of carbon scavenging enzymes were AtmA-dependent. Transcriptomic profiling of the carbon starvation response demonstrated how TOR signaling and the retrograde response, which signals mitochondrial dysfunction, were directly or indirectly influenced by AtmA. The AtmA kinase was also shown to influence a p53-like transcription factor, inhibiting starvation-induced XprG-dependent protease secretion and cell death. Therefore, in response to metabolic stress, AtmA appears to perform a role in the regulation of TOR signaling, involving the retrograde and SnfA pathways. Thus, AtmA may represent a link between mitochondrial function and cell cycle or growth, possibly through the influence of the TOR and XprG function.

Cardiolipin, the main anionic phospholipid in the inner mitochondrial membrane, provides shape, charge and osmotic support to this membrane due to its biophysical properties. In addition, it helps form respiratory supercomplexes and provides functionality to mitochondrial proteins. Defects in the biosynthesis or remodeling of cardiolipin have been related to severe diseases, such as Barth syndrome. Opi1p, a transcriptional repressor for most enzymes in phospholipid biosynthesis found in Saccharomyces cerevisiae, has been demonstrated not to affect the biosynthesis of this mitochondrial phospholipid. However, we found that opi1 deletion compromises mitochondrial metabolism producing severe respiratory defects. The mechanism producing this phenotype was explored and found to be a mitochondrial cardiolipin depletion of almost 50%, resulting in low cytochrome content and high mitochondrial DNA instability. The origin of this low cardiolipin content strongly correlated with the overproduction of inositol, an intrinsic phenotype of this mutation. Overall, our results show that adequate regulation of phospholipid synthesis is essential for the maintenance of mitochondrial function.

Mitochondrial cholesterol has been reported to be increased under specific pathological conditions associated with enhanced oxidative stress parameters. In this scenario, cholesterol oxidation would be increased, leading to the production of reactive aldehydes, including cholesterol carboxyaldehyde (ChAld). By using SDS micelles as a mitochondrial mimetic model, we have demonstrated that ChAld covalently modifies cytochrome c (cytc), a protein known to participate in electron transport and apoptosis signaling. This mimetic model induces changes in cytc structure in the same way as mitochondrial membranes do. Tryptic digestion of the cytc-ChAld adduct followed by MALDI-TOF/TOF analyses revealed that modifications occur at Lys residues (K22) localized at cytc site L, a site involved in protein-protein and protein-membrane interactions. Interestingly, ChAld ligation prevented cytc detachment from liposomes even under high ionic strength conditions. Overall, it can be concluded that ChAld ligation to Lys residues at site L creates a hydrophobic tail at cytc, which promotes cytc anchoring to the membrane. Although not investigated in detail in this study, cytc adduction to cholesterol derived aldehydes could have implications in cytc release from mitochondria under apoptotic stimuli.

Fil: Cardinali, Daniel P. Pontificia Universidad Católica Argentina. Facultad de Ciencias Médicas; Argentina Fil: Pagano, Eleonora S. Pontificia Universidad Católica Argentina. Facultad de Ciencias Médicas; Argentina Fil: Scacchi Bernasconi, Pablo A. Pontificia Universidad Católica Argentina. Facultad de Ciencias Médicas; Argentina Fil: Reynoso, Roxana. Pontificia Universidad Católica Argentina. Facultad de Ciencias Médicas; Argentina Fil: Scacchi, Pablo. Pontificia Universidad Católica Argentina. Facultad de Ciencias Médicas; Argentina Abstract: Cell death and survival are critical events for neurodegeneration, mitochondria being increasingly seen as important determinants of both. Mitochondrial dysfunction is considered a major causative factor in Alzheimer’s disease (AD), Parkinson’s disease (PD) and Huntington’s disease (HD). Increased free radical generation, enhanced mitochondrial inducible nitric oxide (NO) synthase activity and NO production, and disrupted electron transport system and mitochondrial permeability transition, have all been involved in impaired mitochondrial function. Melatonin, the major secretory product of the pineal gland, is an antioxidant and an effective protector of mitochondrial bioenergetic function. Both in vitro and in vivo, melatonin was effective to prevent oxidative stress/nitrosative stress-induced mitochondrial dysfunction seen in experimental models of AD, PD and HD. These effects are seen at doses 2-3 orders of magnitude higher than those required to affect sleep and circadian rhythms, both conspicuous targets of melatonin action. Melatonin is selectively taken up by mitochondria, a function not shared by other antioxidants. A limited number of clinical studies indicate that melatonin can improve sleep and circadian rhythm disruption in PD and AD patients. More recently, attention has been focused on the development of potent melatonin analogs with prolonged effects which were employed in clinical trials in sleep-disturbed or depressed patients in doses considerably higher than those employed for melatonin. In view that the relative potencies of the analogs are higher than that of the natural compound, clinical trials employing melatonin in the range of 50-100 mg/day are needed to assess its therapeutic validity in neurodegenerative disorders.

Durante la isquemia-reperfusión ocurre una caída del pH intracelular que provoca la activación de mecanismos alcalinizantes entre los que se encuentra el co-transportador Na+-HCO3- (NBC). La activación de dichos transportadores lleva al aumento del Na+ intracelular y secundariamente a la sobrecarga de Ca2+. Objetivo: Examinar la participación de la isoforma 1 del co-transportador NBC electrogénico (NBCe1) en el daño producido en la IR, evaluando el estado mitocondrial y las vías de señalización intracelular involucradas., Facultad de Ciencias Médicas

During an ischemic event, bicarbonate and CO2 concentration increase as a consequence of O2 consumption and lack of blood flow. This event is important as bicarbonate/CO2 is determinant for several redox and enzymatic reactions, in addition to pH regulation. Until now, most work done on the role of bicarbonate in ischemia-reperfusion injury focused on pH changes; although reperfusion solutions have a fixed pH, cardiac resuscitation protocols commonly employ bicarbonate to correct the profound acidosis associated with respiratory arrest. However, we previously showed that bicarbonate can increase tissue damage and protein oxidative damage independent of pH. Here we show the molecular basis of bicarbonate-induced reperfusion damage: the presence of bicarbonate selectively impairs mitophagy, with no detectable effect on autophagy, proteasome activity, reactive oxygen species production or protein oxidation. We also show that inhibition of autophagy reproduces the effects of bicarbonate in reperfusion injury, providing additional evidence in support of this mechanism. This phenomenon is especially important because bicarbonate is widely used in resuscitation protocols after cardiac arrest, and while effective as a buffer, may also contribute to myocardial injury.

[cat] Actualment en el nostre medi només l’1-2% dels fills de mares infectades pel VIH contreuen la infecció, gràcies a les mesures preventives aplicades, fonamentades sobretot en l'ús de fàrmacs antiretrovirals. Malgrat que el benefici que aporten aquests fàrmacs és inqüestionable, se sap que generen una càrrega d'efectes secundaris en els fills, ja que els reben durant la seva vida fetal i les primeres setmanes de vida i se'n desconeix la seva potencial repercussió a llarg termini. La tesi consisteix en 5 articles científics que pretenen objectivar què determina aquesta toxicitat i indagar en els mecanismes mitjançant els quals es genera. La toxicitat hematològica en els pacients pediàtrics exposats no infectats consisteix principalment en anèmia macrocítica les primeres setmanes de vida, habitualment subclínica i sempre autolimitada; i neutropènia. En els treballs realitzats observem que ni la reducció de la durada de la profilaxi neonatal amb zidovudina de sis a quatre setmanes ni l’ús durant la gestació de fàrmacs antiretrovirals de nova generació no disminueixen de forma significativa la incidència ni la gravetat de l’anèmia, que sembla relacionar-se sobretot a l´ús de zidovudina durant la gestació, molt habitual en tots els subgrups estudiats. D’altra banda, varis treballs apunten que la clínica derivada de disfunció mitocondrial en aquest col·lectiu de pacients podria ser més freqüent que en la població general. Fins l’actualitat escassos treballs han aportat dades sobre el metabolisme mitocondrial en pacients exposats no infectats, amb resultats contradictoris. En la nostra activitat de recerca, determinem per primera vegada de forma longitudinal els paràmetres mitocondrials, tant genètics com funcionals en els pacients exposats no infectats, observant una clara i persistent disfunció (disminució de l’activitat del complex IV de la cadena respiratòria mitocondrial), que és inversament proporcional al nivell d’ADN mitocondrial fins als sis mesos de vida. Observem també una disminució de les activitats enzimàtiques combinades (CII+CIII i CI+CIII+CIV de la cadena respiratòria mitocondrial) tant en les gestants com en els seus nadons a les sis setmanes de vida, i una correlació positiva entre els paràmetres materns i dels seus fills. Finalment, en analitzar conjuntament les dades de metabolisme mitocondrial i toxicitat hematològica, no observem una correlació clara entre elles. A les sis setmanes de vida, els pacients amb anèmia moderada o greu presenten menor activitat del CIV de la cadena respiratòria mitocondrial, fet que suggereix que la disfunció mitocondrial podria explicar almenys parcialment aquesta anèmia., [spa] Nowadays in developed countries, only 1-2% of infants born of HIV-infected women become infected thanks to preventive measures, mainly to antiretroviral drugs use. Although the benefits of these drugs are unquestionable, the burden of side effects they produce in otherwise healthy children is of concern. This doctoral thesis consists of five papers about the mechanisms and determinants of mitochondrial and hematological toxicity among HIV and antiretrovirals exposed uninfected infants. Several relevant results are obtained. Regarding mitochondrial disturbances, enzymatic dysfunction of complex IV of mitochondrial respiratory chain is observed longitudinally during the first year of life, negatively correlated to mitochondrial DNA content the first 6 months. Lower CII+CIII and CI+CIII+CIV activities are observed among HIV-infected mothers and their infants, with positive correlation between maternal and neonatal parameters. Respecting haematological data, no reduction on incidence of anemia or neutropenia is observed when shorter neonatal prophylaxis or new generation antiretroviral drugs are used. Zidovudine is the drug most strongly related to haematological toxicity. At 6 weeks of life, lower CIV activity is observed among infants with moderate to severe anemia, suggesting that mitochondrial impairment could partially explain this observed anemia.

Background Cell senescence is a process of central importance to the understanding of aging as well as to the development of new drugs. It is related with genomic instability, which has been shown to occur in the presence of autophagy deficiency. Yet, the mechanism that triggers genomic instability and senescence from a condition of autophagy deficiency remains unknown. By analyzing the consequences of treating human keratinocytes (HaCaT) with the pentacyclic triterpenoid Betulinic Acid (BA) we were able to propose that cell senescence can develop as a response to parallel damage in the membranes of mitochondria and lysosome. Methods We performed biochemical, immunocytochemical and cytometric assays after challenging HaCaT cells with BA. We also evaluated membrane leakage induced by BA in liposomes and giant unilamellar vesicles. Results By destabilizing lipid bilayers of mitochondria and lysosomes, BA triggers the misbalance in the mitochondrial-lysosomal axis leading to perceived autophagy impairment, lipofuscinogenesis, genomic instability and cell senescence. The progressive accumulation of mitochondria and lipofuscin, which comes from imperfect mitophagy triggered by BA, provides a continuous source of reactive species further damaging lysosomes and leading to cell aging. Conclusions This work reveals that the initial trigger of cell senescence can be the physical damage in the membranes of lysosomes and mitochondria. General significance This concept will help in the search of new drugs that act as senescence-inductors. BA is under evaluation as chemotherapeutic agent against several types of tumors and induction of cell senescence should be considered as one of its main mechanisms of action.

Genetic mitochondrial disorders are responsible for the most common inborn errors of metabolism, caused by mutations in either nuclear genes or in mitochondrial DNA. This article presents the prokaryotic origin of the organelle and the relation between nuclear and mitochondrial genomes, as well as current evolutionary models for such mechanisms. It also addresses the structure of mitochondrial genes, their expression pattern, clinical features of gene defects, risk of transmission and current techniques to avoid these events in assisted human reproduction. Finally, it discusses the ethical implications of these possibilities.RESUMOAs doenças genéticas mitocondriais são responsáveis pelos erros inatos do metabolismo mais comuns, causados por mutações tanto em genes nucleares como no DNA mitocondrial. Este artigo apresenta a origem procariótica dessa organela, e a relação entre os genomas nuclear e mitocondrial, bem como modelos evolutivos correntes para esses mecanismos. Também trata da estrutura dos genes mitocondriais, seu padrão de expressão, características clínicas de defeitos genéticos, riscos de transmissão e técnicas atualmente utilizadas para evitar esses eventos em reprodução humana assistida. Finalmente, discute as implicações éticas dessas possibilidades.

Genetic mitochondrial disorders are responsible for the most common inborn errors of metabolism, caused by mutations in either nuclear genes or in mitochondrial DNA. This article presents the prokaryotic origin of the organelle and the relation between nuclear and mitochondrial genomes, as well as current evolutionary models for such mechanisms. It also addresses the structure of mitochondrial genes, their expression pattern, clinical features of gene defects, risk of transmission and current techniques to avoid these events in assisted human reproduction. Finally, it discusses the ethical implications of these possibilities. RESUMO As doenças genéticas mitocondriais são responsáveis pelos erros inatos do metabolismo mais comuns, causados por mutações tanto em genes nucleares como no DNA mitocondrial. Este artigo apresenta a origem procariótica dessa organela, e a relação entre os genomas nuclear e mitocondrial, bem como modelos evolutivos correntes para esses mecanismos. Também trata da estrutura dos genes mitocondriais, seu padrão de expressão, características clínicas de defeitos genéticos, riscos de transmissão e técnicas atualmente utilizadas para evitar esses eventos em reprodução humana assistida. Finalmente, discute as implicações éticas dessas possibilidades.

Tese de doutoramento em Biociências, na especialidade de Toxicologia, apresentada ao Departamento de Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra After a literature review to find relevant research on the occurrence, fate and effects of azoxystrobin, the world’s leading agricultural fungicide, in aquatic ecosystems, strengths and gaps were identified in the database. Data revealed that validated analytical methods for complex matrices are very limited and knowledge of base-line values of azoxystrobin in marine ecosystems is lacking. The review also showed that there are very few data on azoxystrobin toxicity to different aquatic organisms, especially in what concerns marine organisms. Further work is also required regarding the effects of exposure to multi-stressors. To successfully determine pesticide levels in sediment, macrophytes and aquatic animals, adequate analytical methodologies were developed and validated. The established methodology applies Selective Pressurised Liquid Extraction (SPLE) followed by on-line solid phase extraction and Ultra Performance Liquid Chromatography-tandem Mass Spectrometry (on-line SPE-UPLC-MS/MS). This cutting-edge research methodology uses a small amount of sample, is time saving and reduces the use of organic solvents, in compliance with Green Chemistry principles. The seasonal and spatial occurrence and fate of the pesticides atrazine, azoxystrobin, bentazon, λ-cyhalothrin, penoxsulam and terbuthylazine were investigated in the Mondego estuary (Portugal). Quantified concentrations were determined mostly during summer. Azoxystrobin presented the highest detection frequency and atrazine the second highest frequency. Bentazon concentrations in surface water were considerably higher than those reported for other countries. All the prospected pesticides were bioaccumulated by the bivalve Scrobicularia plana and s-triazine pesticides were measured in both seaweeds Ulva spp. and Gracilaria gracilis. Acknowledging these data, developing and establishing allowable pesticide tolerance values for edible marine species is recommended. The present project reviewed more than four decades of published research papers in which the crab Carcinus maenas was used as an experimental test organism. This survey indicates that Carcinus sp. is sensitive to a wide range of aquatic pollutants and that its biological responses are linked to exposure concentrations or doses. Current scientific knowledge regarding the biology and ecology of Carcinus sp. and the extensive studies on ecotoxicology found for the present review recognise this crab as a reliable marine model for routine testing in ecotoxicology research and environmental quality assessment, especially in what concerns the application of the biomarker approach. An ecologically relevant study was conducted to evaluate the responses of C. maenas to temperature and azoxystrobin. Superoxide dismutase and glutathione S-transferase activities, mitochondrial oxygen consumption rates and protein content, as well as the Coupling Index were determined. Results provided evidence that crabs’ responses to cope with low temperatures were more effective than their responses to cope with high temperatures, which are expected in future climate projections. Moreover, crabs are capable of handling environmental concentrations of azoxystrobin. However, the Coupling Index showed that combined stress factors unbalance crabs’ natural capability to handle a single stressor. The present project also aimed at identifying, among six cell lines, a sensitive cell line whose azoxystrobin in vitro median inhibitory concentration (IC50) better matches the azoxystrobin in vivo short-term Sparus aurata median lethal concentration (LC50). Identical absolute sensitivities were attained for both in vitro and in vivo assays for H9c2 cells by the sulforhodamine B colorimetric assay. We concluded that this H9c2 cell-based assay is reliable and represents a suitable ethical alternative to conventional fish assays for azoxystrobin. This study determined and validated an aquatic Predicted No-Effect-Concentration (PNEC) value for azoxystrobin. The assessment factor and species sensitive distribution approaches were applied to freshwater and marine toxicity datasets. After comparing the PNEC values estimated in the present study to all the laboratory-derived toxicity information available for azoxystrobin, PNEC values derived using the assessment factor method were considered overprotective and a PNEC of 1.0 µg L-1 was recommended for azoxystrobin in the aquatic environmental compartment. Finally, azoxystrobin environmental risk characterisation could be determined as the Risk Quotient (RQ), the ratio of measured environmental concentrations and its PNEC value. Therefore, since the maximum concentration of azoxystrobin measured in the Mondego estuary was 0.07 µg L-1, it is possible to conclude that here this pesticide currently poses low risk to aquatic organisms (RQ

Studies have demonstrated the importance of mitochondria to sperm functionality, as the main source of ATP for cellular homoeostasis and motility. However, the role of mitochondria on sperm metabolism is still controversial. Studies indicate that, for some species, glycolysis may be the main mechanism for sperm energy production. For ram sperm, such pathway is not clear. Thus, we evaluated ram sperm in response to mitochondrial uncoupling and glycolysis inhibition aiming to assess the importance of each pathway for sperm functionality. Statistical analysis was performed by the SAS System for Windows, using the General Linear Model Procedure. Data were tested for residue normality and variance homogeneity. A p

Las mitocondrias son orgánulos citoplasmáticos con genoma propio presentes en casi todas las células eucariotas, y cuya función principal está asociada a la producción continua de energía a través de un proceso que se denomina respiración celular. En el interior de la matriz mitocondrial se encuentra el genoma mitocondrial, que es una molécula circular bicatenaria de 16569 pares de bases (pb) en la que podemos distinguir dos regiones principales, la región codificante y la región control, esta última con una tasa de mutación mucho más alta que la anterior y dividida a su vez en una región hipervariable 1 y una región hipervariable 2. Desde el punto de vista genético, el ADN mitocondrial (ADNmt) posee una serie de características especiales, como son un alto número de copias por célula, ausencia de recombinación, herencia exclusivamente materna y alta tasa de mutación, que le aportan un carácter distintivo y que le otorgan un gran valor como marcador molecular, sobre todo en los ámbitos de la genética forense y genética de poblaciones. Sin embargo, alguna de las características que confieren al ADNmt ese carácter diferencial, paradójicamente puede suponer a su vez una limitación. Por ejemplo, su modo de herencia nos permite por un lado llevar a cabo reconstrucciones filogenéticas de un modo relativamente sencillo, pero por otro, solamente ofrece una visión demográfico-­‐poblacional desde una perspectiva materna, perspectiva que no tiene que coincidir necesariamente con el proceso demográfico global de la población. Desde el punto de vista forense, la herencia uniparental del ADNmt limita su utilidad a la identificación de linajes maternos y no permite la identificación individual.

Human metabolic diseases can be mimicked in rodents by using dietary interventions such as high fat diets (HFD). Nonalcoholic fatty liver disease (NAFLD) develops as a result of HFD and the disease may progress in a manner involving increased production of oxidants. The main intracellular source of these oxidants are mitochondria, which are also responsible for lipid metabolism and thus widely recognized as important players in the pathology and progression of steatosis. Here, we review publications that study redox and bioenergetic effects of HFD in the liver. We find that dietary composition and protocol implementations vary widely, as do the results of these dietary interventions. Overall, all HFD promote steatosis, changes in β-oxidation, generation and consequences of oxidants, while effects on body weight, insulin signaling and other bioenergetic parameters are more variable with the experimental models adopted. Our review provides a broad analysis of the bioenergetic and redox changes promoted by HFD as well as suggestions for changes and specifications in methodologies that may help explain apparent disparities in the current literature., Graphical abstract fx1, Highlights • High fat diets (HFDs) induce steatosis, even with no weight changes . • HFDs activate β-oxidation. • HFDs promote oxidative imbalance.

Mutations in the POLG gene, encoding the mitochondrial DNA (mtDNA) polymerase subunit gamma-1, have been identified in severe mtDNA depletion syndromes and mtDNA deletion disorders which include ataxia neuropathy spectrum disorders and AR and AD forms of progressive external ophthalmoplegia (PEO) and PEO-plus disorders. We report on a family with exercise intolerance. The proband was a 50-year-old man with severe muscle pain and premature fatigue after exercise of mild to moderate intensity. Serum CK ranged from 400 to 4500 U/L. Sin financiación 2.969 JCR (2016) Q2, 72/194 Clinical Neurology, 124/259 Neurosciences 1.421 SJR (2016) Q1, 69/380 Neurology (clinical), 32/313 Pediatrics, Perinatology and Child Health; Q2, 39/103 Genetics (clinical), 44/166 Neurology No data IDR 2016 UEM

La importancia de las estructuras que delimitan las células eucariotas se asimila en cuanto se determinan las funciones de la membrana plasmática y de la pared celular. El citosol y el citoesqueleto son otras de las estructuras trabajadas en este tema, muy importantes también en relación a su composición y funciones contráctiles y de motilidad celular. Se interconectan los diferentes orgánulos del sistema endomembranoso y orgánulos no membranoso para comprender el funcionamiento celular como un todo y la motilidad celular en donde intervienen algunas de las estructuras celulares explicadas. The importance of the structures that delimit eukaryotic cells is treated as functions of the plasma membrane and the cell wall is determined. Cytosol and cytoskeleton are other structures worked on this issue, very important also in relation to its composition and contractile function and cell motility. Different organelles of endomembrane system and not membranous organelles are relate to understand the functioning of cells as a whole and cell motility involved where some of the cellular structures explained.

[spa] Esta tesis se basa en diagnóstico de pacientes con deficiencia de 2 metil 3 hidroxibutiril CoA deshidrogenasa (MHBD o HSD10) y en el estudio de la fisiopatología de esta enfermedad y de su implicación en la Enfermedad de Alzheimer (EA). El desarrollo de un método cuantitativo de metabolitos clave en orina y un método de análisis enzimático de la proteína HSD10 en fibroblastos cultivados, así como el análisis molecular, de los niveles de HSD10 y de los estudios patogenicidad de las mutaciones encontradas, nos ha permitido el diagnóstico de 6 pacientes y 8 portadoras, que representan el 37% de los pacientes descritos en la literatura. La herencia de esta enfermedad se halla ligada al cromosoma X. Se había descrito que el gen HSD17B10, que codifica para la proteína HSD10, se escapaba de la inactivación del cromosoma X. Sin embargo, el fenotipo bioquímico y clínico de nuestros pacientes no apoyaban este hallazgo. Mediante estudios de expresión de HSD17B10 por PCR cuantitativa se demostró que HSD17B10 no escapaba a la inactivación del cromosoma X. HSD10 es una proteína mitocondrial multifuncional que además de su papel en el metabolismo de la isoleucina y de los ácidos grasos, desempeña otras funciones en el metabolismo de hormonas esteroideas sexuales, esteroides neuroactivos, en la detoxificación de aldehídos citotóxicos y en la metabolización de cardiolipina peroxidada. La clínica neurológica de los pacientes con deficiencia de HSD10 difiere de otras deficiencias de la vía metabólica de la isoleucina. Por ello, realizamos estudios de microarrays de expresión. Obtuvimos una expresión significativamente diferente en 31 genes. Los resultados se confirmaron mediante PCR cuantitativa. La diferencia de expresión de estos genes podrían explicar los síntomas clínicos de los pacientes con deficiencia de HSD10. La proteína HSD10 parece estar implicada en la EA, debido a su interacción con el péptido β amiloide (pßA), habiéndose sugerido que dicha interacción es una de las causas de la disfunción mitocondrial en la EA. Para determinar la implicación de HSD10 en la EA se realizaron una serie de estudios en homogenados de cerebro de pacientes con EA y controles. Encontramos una disminución de la actividad de HSD10 en homogenados de cerebro de pacientes con EA y esta disminución no se debía a una disminución de los niveles de proteína, ya que el wester blot no reveló diferencias entre pacientes y controles. Se observó también que la actividad HSD10 disminuye con la progresión de la enfermedad. Por otro lado, comprobamos que la incubación de homogenados de cerebro control con pßA provocaba una inhibición de la actividad HSD10, que se rescataba al incubar con elevadas concentraciones de NAD +. Esta observación también fue corroborada en la EA. Este hecho se podía explicar por una interacción competitiva entre NAD+ y pßA, ya que ambos interaccionan con HSD10 en la misma posición aminoacídica. Por ello, al incubar con elevadas concentraciones NAD+ se inhibe la interacción pßA HSD10. Por tanto, la medición de la actividad HSD10 podría ser utilizada como diana terapéutica para la EA. Con esta finalidad, obtuvimos un sistema “in vitro” con la valoración de la proteína recombinante HDSD10 isoforma 1, que es la que expresa mayoritariamente en cerebro humano, antes y después de la incubación con pßA, con y sin adición de NAD+. Los resultados obtenidos fueron los mismos que al utilizar homogenados de cerebro humano. Por lo tanto, quedaba validado un sistema para cribar moléculas terapéuticas que pudieran inhibir la interacción pßA HSD10 y restablecer la actividad HSD10. Se probaron 117 compuestos de una librería peptídica, Coenzima Q10 y otros antioxidantes, pero no se consiguió rescatar la actividad HSD10. La actividad enzimática sólo se consiguió rescatar con NAD+ . Así, proponemos que los precursores de NAD+ podrían ser considerados una terapia coadyuvante en la EA. Estudios recientes apoyan nuestros resultados, ya que la administración de precursores de NAD+ a ratones modelo para la EA, mejoran la disfunción mitocondrial así como la cognición., [eng] This thesis is based on diagnosis of patients with 2 methyl 3 hydroxybutyryl CoA dehydrogenase (MHBD or HSD10) deficiency and the study of its pathophysiology and its involvement in Alzheimer's Disease (AD). The development of different methods for quantification of key metabolites in urine and for enzymatic analysis in cultured fibroblasts, as well as the molecular studies performed, has allowed us the diagnosis of 6 patients and 8 carriers, which represents 37% of the patients described in the literature. Expression studies of HSD17B10 demonstrated that this gen not escapes inactivation of the X chromosome, contrary to that published by other authors. The neurological symptoms of patients with deficiency HSD10 differs from other deficiencies in the isoleucine's metabolic pathway, maybe due to the alteration of other functions involved in both, brain and mitochondrial homeostasis. The expression "microarray" studies performed revealed significantly different expression in 31 genes, which could explain the clinical symptoms of the patients. The HSD10 protein appears to be involved in AD, due to their interaction with β amyloid peptide (Aβ). Studies in brain homogenates of AD patients showed decreased HSD10 activity from the early stages of the disease, which could explain the early mitochondrial dysfunction observed in AD. Furthermore, the Aβ incubation caused inhibition of HSD10 activity in control brain homogenates, which was rescued by incubating with high NAD+ concentrations. This observation was also corroborated in AD. This fact could be explained by a competitive interaction that occurs between NAD+ and Aβ. Therefore, the measurement of HSD10 activity could be used as a therapeutic target for AD. To this end, we obtained "in vitro" system to the evaluation of the HDSD10 isoform 1 recombinant protein, which is the mainly expressed in human brain, before and after incubation with Aβ, with and without addition of NAD+. The results were the same as when using human brain homogenates. Therefore, the system was validated for screening therapeutic molecules that could inhibit the Aβ HSD10 interaction and restore HSD10 activity. Among 117 compounds tested, only NAD+ was able to rescue HSD10 activity. Thus, we propose that the precursors of NAD+ could be considered an adjunctive therapy in AD. Recent studies support our results, since administration of NAD precursors mice model for AD, improve mitochondrial dysfunction and cognition.

Producción Científica, The Fas-activated serine/threonine kinase (FASTK) family of proteins has recently emerged as a central regulator of mitochondrial gene expression through the function of an unusual RNA-binding domain named RAP (for RNA-binding domain abundant in Apicomplexans), shared by all six members of the family. Here we describe the role of one of the less characterized members, FASTKD3, in mitochondrial RNA metabolism. First, we show that, in contrast to FASTK, FASTKD2, and FASTKD5, FASTKD3 does not localize in mitochondrial RNA granules, which are sites of processing and maturation of mtRNAs and ribosome biogenesis. Second, we generated FASTKD3 homozygous knock-out cell lines by homologous recombination and observed that the absence of FASTKD3 resulted in increased steady-state levels and half-lives of a subset of mature mitochondrial mRNAs: ND2, ND3, CYTB, COX2, and ATP8/6. No aberrant processing of RNA precursors was observed. Rescue experiments demonstrated that RAP domain is required for FASTKD3 function in mRNA stability. Besides, we describe that FASTKD3 is required for efficient COX1 mRNA translation without altering mRNA levels, which results in a decrease in the steady-state levels of COX1 protein. This finding is associated with reduced mitochondrial complex IV assembly and activity. Our observations suggest that the function of this family of proteins goes beyond RNA processing and ribosome assembly and includes RNA stability and translation regulation within mitochondria., Junta de Castilla y León (grants BIO/ VA20/15 and BIO/VA21/15), Swiss National Science Foundation (grant 310030B_160257/1)

O alto consumo de dietas hiperpalatéveis (HP) ricas em lipídios e carboidratos simples associado a hábitos sedentários são fatores que contribuem para o desenvolvimento de sobrepeso e obesidade. Estas alterações, promovem um estado proinflamatório, causam resistência periférica à insulina e ainda prejudicam o metabolismo cerebral. A glicose é considerada o principal substrato energético para o cérebro. No entanto, em condições de alta demanda energética outros substratos como corpos cetônicos e lactato, por exemplo, podem servir como alternativa energética. A teoria da lançadeira de lactato astrócito-neurônio (do inglês ANLS) propõe que o aumento da captação da glutamato pelos astrócitos aumenta a atividade glicolitica e a produção de lactato, que por sua vez é exportado dos astrócitos para o espaço extracelular via transportadores de monocarboxilados (MCTs) do tipo 1 e 4. Os transportadores MCT do tipo 2 presentes nos neurônios internalizam o lactato para que ele seja convertido em piruvato. A enzima piruvato desidrogenase (PDH) converte o piruvato em acetil-CoA dentro da matriz mitocondrial. Sendo assim, o lactato e outras substâncias em equilíbrio com o piruvato são direcionados para o metabolismo oxidativo. Assim, a PDH conecta o metabolismo citosólico e mitocondrial. Por outro lado, o aumento do metabolismo oxidativo é associado com o aumento de vazamento de prótons e com a produção de peróxido de hidrogênio pela mitocôndria. Consequentemente, pode haver uma menor produção de ATP pela mitocôndria. Neste estudo, os efeitos de uma dieta hiperpalatável (HP) e do exercício físico em parâmetros metabólicos no hipocampo e na memória foram investigados. Foram avaliados: os níveis de lactato, o imunoconteúdo dos MCTs e da PDH e atividade mitocondrial no hipocampo de camundongos. Camundongos C57BJ machos com 30 dias receberam uma dieta HP durante cinco meses e, praticaram atividade física voluntária durante o último mês. Os grupos foram: CDS (dieta controle sedentário), CDE (dieta controle exercício), HPS (dieta HP sedentário) e HPE (dieta HP exercício), (n=15 animais/grupo). Os resultados mostraram um aumento no imunoconteúdo de MCT 1 e 4 causados pela dieta e pelo exercício físico. O exercício físico causou um aumento na expressão de PDH. A expressão do MCT2 não apresentou diferença significativa entre os grupos. Não foram observadas alterações na memória no teste do Y-maze. No entanto, houve um aumento no nível de lactato em microdialisado de hipocampo no grupo HPE após a exposição ao teste. Não foi observada diferença significativa no metabolismo mitocondrial basal, e nem no potencial de membrana mitocondrial. A ativação do complexo II mitocondrial pelo succinato aumentou a produção de H2O2 no grupo HPS. Esse efeito foi revertido pelo exercício físico. Os resultados demonstram que o exercício e a dieta HP aumentaram a expressão dos MCTs 1 e 4, enquanto que a expressão de PDH apenas aumentou pelo exercício. Isto sugere uma adaptação da maquinaria metabólica hipocampal em resposta a estes estímulos ambientais. The high consumption of saturated fats and simple sugar diets associated with sedentary habits are factors that contribute to the development of overweight and obesity. These consequences produce a pro-inflammatory state, resulting in periphery insulin resistance and damage the cerebral metabolism. The glucose is considered to be the main energetic substrate to the brain, other particular substrates, like ketonic bodies and lactate, may work as alternative energy sources during moments of high energetic demand. The theory of Astrocyte-Neuron Lactate Shuttle suggests that the increased uptake of glutamate by the astrocyte enhance the glycolytic activity and the production of lactate, which is exported from the astrocyte to the extracellular space via monocarboxylate transporters (MCTs) type 1 and 4. The MCTs type 2 residing inside the neurons accumulates lactate, which is converted into pyruvate. Furthermore, the dehydrogenase pyruvate enzyme (PDH) convert the pyruvate to Acetyl-CoA inside the mitochondrial matrix. Thus, the lactate and other substances in equilibrium are directed to the oxidative metabolism. This way, the PDH connects the cytosolic and mitochondrial metabolism. On the other hand, the increased oxidative metabolism is associated with the leakage of protons and with the hydrogen peroxide production by the mitochondria. Consequently, the ATP production might be reduced. In this study, the effects of a palatable diet and physical exercise in metabolic parameters in the hippocampus and memory were investigated. Were evaluated: lactate levels, the expression of MCTs and PDH and mitochondrial activity in the hippocampus. Male C57BJ mice with 30 days received a HP diet for five months and practiced voluntary physical activity during the last month. The groups were: CDS (control diet sedentary), CDE (control diet exercise), HPS (HP diet sedentary) and HPE (HP diet exercise), (n = 15 animals / group). The results showed an increase in immunocontent of MCT 1 and 4 caused by diet and exercise. The physical exercise caused an increase in PDH expression. The expression of MCT2 had no relevant impact among the groups. No changes were observed in memory in the Y-maze test even though it was observed an increase in lactate levels in hippocampal microdialysate of the HPE group after the exposure to test. There was no significant difference in basal mitochondrial metabolism nor in the mitochondrial membrane potential. The activation of the mitochondrial complex II by succinate increased H2O2 production in the HPS group. This effect was reversed by exercise. The results show that the exercise and the HP diet increased the expression of MCTs 1 and 4, while the PDH expression only increased by exercise. This suggests an adaptation of the hippocampal metabolic machinery in response to these environmental stimuli.

As deficiências da 3-hidroxiacil-CoA desidrogenase de cadeia longa (LCHAD) e da proteína trifuncional mitocondrial (MTP) são defeitos hereditários na oxidação de ácidos graxos. Os pacientes apresentam acúmulo de ácidos graxos hidroxilados de cadeia longa (LCHFA), especialmente os ácidos 3- hidroxitetradecanoico (3HTA) e 3-hidroxipalmítico (3HPA), no sangue e outros tecidos. A sintomatologia é bastante variada, incluindo cardiomiopatia severa e sintomas musculares como fraqueza, dor e episódios recorrentes de rabdomiólise, assim como hepatopatia, retinopatia, hipotonia, neuropatia periférica, atraso no desenvolvimento e na fala, podendo levar a morte ainda na infância. Considerando que os mecanismos patogênicos do dano aos tecidos musculares cardíaco e esquelético apresentados por esses pacientes ainda não estão esclarecidos, o presente trabalho teve como objetivo investigar os efeitos in vitro do 3HTA e 3HPA sobre importantes parâmetros da bioenergética mitocondrial, tais como os parâmetros respiratórios estado 3, estado 4, razão de controle respiratório (RCR) e estado desacoplado, bem como o potencial de membrana (ΔΨm), o inchamento, a liberação de citocromo c, a capacidade de retenção de Ca2+ e o conteúdo do NAD(P)H em mitocôndrias isoladas de músculo cardíaco e esquelético de ratos jovens. Inicialmente, observamos que o 3HTA e 3HPA em baixas concentrações (10-30 UM) aumentaram o estado 4 e diminuíram o RCR em mitocôndrias de músculo esquelético, indicando um efeito desacoplador. Quando em concentrações mais elevadas (50-100 UM), esses ácidos graxos diminuíram o estado 4, o estado 3 e o estado desacoplado da respiração mitocondrial, característicos de inibidores metabólicos. Ainda observamos que o 3HPA foi capaz de produzir efeitos semelhantes sobre a respiração mitocondrial em fibras musculares permeabilizadas, validando os resultados obtidos em mitocôndrias isoladas. Além disso, demonstramos que o 3HPA e o 3HTA (30 UM) diminuíram marcadamente o ΔΨm, o conteúdo de NAD(P)H, a capacidade de retenção de Ca2+ e a produção de ATP, além de induzirem inchamento, em mitocôndrias obtidas de ambos os tecidos e suplementadas com Ca2+. Esses efeitos foram prevenidos por ciclosporina A e ADP, assim como pelo rutênio vermelho, indicando o envolvimento do PTP e do Ca2+, respectivamente. O fato de termos verificado que o 3HPA aumentou marcadamente a fluidez das membranas mitocondriais em músculo esquelético indica que esse mecanismo pode estar envolvido no prejuízo da homeostase mitocondrial causado por esses compostos. Finalmente, verificamos que o análogo dicarboxílico do 3HTA, o ácido 3-hidroxitetradecanodioico, que também se acumula nos pacientes, não alterou os parâmetros avaliados, indicando uma ação seletiva para os ácidos monocarboxílicos. Analisando nossos resultados em conjunto, demonstramos que os principais LCHFA acumulados nas deficiências da LCHAD e MTP prejudicam a homeostase mitocondrial em músculo cardíaco e esquelético. Presumimos que esse mecanismo possa explicar, pelo menos em parte, a cardiomiopatia severa, os sintomas e alterações musculares que acometem os pacientes afetados. Long-chain 3-hydroxy-acyl-CoA dehydrogenase (LCHAD) and mitochondrial trifunctional protein (MTP) deficiencies are inborn errors of fatty acid oxidation. Affected patients present accumulation of long-chain hydroxylated fatty acids (LCHFA), particularly 3-hydroxytetradecanoic (3HTA) and 3-hydroxypalmitic (3HPA) acids, in blood and other tissues. The symptomatology is varied, including severe cardiomyopathy and muscular symptoms such as weakness, muscle pain and recurrent episodes of rhabdomyolysis, as well as hepatopathy, retinopathy, hypotonia, peripheral neuropathy, speech and development delay, leading to premature death. Considering that the pathogenesis of cardiac and skeletal muscle damage presented by the patients are still not established, the aim of the present work was to investigate the in vitro effects of 3HTA and 3HPA on important parameters of mitochondrial bioenergetics, namely the respiratory parameters state 3, state 4, respiratory control ratio (RCR) and uncoupled respiration, as well as mitochondrial membrane potential (ΔΨm), swelling, Ca2+ retention capacity and NAD(P)H redox state in cardiac and skeletal muscle mitochondria isolated from young rats. Initially, we observed that 3HTA and 3HPA at lower concentrations (10-30 UM) increased state 4 and decreased RCR in skeletal muscle mitochondria, indicating an uncoupling effect. At higher concentrations (50-100 UM), these fatty acids decreased state 4, state 3 and uncoupled respiration, suggesting metabolic inhibition. Furthermore, we observed that 3HPA was capable to provoke similar effects on mitochondrial respiration in permeabilized skeletal muscle fibers, validating the results obtained in isolated mitochondria. We also demonstrated that 3HPA and 3HTA (30 UM) strongly decreased the ΔΨm, NAD(P)H content, Ca2+ retention capacity and ATP production, besides inducing swelling, in mitochondria obtained from both tissues and supplemented with Ca2+. These effects were prevented by cyclosporin A and ADP, as well as by ruthenium red, indicating the involvement of mitochondrial permeability transition and Ca2+, respectively. The fact that 3HPA strongly increased the mitochondrial membrane fluidity in skeletal muscle indicates that this mechanism may be involved in the mitochondrial bioenergetics impairment caused by these compounds. Finally we verified that the 3HTA dicarboxylic analogue, 3-hydroxytetradecanodioic acid, which also accumulates in the affected patients, did not alter the tested parameters, indicating a selective action of the monocarboxylic acids. Taken together, we demonstrated that the major LCHFA accumulated in LCHAD and MTP deficiencies impair mitochondrial homeostasis in cardiac and skeletal muscle. We presume that these mechanisms may explain, at least in part, the severe cardiomyopathy, symptomatology and muscle alterations characteristics of the patients affected by these disorders.

Tesis (Ingeniero en Biotecnología) Las mitocondrias son organelos esenciales para las células. Éstas cumplen variadas e importantes funciones como: el metabolismo de hierro y cobre, producción de hemo y también producción de energía en forma de ATP para la célula. Si las mitocondrias fallan, las células entran en apoptosis estimulado por la acción de las especies reactivas de oxígeno (ROS), que causan daños en la estructura mitocondrial, en su DNA y en sus proteínas. Por esto, la mitocondria tiene métodos de protección contra daños, entre ellos, los sistemas antioxidantes, proteasas triple A, ciclos de biogénesis y autofagia; y por último, el sistema de respuesta a proteínas mal plegadas mitocondrial (UPRmt). En mamíferos se sabe que este sistema, que funciona a través de proteínas chaperonas y proteasas, es activado por un heterodímero compuesto por la proteína homologa a C/EBP (CHOP) y la proteína que interactúa con CCAAT β (C/EBP-β), luego de que la proteína quinasa c-Jun N-Terminal (JNK2) sensa estrés oxidativo en el citosol Uno de los tipos celulares mamíferos que más estrés oxidativo debe soportar, es el eritroide, donde la producción de hemoglobina, causa un gran aumento de ROS en las células. Cuando la producción de hemoglobina comienza, la expresión de CHOP y C/EBP-β aumentan simultáneamente, así como las proteasas y chaperonas mitocondriales que éstas activan, como a la subunidad proteolítica de la proteasa Clp ATP-dependiente (ClpP) y la proteína Heat-shock 60 (Hsp60). Dada la correlación entre la expresión de las proteínas del UPRmt y la producción de hemoglobina; se puede decir que la el sistema UPRmt es esencial para la diferenciación eritroide. Es por esto que se investiga, en este trabajo, la participación del sistema UPRmt en la eritropoyesis. Durante esta investigación, se determinó que la expresión de CHOP y C/EBP-β, ClpP y Hsp60 aumenta a medida que las células hematopoyéticas se diferencian. También se logró determinar que al silenciar, con RNA de interferencia, las proteínas involucradas en el sistema mitocondrial de proteínas mal plegadas (UPRmt), las células fueron incapaces de alcanzar las etapas finales de la diferenciación. Mientras que a nivel mitocondrial, se provocan bajas en la respiración celular y en el potencial de membrana, aceleración del recambio mitocondrial, aumentos en el estrés oxidativo y en la apoptosis. Esto hace concluir que cuando aumenta el estrés en la mitocondria, la participación del heterodímero CHOP-C/EBP-β en la activación del UPRmt es esencial para la diferenciación eritroide. Mitochondria are essential organelles for the cells, these organelles performs varied and important functions like: Copper and iron metabolism, heme production, and also energy production in form of ATP, for the cell. If mitochondria fail, cells undergo apoptosis, which occurs by the action of reactive oxygen species (ROS) that cause damage in the mitochondrial structure, DNA and proteins. Because of this, mitochondria have protection methods against damage, such as the antioxidant systems, the triple A proteases, the biogenesis and autophagy cycle and ultimately, the mitochondrial unfolded protein response (UPRmt). In mammals, is known that this system, working through chaperones and proteases, is activated by a heterodimer composed of the C/EBP homolog protein (CHOP) and CCAAT-β interacting protein (C/EBP-β), after the c-Jun N-Terminal protein kinase 2 (JNK2) senses oxidative stress in the mitochondrial matrix. One of the cell types that bears most of oxidative stress is the erythroid type, where the production of hemoglobin causes a great increase in intracellular ROS. When the production of hemoglobin starts, the expression of CHOP and C/EBP-β increases simultaneously, as well as the mitochondrial proteases like ATP-dependent Clp protease proteolytic subunit (ClpP) and chaperones like Heat shock protein 60 (Hsp60). Given this correlation between expression of the UPRmt proteins and the hemoglobin production; it can be said that the UPRmt system is essential for erythroid differentiation. For this reason this work studies the participation of the UPRmt system in erythropoiesis. During this investigation it was determined that expression of CHOP and C/EBP-β and also the expression of ClpP and Hsp60, increases when hematopoietic cells differentiate. Also we determined that when the proteins involved in the UPRmt are silenced with siRNA, the cells were incapable of reaching the final phases of differentiation. At the mitochondrial level, silencing caused low respiration index, and low membrane potential, as well as faster mitochondrial turnover, increased oxidative stress and increased apoptosis. This make conclude when there is an increase in mitochondria stress, the participation of the heterodimer CHOP-C/EBP-β in the activation of the UPRmt is essential for erythroid differentiation.

The research leading to these results was funded by the Spanish Ministry of Health (FIS-PI11/00610, FIS-PI041436, FIS-PI081151, FIS-PI042018, and FIS-PI09/02311), the European Union (EU) (FP7-ENV-2011 cod 282957 and HEALTH.2010.2.4.5-1), the Instituto de Salud Carlos III (Red INMA G03/176, CB06/02/0041, FIS-FEDER 03/1615, 04/1509, 04/1112, 04/1931, 05/1079, 05/1052, 06/1213, 07/0314, 09/02647, 11/01007, 11/02591, CP11/00178, FIS-PI06/0867, and FIS-PS09/00090), the Conselleria de Sanitat Generalitat Valenciana, the Generalitat de Catalunya-CIRIT (1999SGR 00241), the Obre Social Cajastur, the Universidad de Oviedo, the Department of Health of the Basque Government (2005111093 and 2009111069), and the Provincial Government of Gipuzkoa (DFG06/004 and DFG08/001). The ENVIRONAGE cohort is supported by the EU Program “Ideas” (ERC-2012-StG 310898) and by the Flemish Fund for Scientific Research (FWO 1516112N and G073315N)., Clemente, D.B.P., Casas, M., Vilahur, N., Begiristain, H., Bustamante, M., Carsin, A., Fernández, M.F., Fierens, F., Gyselaers, W., Iñiguez, C., Janssen, B.G., Lefebvre, W., Llop, S., Olea, N., Pedersen, M., Pieters, N., Marina, L.S., Souto, A., Tardón, A., Vanpoucke, C., Vrijheid, M., Sunyer, J., Nawrot, T.S.

A Doença de Alzheimer (DA) é a mais comum desordem neurodegenerativa e a principal causa de demência re lacionada a idade. Os números em crescimento de pessoas afetadas pela DA estão diretamente ligados ao aumento na expectativa de vida da população mundial. A DA, por se tratar de uma doença multifatorial, envolve fatores genéticos, moleculares e ambientais.As principais alterações fisiopatológicas, hoje alvos de estudos, são os emaranhados neurofibrilares, constituídos pela proteína tau hiperfosforilada e as placas senis, compostas pelo peptídeo beta-amilóide (Aβ). O peptídeo Aβ tem sido considerado o principal responsável pelos processos de disfunção sináptica, morte neuronal e consequente declínio cognitivo dos pacientes com DA. Diversos processos estão envolvidos na formação deste peptídeo, que uma vez formado desencadeia uma cascata de eventos que envolvem alterações na sinalização celular, ativação da neuroinflamação e geração de um desbalanço na produção de espécies reativas de oxigênio nas organelas presentes nos neurônios culminando na disfunção sináptica e morte celular. Apesar do intenso estudo sobre a fisiopatologia da DA observado nas últimas décadas, ainda não existe o conhecimento completo sobre as causas e sobre o mecanismo que levam a progressão da doença. Os receptores nicotínicos estão presentes no sistema nervoso e, de uma forma geral, são responsáveis pela liberação de neurotransmissores importantes para uma diversidade de processos cognitivos. O estresse oxidativo e a disfunção mitocondrial estão correlacionados a um ciclo vicioso de desequilíbrio que desempenha um importante papel na patogênese da DA. Por isto, neste trabalho procuramos avaliar o efeito da nicotina em um modelo in vitro de toxicidade do peptideo Aß em cultura organotípica de hipocampo de ratos, bem como avaliar os efeitos desta droga sobre as proteínas relacionadas com a neurodegeneração, AKT e GSK-3ß . Além disso, avaliamos as alterações mitocondriais que a nicotina ocasionou em culturas organotipicas. Para isso, as culturas foram expostas ao peptideo Aß e á nicotina concomitantemente por um período de 48h. A morte celular foi aval iada a partir da incorporação do iodeto de propídeo, um marcador de morte de células principalmente em processo de necrose. As cul turas expostas ao peptídeo Aß quando tratadas com nicotina não apresentaram prevenção da morte celular. A nicotina não teve efeito sobre a fosforilação das proteínas AKT e GS K-3ß. através da análise obtida por Western blotting. Entretanto, na análise real izada por citometria de fluxo, utilizando as sondas mitocondriais MitoTracker® e MitoSOX®, observamos um aumento na produção do ânion superóxido, bem como uma diferença na massa mitocondrial nas culturas expostas ao peptideo com nicotina. Alzheimer's Disease (AD) is the most common neurodegenerative disorder and the leading cause of age-related dementia. The growing numbers of people affected by AD are directly linked to the increase in life expectancy of the world population. The AD, because it is a multifactorial disease, involving genetic, molecular and environmental factors. The main pathophysiological changes, today object of studies, are the neurofibrillary tangles formed by the hyperphosphorylated tau protein and the senile plaques, composed of the betaamyloid peptide (Aß). The Aß peptide has been considered the main responsible for synaptic dysfunction processes, neuronal death and consequent cognitive decline in AD patients. Several processes are involved in the formation of this peptide, which once formed triggers a cascade of events that involve alterations in cell signaling, activation of neuroinflammation and generate an imbalance in the production of reactive oxygen species in the organelles present in neurons culminating in synaptic dysfunction and cell death. Despite the intense study of the pathophysiology of AD observed in recent decades, there is still no complete knowledge about the causes and the mechanisms that lead to disease progression. Nicotinic receptors are present in the nervous system and, in general, are responsible for the release of neurotransmitters important for a variety of cognitive processes. Oxidative stress and mitochondrial dysfunction is correlated to a vicious circle of imbalance plays an important role in the pathogenesis of AD. Therefore, in this study we evaluated the effect of nicotine on a model in vitro toxicity of Aß peptide in organotypic culture in rat hippocampus, and to evaluate the effects of this drug on proteins related to neurodegeneration, Akt and GSK-3β. In addition, we evaluated the mitochondrial alterations that nicotine caused in organotypic cultures. For this, the cultures were exposed to the Aß-peptide and nicotine concomitantly for a period of 48h. Cell death was evaluated from the incorporation of propidium iodide, a marker of cell death mainly in necrosis process. Cultures exposed to Aß peptide when treated with nicotine did not show any preventing cell death. Nicotine had no effect on the phosphorylation of Akt and GSK-3β protein, obtained through the analysis by Western blotting. However, the analysis performed by flow cytometry using the mitochondrial probe MitoTracker® and MitoSOX®, an increase in the production of superoxide anion as well as a difference in mitochondrial mass in peptide cultures exposed to nicotine.

Premio extraordinario de Trabajo Fin de Máster curso 2014-2015. Investigación Biomédica Traslacional Las gotas lipídicas (LDs) son orgánulos intracelulares que coordinan el almacenamiento y la movilización de los lípidos, siendo ambos procesos especialmente importantes en las células especializadas en la gestión de estos compuestos, los adipocitos. En estos procesos participan activamente otros orgánulos celulares, en interacción con las LDs, como el retículo endoplásmico (RE), las mitocondrias y los peroxisomas. Concretamente, existen zonas de contacto entre el RE y las mitocondrias, denominadas membranas asociadas a mitocondrias (MAMs), en las cuáles tiene lugar el transporte no vesicular de lípidos en ambos sentidos. Por este motivo, se propone que este sistema sirva, también, para suministrar lípidos a las LDs así como para enviar lípidos hacia las mitocondrias para su β-oxidación o para llevar a cabo etapas intermedias en la síntesis de triglicéridos y fosfolípidos. De esta forma, se garantiza el turnover necesario a la hora de acumular lípidos en las LDs. Análisis proteómicos de LDs previos han identificado a la GTPasa de bajo peso molecular Rab18 como un componente de la superficie de la LD. Rab18 está involucrada en la regulación de los procesos de lipogénesis mediada por insulina y en la lipólisis inducida por estímulos β- adrenérgicos, probablemente facilitando la interacción de las LDs con membranas del RE y el intercambio de lípidos entre estos compartimentos. En este trabajo, se ha estudiado la localización intracelular de Rab18 en relación con mitocondrias y con las regiones especializadas de la membrana del RE que interaccionan con ellas, las MAMs. Mediante estudios bioquímicos y de imagen, se muestra que Rab18 está presente en toda la superficie de las LDs así como en áreas concretas de contacto con las mitocondrias que podrían corresponder con las MAMs. En conjunto, estos datos apoyan la idea de que Rab18 podría facilitar el acercamiento y/o anclaje de LDs y mitocondrias a MAMs en adipocitos 3T3-L1. Además, el presente trabajo ha contribuido al desarrollo y validación de un método optimizado para establecer el lipidoma de LDs aisladas de adipocitos 3T3-L1, que permitirá conocer con detalle la composición lipídica de estos orgánulos y determinar los cambios que tienen lugar en células en respuesta a cambios en los niveles de expresión de Rab18. Los resultados obtenidos en el presente trabajo ayudarán a comprender mejor la función específica de esta GTPasa en relación al metabolismo lipídico y a los cambios adaptativos que experimenta en condiciones patológicas como obesidad y resistencia a insulina.

O cobre (Cu) é um metal essencial à vida, porém é toxico quando em altas concentrações. Seus efeitos incluem redução de crescimento e alteração no metabolismo energético. Portanto, o objetivo do presente estudo foi avaliar os efeitos da exposição crônica ao Cu sobre o metabolismo mitocondrial e a regulação do eixo somatotrópico em diferentes tecidos do teleósteo Poecilia vivipara através da avaliação da expressão dos genes que codificam o GH, GHR1, GHR2, IGF1, IGF2, COX I, COXII, COXIII e ATP5A1, bem como através da análise da concentração de GH e GHR. Peixes recém-nascidos (

En las plantas, el término “senescencia” se utiliza para aludir al deterioro estructural y funcional que sufren muchos órganos en la fase terminal de su desarrollo. En particular, en las especies anuales todos los órganos vegetativos atraviesan un proceso generalizado de senescencia durante el final del desarrollo reproductivo (“senescencia monocárpica”). Se ha estudiado extensamente la senescencia de las hojas y flores, pero se ha desatendido la de otros órganos como las raíces. Como resultado del énfasis puesto en la senescencia foliar, la mayoría de los estudios se han focalizado en el desmantelamiento de los cloroplastos. Son escasos los estudios sobre las modificaciones que ocurren durante el envejecimiento en otras organelas, particularmente las mitocondrias. El objetivo general de la presente tesis fue determinar modificaciones funcionales y estructurales de mitocondrias durante la senescencia de hojas y raíces. De acuerdo a los resultados obtenidos, Arabidopsis thaliana sería un caso intermedio entre una planta monocárpica y una planta de crecimiento continuo, o por lo menos no presentó los síntomas de senescencia de una planta monocárpica típica. Estos resultados condujeron a realizar ensayos también en una especie monocárpica típica, como la soja (Glycine max). El patrón de cambio en el metabolismo radical a lo largo del desarrollo reproductivo fue similar en Arabidopsis thaliana y en soja. Esto indicaría que la actividad radical no está ligada a un proceso de senescencia como el que sufren los tejidos aéreos, y que es moderadamente distinto en Arabidopsis y soja. Las raíces mantuvieron su actividad metabólica y acumularon biomasa aún durante el período de crecimiento de las semillas. En plantas desfrutadas (plantas de soja a las cuales se les extrajo los frutos) las raíces parecerían ser el destino de fotoasimilados más importante, ya que incrementaron su biomasa 4,7 veces respecto del estadio inicial (es decir, en 54 días). La falta de compensación entre la cantidad de tejido producido y la actividad metabólica en estos tratamientos serían indicadores de desajustes metabólicos, y la AOX (Oxidasa Alternativa) podría cumplir una función de “alivio” de las presumiblemente grandes cantidades de productos reducidos de la fotosíntesis (fotosintatos) presentes en las raíces de las plantas desfrutadas. La disminución de la cantidad de proteína total y mitocondrial hacia las etapas finales del período de crecimiento de las semillas, podría indicar que existen procesos de exportación de N desde las raíces. El decaimiento de la actividad respiratoria de las raíces posterior a la muerte de las hojas, sugeriría que las raíces experimentan un envejecimiento por falta de fotoasimilados. Para estudiar este tema se realizaron ensayos en plantas inducidas a senescer en oscuridad. En plantas de Arabidopsis que fueron sometidas a oscuridad contínua aumentó la proporción de biomasa de las rosetas, respecto de las raíces. En las raíces decrecieron la actividad respiratoria, la cantidad de proteína mitocondrial y el número total de mitocondrias. Las mitocondrias de menor tamaño son la que se encontraron en mayor abundancia y las que más cambiaron en su cantidad debido a los tratamientos de oscuridad, por lo que parecería ser las que más contribuyen a la actividad respiratoria radical. En oscuridad disminuyó más marcadamente el número de mitocondrias activas que el de mitocondrias totales, lo que parece indicar que existieron conjuntos de mitocondrias con actividades basales o en “standby”. El contenido de azúcares solubles disminuyó, y esto podría explicar las caídas de las tasas metabólicas por un efecto de hambreado. El aumento de la respiración radical por el agregado temporario de glucosa confirmaría esta hipótesis. El estudio con mutantes KO en la vía autofágica (atg5-KO) de Arabidopsis thaliana no arrojó resultados concluyentes respecto de la participación de esta vía en la degradación de las mitocondrias radicales. De acuerdo a los datos obtenidos se puede concluir que se cumplió con el objetivo planteado en términos generales. También se ha aportado información respecto a temas poco estudiados como la fisiología de las raíces y las mitocondrias en el contexto de la senescencia., Facultad de Ciencias Naturales y Museo

La captación de calcio mitocondrial esta regulada por diferentes proteínas entre las que se encuentran las formadoras del complejo del uniportador de calcio mitocondrial, MCU, MICU1 y MICU2. MCU es la proteína formadora del poro del canal mientras que MICU1 y MICU2 son proteínas reguladoras. En esta tesis se ha estudiado el papel específico de estas dos proteínas reguladoras mediante la generación de clones estables con estas proteínas silenciadas. Estos clones han sido utilizados para estudiar la dinámica de calcio celular con Aequorina, una fotoproteína sensible a calcio. De este modo se han realizado medidas de calcio en mitocondria, retículo endoplásmico y citosol que han permitido encontrar la función especifica de MICU1 y MICU2 en la captación de calcio mitocondrial. MICU1 actúa como sensor de calcio cuando la concentración de calcio citosólico es baja y como activador del canal cuando la concentración del ion es alta. En cambio MICU2 actual como inhibidor del canal cuando la concentración de calcio en el citosol es baja., Departamento de Bioquímica y Biología Molecular y Fisiología

Producción Científica, Aging is associated to cognitive decline and susceptibility to neuron death, two processes related recently to subcellular Ca2+ homeostasis. Memory storage relies on mushroom spines stability that depends on store-operated Ca2 + entry (SOCE). In addition, Ca2+ transfer from endoplasmic reticulum (ER) to mitochondria sustains energy production but mitochondrial Ca2+ overload promotes apoptosis. We have addressed whether SOCE and ER-mitochondria Ca2+ transfer are influenced by culture time in long-term cultures of rat hippocampal neurons, a model of neuronal aging. We found that short-term cultured neurons show large SOCE, low Ca2+ store content and no functional coupling between ER and mitochondria. In contrast, in long-term cultures reflecting aging neurons, SOCE is essentially lost, Stim1 and Orai1 are downregulated, Ca2+ stores become overloaded, Ca2+ release is enhanced, expression of the mitochondrial Ca2+ uniporter (MCU) increases and most Ca2 + released from the ER is transferred to mitochondria. These results suggest that neuronal aging is associated to increased ER-mitochondrial cross talking and loss of SOCE. This subcellular Ca2+ remodeling might contribute to cognitive decline and susceptibility to neuron cell death in the elderly., Ministerio de Economía, Industria y Competitividad (grants BFU2012-37146 and BFU2015-70131R), Junta de Castilla y León (grants VA145U13 and BIO/VA33/13)

The present work was focused in the study of mitochondrial function during sepsis. Moreover, was evaluated the properties of melatonin against mitochondrial failure during sepsis. The objectives of this research were: 1. To determine the mitochondrial bioenergetics in liver and heart of mice at early and late phases of sepsis. 2. To identify mitochondrial substrate-dependent behavior during septic process in these tissues. 3. To assess the protective effect of melatonin administration to mice on mitochondrial function of liver and heart of septic mice. 4. To evaluate the mechanistics effects of melatonin on mitochondria using different substrate combinations in liver and heart of septic mice., The present thesis reports on the study of mitochondrial function (of 3 mo. C57BL/6 mice) in liver and heart mouse at 8 and 24 h of sepsis induction by cecal ligation and puncture (CLP). Mainly, mitochondrial function was evaluated assessing mitochondrial respiration by high resolution respirometry (HRR). Other bioenergetic parameters including cytochrome a + a3, b, and c + c1 content, mitochondrial mass, and mitochondrial supercomplexes (SC) formation, were analyzed. Regarding to an effective treatment, we assess melatonin, a recognize molecule with important pleiotropic functions., Tesis Univ. Granada. Programa Oficial de Doctorado en: Biomedicina, Durante la realización del siguiente trabajo, la Lda. Carolina Anneliese Doerrier Velasco fue financiada por las siguientes fuentes: RETICEF (2009-2011); Beca del programa de doctorado en Biomedicina, Contrato de técnico de apoyo investigación FIBAO (2011-2013); Proyecto P07-CTS-03135 de la Junta de Andalucíal, Consejería de Innovación, Ciencia y Empresa; Proyecto PI08-1664 de ISCIII.

Capybara is the largest rodent in the world and displays a seasonally dependent herbivore feeding behavior. Here, we present an anatomical contribution for understand this fact, by light, scanning, and transmission electron microscopy methodologies for tongue tissue analysis. The histological preparations revealed filiform, fungiform, vallate, and foliate papillae on the dorsal mucosa of the capybara tongue. The epithelial layer exhibited a lining of keratinized stratified squamous epithelial cells. The lamina propria was characterized by a dense connective tissue composed of the primary and secondary papillar projections. We also revealed the original aspects of the connective papillae. The shapes of the papillae varied by region of the tongue, and filiform, fungiform, vallate, and foliate papillae and subjacent layers of muscular fibers were observed. Pyriform taste buds occupying the epithelial layer of fungiform, vallate and foliate papillae were identified and the intracellular components of the taste buds and the intracorpuscular amyelinated nerve fibers were observed. The taste buds were characterized by the distribution of granular endoplasmic reticulum throughout the perinuclear area, the Golgi apparatus, and mitochondrial assemblies of various distinct diameters. Mitochondrial accumulation was also observed in the collagen bundle-surrounded amyelinated nerve fibers beside the basal cells. Therefore, these peculiar anatomical descriptions may contribute to understanding the adaptation of the feeding behavior of capybaras in a seasonally changing environment.

We aim in this study to characterize the effect of cations and polycations on the formation of hybrid bilayer membranes (HBMs), especially those that mimic the inner mitochondrial membrane (IMM), with a proper composition of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and cardiolipin (CL) adsorbed on an alkanethiol monolayer. HBMs are versatile membrane mimetics that show promising results in sensor technology. Its formation depends on the fusion of vesicles on hydrophobic surfaces, a process that is not well understood at the molecular level. Our results showed to which extend and in which condition the presence of cations and polycations facilitate the formation of HBMs. The required time for lipid layer formation was reduced several times and the lipid layer reaches the expected thickness of 19.5±1.8 Ǻ, in contrast to only 2±1.5 Ǻ usually observed in the absence of cations. In the presence of specific concentrations of spermine and Ca(2+) the amount of adsorbed phospholipids on the thiol layer increased nearly 70% compared to that observed when Na(+) was used at concentrations 10 times higher. Divalent cations and polycations adsorb specifically on the lipid headgroups destabilizing the hydration forces, facilitating the process of vesicle fusion and formation of lipid monolayers. The concepts and conditions described in the manuscript will certainly help the development of the field of membrane biosensors.

CHCHD10-related diseases include mitochondrial DNA instability disorder, frontotemporal dementia-amyotrophic lateral sclerosis (FTD-ALS) clinical spectrum, late-onset spinal motor neuropathy (SMAJ), and Charcot-Marie-Tooth disease type 2 (CMT2). Here, we show that CHCHD10 resides with mitofilin, CHCHD3 and CHCHD6 within the mitochondrial contact site and cristae organizing system (MICOS) complex. CHCHD10 mutations lead to MICOS complex disassembly and loss of mitochondrial cristae with a decrease in nucleoid number and nucleoid disorganization. Repair of the mitochondrial genome after oxidative stress is impaired in CHCHD10 mutant fibroblasts and this likely explains the accumulation of deleted mtDNA molecules in patient muscle. CHCHD10 mutant fibroblasts are not defective in the delivery of mitochondria to lysosomes suggesting that impaired mitophagy does not contribute to mtDNA instability. Interestingly, the expression of CHCHD10 mutant alleles inhibits apoptosis by preventing cytochrome c release., We acknowledge Pasteur-IRCAN Cellular and Molecular Imaging platform (PICMI). This work was made possible by grants to VP-F from the Fondation pour la Recherche Medicale (FRM) and the Association Francaise contre les Myopathies (AFM), to HS from National Institutes of Health (GM089853) and to J-ER from la Fondation ARC pour la Recherche sur le Cancer. PYWM is supported by a Clinician Scientist Fellowship Award (G1002570) from the Medical Research Council (UK) and also receives funding from Fight for Sight (UK) and the UK National Institute of Health Research (NIHR) as part of the Rare Diseases Translational Research Collaboration. CV-B is supported by the Spanish Instituto de Salud Carlos III (CP11/00046). MP is granted by an FRM fellowship (DEA20130727390).

The ruthenium compound [Ru2Cl(Ibp)4] (or RuIbp) has been reported to cause significantly greater inhibition of C6 glioma cell proliferation than the parent HIbp. The present study determined the effects of 0–72 h exposure to RuIbp upon C6 cell cycle distribution, mitochondrial membrane potential, reactive species generation and mRNA and protein expression of E2F1, cyclin D1, c-myc, pRb, p21, p27, p53, Ku70, Ku80, Bax, Bcl2, cyclooxygenase 1 and 2 (COX1 and COX2). The most significant changes in mRNA and protein expression were seen for the cyclin-dependent kinase inhibitors p21 and p27 which were both increased (p < 0.05). The marked decrease in mitochondrial membrane potential (p < 0.01) and modest increase in apoptosis was accompanied by a decrease in anti-apoptotic Bcl2 expression and an increase in pro-apoptotic Bax expression (p < 0.05). Interestingly, COX1 expression was increased in response to a significant loss of prostaglandin E2 production (p < 0.001), most likely due to the intracellular action of Ibp. Future studies will investigate the efficacy of this novel ruthenium–ibuprofen complex in human glioma cell lines in vitro and both rat and human glioma cells growing under orthotopic conditions in vivo.

Protoporphyrin (Pp IX) derivatives were prepared to study the relationship between photosensitizer structure and photoactivity, with an emphasis on understanding the role of membrane interactions in the efficiency of photosensitizers used in photodynamic therapy (PDT). The synthetic strategies described here aimed at changing protoporphyrin periferic groups, varying overall charge and oil/water partition, while maintaining their photochemical properties. Three synthetic routes were used: (1) modification of Pp IX at positions 31 and 81 by addition of alkyl amine groups of different lengths (compounds 2–5), (2) change of Pp IX at positions 133 and 173, generating alkyl amines (compounds 6 and 7, a phosphate amine (compound 8, and quarternary ammonium compounds (compounds 9 and 10), and (3) amine-alkylation of Hematoporphyrin IX (Hp IX) at positions 31, 81, 133 and 173(compound 12). Strategy 1 leads to hydrophobic compounds with low photocytotoxicity. Strategy 2 leads to compounds 6–10 that have high levels of binding/incorporation in vesicles, mitochondria and cells, which are indicative of high bioavailability. Addition of the phosphate group (compound 8), generates an anionic compound that has low liposome and cell incorporation, plus low photocytotoxicity. Compound 12 has intermediate incorporation and photocytotoxic properties. Compound modification is also associated with changes in their sub-cellular localization: 30% of 8 (anionic) is found in mitochondria as compared to 95% of compound 10 (cationic). Photocytotoxicity was shown to be highly correlated with membrane affinity, which depends on the asymmetrical and amphiphilic characters of sens, as well as with sub-cellular localization.

7,8-Dihydro-8-oxoguanine DNA glycosylase (OGG1) is a major DNA glycosylase involved in base-excision repair (BER) of oxidative DNA damage to nuclear and mitochondrial DNA (mtDNA). We used OGG1-deficient (OGG1−/–) mice to examine the possible roles of OGG1 in the vulnerability of neurons to ischemic and oxidative stress. After exposure of cultured neurons to oxidative and metabolic stress levels of OGG1 in the nucleus were elevated and mitochondria exhibited fragmentation and increased levels of the mitochondrial fission protein dynamin-related protein 1 (Drp1) and reduced membrane potential. Cortical neurons isolated from OGG1−/– mice were more vulnerable to oxidative insults than were OGG1+/+ neurons, and OGG1−/– mice developed larger cortical infarcts and behavioral deficits after permanent middle cerebral artery occlusion compared with OGG1+/+ mice. Accumulations of oxidative DNA base lesions (8-oxoG, FapyAde, and FapyGua) were elevated in response to ischemia in both the ipsilateral and contralateral hemispheres, and to a greater extent in the contralateral cortex of OGG1−/– mice compared with OGG1+/+ mice. Ischemia-induced elevation of 8-oxoG incision activity involved increased levels of a nuclear isoform OGG1, suggesting an adaptive response to oxidative nuclear DNA damage. Thus, OGG1 has a pivotal role in repairing oxidative damage to nuclear DNA under ischemic conditions, thereby reducing brain damage and improving functional outcome.

Aims In the present work we investigated the in vitro effect of cis -4-decenoic acid, the pathognomonic metabolite of medium-chain acyl-CoA dehydrogenase deficiency, on various parameters of bioenergetic homeostasis in rat brain mitochondria. Main methods Respiratory parameters determined by oxygen consumption were evaluated, as well as membrane potential, NAD(P)H content, swelling and cytochrome c release in mitochondrial preparations from rat brain, using glutamate plus malate or succinate as substrates. The activities of citric acid cycle enzymes were also assessed. Key findings cis -4-decenoic acid markedly increased state 4 respiration, whereas state 3 respiration and the respiratory control ratio were decreased. The ADP/O ratio, the mitochondrial membrane potential, the matrix NAD(P)H levels and aconitase activity were also diminished by cis -4-decenoic acid. These data indicate that this fatty acid acts as an uncoupler of oxidative phosphorylation and as a metabolic inhibitor. cis -4-decenoic acid also provoked a marked mitochondrial swelling when either KCl or sucrose was used in the incubation medium and also induced cytochrome c release from mitochondria, suggesting a non-selective permeabilization of the inner mitochondrial membrane. Significance It is therefore presumed that impairment of mitochondrial homeostasis provoked by cis -4-decenoic acid may be involved in the brain dysfunction observed in medium-chain acyl-CoA dehydrogenase deficient patients.