Dissertationen zum Thema „Adaptation mitochondriale“

Malgré la quasi-normalisation de sa fonction cardiaque, le sujet transplanté cardiaque présente généralement une limitation à l'effort physique. Ce handicap a été attribué en partie à des altérations du métabolisme énergétique musculaire périphérique, dont les causes précises restent encore à élucider. Cependant, le traitement immunosuppresseur (la ciclosporine en particulier) et le déconditionnement physique, ont été fortement suspectés d'être à l'origine des anomalies musculaires du transplanté. L'objectif de ce travail de thèse était donc de déterminer les parts respectives de la ciclosporine et du déconditionnement dans cette limitation physique. Pour ce faire, nous avons étudié les caractéristiques phénotypiques et fonctionnelles de leurs muscles squelettique et cardiaque, et évalué les effets d'un entraînement en endurance sur ces divers paramètres. Nos résultats démontrent que les capacités oxydatives musculaires du sujet transplanté cardiaque ne varient pas significativement près d'un an après transplantation et demeurent similaires à celles de sujets sédentaires sains appariés en âge. Cependant, in vitro, la CsA (Sandimmun), entraîne une diminution de ces capacités oxydatives, entièrement attribuable au composant majeur de son excipient, le Crémophor. Cet effet délétère est tissue-spécifique et s'exerce via une altération spécifique de l'activité fonctionnelle de certains complexes de la chaîne respiratoire mitochondriale. Par ailleurs, l'administration chronique de la molécule active de CsA, sans son excipient, entraîne au contraire, une augmentation de la capacité oxydative du muscle squelettique lent oxydatif. L'entraînement en endurance de ces sujets induit une augmentation significative de la capacité oxydative du muscle squelettique (dont la valeur devient même similaire à celle du sujet sain sportif) associée à une activation fonctionnelle de la créatine kinase mitochondriale. L'ensemble de notre travail suggère que la fonction mitochondriale musculaire et ses mécanismes de régulation soient préservés in vivo, malgré le traitement immunosuppresseur clinique. D'autres acteurs, extramitochondriaux, tels que le réseau capillaire et certaines voies de signalisation impliquées dans la transition phénotypique musculaire, seraient les cibles préférentielles des immunosuppresseurs
Despite a near-normalization of their cardiac function, heart transplant recipient displayed a limited physical capacity, at maximal exercise. This physical limitation has been in part attributed to altered energetical metabolism in peripheral muscle, which precise mechanisms are still undetermined. However, immunosuppressive treatment, particularly cyclosporin A, and deconditionning have been closely suspected to be the major causes of muscular abnormalities after cardiac transplantation. The aim of this study was to determine the respective contribution of cyclosporin A and deconditionning in physical limitation of heart transplant recipient. To this end, we have characterized the phenotypic and functional properties of their skeletal and cardiac muscles, and have determined the effects of an endurance training program on these various parameters. Our results demonstrate that the intrinsic properties of muscular mitochondria, remain normal in heart transplant recipient, long after transplantation, either at the level of quantitative functional characteristics than at their adaptative mechanisms of regulation of cellular energy production and transfer. This study suggests that the muscular mitochondrial function and its adaptative mechanisms are not directly affected in vivo by clinical immunosuppressive treatment. Others non-mitochondrial actors such as the capillary network and some signaling pathways implicated in phenotypic muscular transition, should represent preferential targets of immunosuppressive drugs in peripheral muscle of heart transplant recipient

Le cancer du sein triple négatif (TNBC) est un sous-type particulièrement agressif du cancer du sein, traité principalement par chimiothérapie. Cependant, environ 50% des patients connaissent une rechute avec métastases dans les 3 à 5 ans suivant le traitement. Afin de mieux comprendre l'évasion post-chimiothérapie et la formation de métastases des cellules cancéreuses TNBC, nous avons établi des modèles de cellules TNBC en traitant les cellules SUM159-PT et MDA-MB-231 avec de l'épirubicine, du cyclophosphamide et du paclitaxel, simulant des protocoles cliniques. Nous nous sommes initialement concentrés sur l'adaptation mitochondriale de ces cellules persistantes. Les cellules MDA-MB-231 ont montré une sensibilité réduite à la chimiothérapie, associée à une phosphorylation oxydative accrue et à des intermédiaires du cycle de l'acide tricarboxylique modifiés. En revanche, les cellules SUM159-PT ont conservé leur sensibilité. Le ciblage du métabolisme mitochondrial du pyruvate avec le UK-5099 a resensibilisé les cellules persistantes MDA-MB-231 aux agents thérapeutiques. Les cellules persistantes ont montré une migration, une invasion et une survie accrues en culture en suspension, les cellules SUM159-PT présentant une adhésion accrue aux cellules endothéliales. Des études de xénogreffe in vivo ont confirmé ces observations, mettant l'accent sur une croissance cellulaire accrue et une colonisation métastatique dans des organes vitaux, en particulier le cerveau. Le tropisme accru pour le cerveau pourrait s'expliquer par le fait que les cellules TNBC persistantes présentaient une capacité accrue de traverser la barrière hémato-encéphalique, d'envahir le parenchyme cérébral et de croître dans une matrice 3D similaire au cerveau. Dans une deuxième phase de notre étude, nous avons étudié les mécanismes moléculaires facilitant la formation de métastases cérébrales de ces cellules persistantes. L'analyse protéomique a identifié des protéines surexprimées, notamment le COL1A1, fréquemment élevé chez les patients atteints de TNBC. Une augmentation de COL1A1 était corrélée à un mauvais pronostic et à une augmentation de la formation de métastase. L'inhibition de COL1A1 a réduit le potentiel métastatique à la fois in vitro et in vivo, soulignant son potentiel en tant que cible thérapeutique pour prévenir les métastases cérébrales après un traitement par chimiothérapie.L'ensemble de ces résultats offre un aperçu des mécanismes d'adaptation mis en place par les cellules cancéreuses en réponse à la chimiothérapie, et suggère que cibler le métabolisme du pyruvate mitochondrial pourrait contribuer à surmonter les adaptations mitochondriales des cellules de cancer du sein triple négatif. De plus, nos résultats mettent en lumière la manière dont une chimiothérapie combinée et séquentielle peut accroître le potentiel métastatique des cellules TNBC, en particulier vers le cerveau. Nous avons identifié la protéine COL1A1 comme un élément clé favorisant les différentes étapes de formation des métastases cérébrales dans les cellules TNBC résistantes à la chimiothérapie. Des recherches complémentaires sont nécessaires pour élucider les mécanismes détaillés de la surexpression de COL1A1.En utilisant le même schéma thérapeutique, nous avons mis en œuvre un traitement court de 48h, combiné et séquentiel pour évaluer les modifications protéomique précoces dans les vésicules extracellulaires libérées par les cellules TNBC persistantes. Avec cette approche, on a également exploré l'impact de la chimiothérapie sur les facteurs angiocrines des cellules endothéliales, suggérant le rôle du sécrétome induit par la chimiothérapie dans la facilitation des métastases post-chimiothérapie. Bien que ce volet de notre étude en soit à un stade préliminaire, les résultats encouragent à approfondir davantage l'investigation expérimentale
Triple negative breast cancer (TNBC) is a highly aggressive breast cancer subtype, primarily treated with chemotherapy. However, approximately 50% of patients experience relapse with metastasis within 3 to 5 years post-treatment. To gain insight into the post-chemotherapy escape and metastasis formation of TNBC cancer cells, we established TNBC cell models by treating SUM159-PT and MDA-MB-231 cells with epirubicin, cyclophosphamide, and paclitaxel. simulating clinical protocols. We initially focused on the mitochondrial adaptation of these chemo-persistent cells. MDA-MB-231 cells showed reduced chemosensitivity, associated with increased oxidative phosphorylation and altered tricarboxylic acid cycle intermediates. In contrast, SUM159-PT cells retained sensitivity. Targeting mitochondrial pyruvate metabolism with UK-5099 re-sensitized persistent cells to therapeutic agents, suggesting a potential strategy to overcome mitochondrial adaptation. Persistent cells exhibited increased migration, invasion, survival in suspension culture, with SUM159-PT cells displaying increased adhesion to endothelial cells. In vivo xenograft studies confirmed these observations, emphasizing increased cell growth and metastatic colonization in vital organs, particularly the brain. The enhanced trophism for brain could be explained by the fact that persistent TNBC cells exhibited increased abilities to transmigrate through BBB, to invade the brain parenchyma and to grow in a brain-like 3D matrix. In a second phase of our study, we investigated the molecular mechanisms facilitating brain metastasis of these persistent cells. proteomic analysis identified upregulated proteins, notably COL1A1, frequently elevated in TNBC patients. Increased COL1A1 correlated with poor prognosis and enhanced metastasis. Inhibition of COL1A1 reduced metastatic potential both in vitro and in vivo, highlighting its potential as a therapeutic target in preventing brain metastasis post chemotherapy treatment.Collectively, these findings provide insight into the adaptive mechanisms employed by cancer cells in response to chemotherapy, and suggest that targeting mitochondrial pyruvate metabolism may help to overcome the mitochondrial adaptations in TNBC cells. Furthermore, our data illuminate how combined and sequential chemotherapy may increase the metastatic potential of TNBC cells, particularly towards the brain. We have pinpointed COL1A1 as a key factor promoting various stages of brain metastasis formation in chemotherapy-resistant TNBC cells. Additional research is required to elucidate the detailed mechanisms behind COL1A1 overexpression.Using the identical drug regimen, we implemented a short, combined, and sequential treatment to replicate initial proteomic alterations in extracellular vesicles released by persistent TNBC cells. This approach also explored the impact of chemotherapy on angiocrine factors from endothelial cells, suggesting the role of the chemo-induced secretome in evading treatment and facilitating metastasis post-chemotherapy. Although this aspect of our study is currently in its early phases, the findings underscore the necessity for further experimental validation

Bien que les statines forment la classe d'hypolipidémiants la plus utilisée, une toxicité musculaire a été reportée, pouvant ainsi provoquer l’apparition d’une myopathie. Dans la première partie, nous avons montré chez l’Homme et l’animal que les statines inhibent directement la chaine respiratoire mitochondriale, et induisent la production de radicaux libres dérivés de l’oxygène (RLO), qui active les voies apoptotiques dans les muscles glycolytiques, alors que les muscles oxydatifs ne sont pas atteints. Nous avons ensuite montré in vitro que le stress réducteur peut engendrer une oxydation mitochondriale, pouvant conduire à une activation de la voie de biogenèse mitochondriale. De plus l’augmentation du contenu mitochondrial induite a permis de protéger les cellules contre l’apoptose induite par les statines. Enfin, nous avons montré in vivo que l’induction des voies de biogenèse mitochondriale est nécessaire à la tolérance des statines dans les muscles oxydatifs. En conclusion, le phénotype mitochondrial, tant au niveau quantitatif que qualitatif, semble être un facteur clé dans l’apparition de la myopathie aux statines
Although statins are the most prescribed class of lipid-lowering agents, adverse muscular toxicity has been reported, which can lead to the appearance of a myopathy. In the first part, we showed in Humans and animals that statins inhibit directly the mitochondrial respiratory chain, and induce the production of reactive oxygen species (ROS), that trigger apoptotic pathways in glycolytic skeletal muscles, whereas oxidative muscles are not impaired. We then showed in vitro that reductive stress can provoke mitochondrial oxidation, that could lead to an activation of mitochondrial biogenesis pathways. Moreover, the consequent increase in mitochondrial content enabled to protect cells against statin-induced apoptosis. Finally, we showed in vivo that the induction of mitochondrial biogenesis is necessary for statin tolerance in oxidative skeletal muscles. In conclusion, mitochondrial phenotype, both quantitatively and qualitatively, seems to be a key factor in the appearance of statin myopathy

Les mécanismes impliqués dans les adaptations du phénotype métabolique musculaire au cours de l’exercice physique restent imparfaitement connus. Nous nous sommes intéressés au concept d’hormèse mitochondriale qui se définit comme un stress métabolique activant les voies de signalisation menant à une activation mitochondriale.En première partie, nous avons validé l’utilisation d’un nouveau système de mesure des échanges gazeux chez le rat au cours de différents exercices sur tapis roulant, et démontré que pour une vitesse de course sous maximale, un exercice en descente sollicite le système cardiovasculaire de façon modérée sans altérer la fonction mitochondriale musculaire, ni augmenter la production de radicaux libres oxygénés.En deuxième partie, nous avons montré qu’un découplage mitochondrial provoqué par un traitement des rats au 2,3-dinitrophénol (DNP) pendant 3 semaines engendre des adaptations métaboliques menant à l’augmentation de la masse mitochondriale du muscle squelettique. Ces animaux ont une capacité à l’exercice diminuée, malgré une augmentation de leur VO2max.Pour finir, nous avons montré qu’un préconditionnement par l’exercice protège la mitochondrie musculaire squelettique des effets délétères de l’ischémie-reperfusion. L’exercice semble activer le métabolisme via un phénomène d’hormèse mitochondriale permettant la protection musculaire. En conclusion, cette thèse nous montre d’une part l’importance de la mitochondrie (aspect quantitatif mais surtout qualitatif) en terme de limitation à l’exercice, et d’autre part nous suggère que l'optimisation du fonctionnement mitochondrial pourrait être une bonne garantie pour pouvoir lutter efficacement contre les stress, notamment oxydatifs, auxquels l'organisme est soumis en (quasi)permanence
The mechanisms regulating the metabolic phenotype adaptations in skeletal muscle during physical exercise is still unknown.We studied the mitochondrial hormesis phenomenon that could be defined as a metabolic stress activating the signaling pathways leading to a mitochondrial stimulation (mitochondrial biogenesis).In the first part, we validated the utilization of a new system determining the gas exchange in rat during a treadmill exhaustive exercise. We showed that a submaximal downhill exercise activate moderately the cardiovascular system, without mitochondrial functional impairments and without any augmentation of the systemic ROS production. In the second part, we showed that a mitochondrial uncoupling following a dinitrophenol treatment during 3 weeks in rats induced some metabolic adaptations leading to a higher mitochondrial mass in skeletal muscle. The exercise capacity of these animals is reduced whereas the maximal oxygen consumption is higher.In the last part, we showed that a preconditioning protocol with an acute exercise protected the skeletal muscle mitochondria from the deleterious effects of ischemia-reperfusion. This exercise seems to activate the muscular metabolism via a phenomenon of mitochondrial hormesis activation, allowing an efficient muscular protection.In conclusion, this thesis shows the importance of the mitochondria in terms of qualitative and quantitative aspects and shows the participation of this organelle in the exercise limitation. Moreover, these works suggest that the optimization of the mitochondrial function could be a good guarantee in order to efficiently fight against oxidative stress at the level of the whole organism

Dans les cellules eucaryotes aerobies, l'energie provient essentiellement de l'atp synthetise par les phosphorylations oxydatives (oxphos) au niveau de l'atpase-atpsynthetase f0-f1. L'atp est alors exporte hors des mitochondries par le translocateur des nucleotides adenyliques (ant). Certaines sous-unites des complexes oxphos sont codees par l'adn mitochondrial (adnmt), les autres par l'adn nucleaire et importees dans les mitochondries par un mecanisme faisant intervenir le potentiel de membrane mitochondrial (pmm) etabli par les oxphos. Nous avons verifie que 2 lignees de cellules rho o humaines sont totalement depourvues d'adnmt, d'arn et de proteines codees par l'adnmt. Par contre, elles importent normalement des proteines d'origine nucleaire dans leurs mitochondries. Comment un pmm suffisant pour cette importation peut s'etablir dans les rho o en l'absence des oxphos ? nous avons montre que les rho o, depourvues des 2 sous-unites du facteur f0 de l'atpase-atpsynthetase codees par l'adnmt, ont un f1 assemble et fonctionnel pour hydrolyser de l'atp. De plus, 2 inhibiteurs specifiques de f1 (aurovertine) ou de l'ant (acide bongkrekique) reduisent la croissance et le pmm des rho o. Ainsi, la survie des rho o necessite un pmm suffisant maintenu par l'importation d'atp 4 cytosolique dans la mitochondrie par l'ant qui l'echange contre de l'adp 3 mitochondrial produit par l'atpase-f1. Nous avons egalement utilise les rho o pour determiner l'origine genetique de pathologies dues a un deficit d'un complexe oxphos. Les cellules de 3 patients atteints du syndrome de leigh avec deficit en cytochrome oxydase (cox) ont ete enucleees et fusionnees avec les rho o. L'activite cox etant restauree dans les cybrides, le deficit est localise sur l'adn nucleaire des patients. Par contre, la fusion a ete inutilisable pour determiner l'origine d'un deficit en complexe iii. En effet, le deficit est rapidement perdu dans les lymphocytes en culture suggerant un defaut heteroplasmique de l'adnmt.

Mitochondrial Ca2+ homeostasis plays a fundamental role in the regulation of several biological processes, ranging from the regulation of ATP production to the control of cell death. Recent studies have identified the multimolecular complex responsible for Ca2+ entry into mitochondria: the mitochondrial calcium uniporter (MCU) complex [1]. It is widely accepted that mitochondria actively participate in the regulation of cellular Ca2+ homeostasis by dictating the spatio-temporal properties of [Ca2+]cyt rises [2]. Mitochondrial calcium uptake, in specific cells, contributes to regulate cellular Ca2+ homeostasis acting as high-capacity fixed buffer, sequestering large amounts of Ca2+ from a subcellular domain[2]. Furthermore, one of the most important roles of mitochondrial Ca2+ uptake is the mitochondrial Ca2+-dependent control of the rate of mitochondrial adenosine triphosphate (ATP) production, the main fuel for sustaining cellular functions [3], [4]. This general picture is particularly relevant in skeletal muscle, a tissue where mitochondria produce most of the ATP required to sustain muscle contraction [3], [5]. It is thus not surprising that skeletal muscle mitochondria display the highest mitochondrial Ca2+ transients, as demonstrated by the measurement of the MCU current by patch-clamp from IMM-derived mitoplasts from different tissues [6]. Moreover, pivotal findings have highlighted the role of mitochondria as key players in the dynamic regulation of crucial signalling pathways in skeletal muscle [7], [8], involved not only in muscle contraction but also in skeletal muscle homeostasis [9], [10]. However, whether skeletal muscle mitochondria act also as a possible high capacity Ca2+ buffer remains a fundamental question on muscle physiology and diseases. Our research investigated the regulatory processes that modulate mitochondrial Ca2+ signalling in skeletal muscle. In detail, to understand the impact of changes in cytosolic Ca2+ concentrations ([Ca2+]cyt) on mitochondrial Ca2+ uptake and muscle physiology, we explored a condition where intra-fiber Ca2+ kinetics have been profoundly altered by removing parvalbumin (PV), one of the crucial cytosolic Ca2+ buffers in skeletal muscle, specifically expressed in fast twitch muscle fibers [11], [12]. To this end, as study tool, we used a PV knockout (KO) mouse model obtained from the laboratory of Prof. Beat Schwaller (Dept. of Medicine, University of Fribourg, Switzerland) [13]. PV plays an important role in skeletal muscle, acting as a temporary Ca2+ buffer (e.g. increasing the relaxation rate of fast twitch muscle contraction) [14]. To investigate the physiological role of PV in muscle fibers and in Ca2+ homeostasis, we investigated cytosolic and mitochondrial Ca2+ transients in PV KO mice compared. We observed that basal [Ca2+]cyt was not affected in PV knockout fibers, but kinetics of Ca2+ transients and Ca2+ clearance were altered. In detail, consistently with the role of PV in buffering cytosolic Ca2+, the time-to-peak and the half-relaxation time was increased in PV KO FDB fibers. Unexpectedly, however, under tetanic stimulation, PV KO FDB muscle fibers showed a decrease in [Ca2+]cyt. To explain this result we asked whether the lack of PV could induce rearrangements of one of the two main Ca2+ stores, the sarcoplasmic reticulum (SR) and mitochondria. SR Ca2+ measurements demonstrated that lack of PV increases SR Ca2+ release during stimulation. Therefore, we concluded that SR is not causative of the effect of PV removal on cytosolic Ca2+ transients. Consistently, we found no difference in the mRNA levels of RyR1, the main Ca2+ releasing channel in muscle, and on the expression of two different isoforms of SERCA in PV KO muscles compared to WT. We then focused our attention on mitochondrial Ca2+ homeostasis. The data obtained demonstrated that the lack of PV induces an increase of mitochondrial Ca2+ uptake and this is accompanied by the induction of the expression of MCU complex components, the channel responsible for Ca2+ entry in mitochondria [1], [2], [15], [16]. In addition, electron microscopy analysis demonstrated that the volume of PV KO mitochondria was doubled compared to WT with an increase of mitochondria associated to the Ca2+ release units (CRUs), suggesting a tight connection of PV expression with mitochondrial morphology and function in muscle cells. Furthermore, to further prove that mitochondria are responsible for cytosolic Ca2+ buffering in fibers lacking PV, we silenced MCU on WT and PV KO FDB fibers and we measured [Ca2+]cyt.. In WT animals, [Ca2+]cyt was not affected by the absence of MCU, while MCU silencing in PV KO fibers resulted in a significant higher [Ca2+]cyt, reinforcing the hypothesis that, while in WT animals mitochondria do not significantly buffer [Ca2+]cyt, mitochondria of fibers lacking PV adapt to buffer [Ca2+]cyt increases. Moreover, since PV is one of the most downregulated “atrogenes”, the genes commonly up- and down-regulated during both disuse and systemic types of atrophy [17], [18] and that mitochondrial Ca2+ controls skeletal muscle trophism [10], the role of PV in the regulation of muscle mass was investigated through denervation experiments. In PV KO muscles, loss of muscle mass caused by denervation is reduced compared to WT fibers, demonstrating that the lack of PV can partially protect muscles from denervation-induced atrophy. Since the effect of PV ablation on denervated muscle was modest and the effect on innervated muscles was negligible, we decided to perform PV acute silencing and overexpression in adult WT tibialis anterior (TA) muscles and we monitored fiber size. We demonstrated that the acute modulation of PV protein controls skeletal muscle size. In detail, we observed an increase of fiber size in PV silenced muscles and coherently, PV overexpressing muscles displayed an atrophic phenotype. Since the regulation of muscle size involves a precise transcriptional program [18], [19], we focused our attention on PGC-1α4, a splicing variant of the PGC-1α gene, that plays a key role in triggering muscle hypertrophy as adaptive response to exercise [20]. Intriguingly, we found an up-regulation of PGC-1α4 mRNA in PV KO skeletal muscles, suggesting the activation of this hypertrophic pathway. Of note, our data are in accordance with previous studies showing that mitochondrial Ca2+ positively regulates skeletal muscle mass by impinging also on PGC-1α4 pathway [10]. Our results show that the lack of PV in skeletal muscle leads to morphological and functional adaptations of mitochondria. In particular, mitochondria of fibers lacking PV, either constitutively or transiently, adapt to take up more Ca2+ to control [Ca2+]cyt increases. Furthermore, we demonstrated that the absence of PV partially counteracts denervation atrophy by triggering the expression of PGC-1α4. Our hypothesis is that PV ablation, leading to an increase of mitochondrial Ca2+ uptake, activates mitochondrial Ca2+-dependent pathways to control skeletal muscle trophism.

L'évolution pondérale des diabétiques de type 2 (DT2) est particulière avec une propension à prendre du poids sous insuline, et à l'inverse des difficulés à en perdre lors des programmes de réduction pondérale. Nos résultats montrent qu'après un an de traitement par insuline la prise de poids est faite préférentiellement de masse maigre et suggèrent qu'elle n'augmente pas le risque vasculaire. Nos résultats montrent également qu'au stade précoce de leur maladie, les DT2 peuvent perdre la même quantité de poids que les obèses et que dans les deux cas il n'a pas d'adaptation du métabolisme énergétique corps entier ou mitochondrial pouvant expliquer le plateau pondéral observé à partir du cinquième mois. Il n'y a pas non plus d'adaptation particulière du métabolisme énergétique chez les DT2, leur métabolisme de base n'étant pas différent de celui des obèses à poids stable comme après perte de poids et l'insulinorésistance n'est pas un déterminant du métabolisme énergétique
Weight change in type 2 diabetic patients (T2D) is distinctive with an inclination towards weight gain with insulin, and conversely a difficulty in weight loss in weight reduction programmes. Our results show that after one year of insulin treatment, resultant weight gain is composed primarly of fat-free mass and it appears not to incrase cardivascular risk. Our results also show that in the early stage of their disease, T2D lose the same amount of weight as obese nondiabetic patients and that in both cases there is non whole body or mitochondrial energy metabolism adaptation that can explain the stabilisation of weight observed from the 5th month onwards. Furthermore, there is no specific adaptation of energy metabolism in T2D as their basal metabolism is not any different to that of obese nondiabetic patients or after weight loss. Insulin resistance is not a determinant of energy metabolism

Les mitochondries sont des organelles qui jouent un rôle clé dans le métabolisme cellulaire en centralisant la production d'ATP à partir de nombreux substrats via la phosphorylation oxydative (OXPHOS). Les réactions enzymatiques impliquées dans ce processus régulent la prolifération, la différenciation, l'activation et l'auto renouvellement cellulaire. Le but de mon travail a été d'identifier le rôle de l'OXPHOS dans l'hématopoïèse et les mécanismes d'adaptation métabolique des cellules sanguines de la moelle, des lymphocytes B et des thymocytes à la dysfonction mitochondriale. L'atout majeur de cette étude est la génération de deux modèles murins déficients pour les protéines mitochondriales AIF ou NDUFS4 dans le système hématopoïétique. Nous avons observé que l'absence de ces protéines entraine des dysfonctions de l'OXPHOS sévère (AIF KO) ou modérée (NDUFS4 KO), entrainant des anomalies dans le développement hématopoïétique. Dans les deux modèles, en réponse au stress métabolique induit par la dysfonction de l'OXPHOS, les cellules de moelle activent la glycolyse anaérobie et la biogenèse mitochondriale tandis que les thymocytes favorisent l'assimilation et la dégradation des acides gras. Cette étude multiparamétrique, incluant des approches in vivo, ex vivo et in vitro, souligne l'importance de l'OXPHOS et du métabolisme mitochondrial dans le développement hématopoïétique
By integrating different biochemical pathways and generating energy in form of ATP, through the electron transfer associated to oxidative phosphorylation (OXPHOS), mitochondria play a key role in cellular metabolism. In the hematopoietic cells, the mitochondrial metabolism appears implicated in proliferation, differentiation, activation and self-renewal regulation. In this context, the aim of my PhD work was to unravel the response of bone marrow (BM) cells, B-cells and thymocytes to OXPHOS dysfunction. To do that, we have developed two original hematopoietic cell-specific murine models deficient in the mitochondrial proteins AIF or NDUFS4. Severe (AIF KO) or moderate (NDUFS4 KO) OXPHOS dysfunction leads to pleiotropic consequences on hematopoietic development, including pancytopenia, BM aplasia, alterations in the development of the B-cell and erythroid lineages and T-cell developmental blockade at the immature stage. Strikingly, in response to OXPHOS dysfunction, BM cells stimulate anaerobic glycolysis and mitochondrial biogenesis, whereas thymocytes favor the assimilation and degradation of fatty acids. Overall my work, which included in vivo, ex vivo and in vitro approaches, underlines the relevance of OXPHOS and mitochondrial metabolism in the development of the hematopoietic cells

The New World Junonia butterflies include well-studied model organisms yet their phylogeny remains unresolved by traditional cox1 DNA barcodes. Sixteen Junonia mitochondrial genomes were sequenced using next generation MiSeq technology. Junonia lemonias, an Old World species, has mitochondrial genome features typical of Ditrysian Lepidoptera, and synteny is maintained throughout Junonia. Analysis of Junonia mitogenomes produced a robust phylogeny that was used with biogeographic information to infer that Junonia crossed the Pacific Ocean to invade the New World on 3 separate occasions. Junonia vestina, a high elevation species from the Andes Mountains, shows high altitude adaptation in the mitochondrial protein coding loci atp6, atp8, cox1, cob, nad1, and nad2, with the strongest effects seen in cox1 and nad1. There is some overlap between these genes with human loci that have disease associations with the same amino acid positions which could help elucidate the function of high elevation mutations in J. vestina.
February 2016

La mitochondrie est un centre de régulation métabolique à la fois intégrateur de signaux (visant à ajuster son fonctionnement selon les besoins énergétiques cellulaires) et initiateur de voies rétrogrades (permettant une réponse cellulaire à des changements d'états fonctionneles de la mitochondrie). Ce travail s'intéresse plus particulièrement au métabolisme oxydatif mitochondrial et aux voies de signalisation activées, dans les cellules HepG2, lors de deux situations de stress énergétique : le découplage mitochondrial constitue un signal conduisant les cellules à développer leur métabolisme oxydatif sans modifier la glycolyse (notamment par activation de la transcription de gènes codant pour des protéines mitochondriales). La mitochondrie est également une des cibles du traitement par glucocorticoïdes, ces hormones induisant à la fois des effets à court terme et à long terme. les effets rapides (modification de l'activité des complexes I, II et III de la chaîne respiratoire mitochondriale) sont non génomiques et impliquent la fixation de la dexamethasone sur un récepteur membranaire. Ces effets sont médiés par l'activation calcium-dépendante de la protéine p38MAPK. Les effets à long terme (augmentation de la capacité de la chaîne respiratoire) sont transcriptionnels et nécessitent le recrutement du récepteur intracellulaire classique aux glucocorticoïdes. Les modifications du fonctionnement de la chaîne respiratoire mitochondriale par les glucocorticoïdes sont induites par le recrutement graduel de différents sites de liaison aux glucocorticîdes (membranaire et intracellulaire).

From birth, throughout the entire lifespan, the myocardium is constituted by an almost fixed number of cardiomyocytes (CMs). Post-natal heart growth occurs through CM hypertrophy, and the cell achieves the adult phenot ype through profound structural, functional and metabolic maturation. Once fully developed, the heart continuously adapts its performance and structure in response to the varying requests of the organism, elicited by changes in intrinsic and environmental conditions. While acute stresses operate through reversible modulation of contractility, CMs subjected to prolonged increase in workload undergo complex structural remodelling, mostly occurring through further growth necessary to sustain the chronic elevation of mechanical load. Depending on the nature, intensity and duration of the hypertrophying stimuli, cardiac remodelling may lead to either the so-called "physiologic" (i.e., in the athletic heart), or "pathologic" hypertrophy (i.e., in pressure overload), the latter resulting, with time, in cardiac dysfunction, heart failure and death. Although the cellular and clinical phenotypes of the two conditions are different, the common tenet is that, in the initial phases, they share the same adaptive mechanisms, including increased sarcomeric deposition and enhanced/preserved contractility, both of which require increased ATP supply. Unsurprisingly, the regulation of mitochondrial function is a critical process for ATP production to match energetic demand during cell growth. Mitochondria are the CM powerhouse, and [Ca2+] operates as a primary dynamic regulator of ATP production. In CMs, Ca2+ influx into the mitochondrial matrix occurs during the systolic elevation in cytosolic Ca2+, and is mediated by the recently identified mitochondrial Ca2+ uniporter complex (MCUC). Mitochondrial Ca2+ uptake is fundamental in the acute modulation of contractility during the fight-or-flight response triggered by β-adrenergic receptor (β-AR) activation. Consistently, deletion of MCU in mice impairs exercise capacity, and reduction of functioning MCU channels in sino-atrial node cells or ventricular CMs, blunts the chronotropic or contractile response, respectively, to β-AR stimulation. Whether changes in the expression of MCUC proteins take place during cardiac diseases and, conversely, the effect of modulating mitochondrial Ca2+ uptake on myocardial remodelling is, at present not known. The aims of this thesis were to: Aim 1) identify the molecular mechanisms involved in the endogenous regulation of MCU; Aim 2) determine whether MCU has a role in physiologic and pathologic cardiac remodelling and Aim 3) develop an experimental model of cultured cardiomyocytes suited for the in vitro characterization of mitochondrial Ca2+ dynamics in prolonged observations. Results. 1. Content of mitochondrial calcium uniporter (MCU) in cardiomyocytes is regulated by microRNA-1 in physiologic and pathologic hypertrophy. In our preliminary experiments, we compared the protein levels of MCU in normal and hypertrophied hearts, and observed that changes in mitochondrial MCU protein density were not accompanied by parallel alteration in its transcriptional levels. This prompted us to investigate whether post-translational regulation of MCU might occur in myocardial remodelling. We thus focused on microRNAs (miRs), which are small, non-coding RNA sequences (18-25 nt) capable of finely tuning the expression of a variety of genes by interfering with either the stability or the translation of mRNA. By bioinformatics analysis that identified several microRNAs predicted to target MCU 3’UTR (untranslated region). Among these, we focused on miR-1 for its muscle specific expression, the critical role in the activation in cardiac hypertrophy, its conserved homology among species, and its specificity for MCU among MCUC members. Luciferase assay confirmed the prediction and identified the specific seed sequences on the MCU gene. Consistently, CMs expressing miR-1 showed decreased MCU protein content, with no alterations in mRNA expression, which resulted in significant reduction in mitochondrial Ca2+ uptake. We thus investigated whether MCU content was modulated in hypertrophic conditions associated to changes in miR-1, including: i) post-natal development, ii) moderate exercise and iii) pressure overload. By comparing neonatal and adult mouse hearts we observed that, in line with its role of repressor of fetal gene program, miR-1 expression increased during postnatal development and, coherently, MCU protein content decreased without alterations at transcriptional level. Moreover, this modulation was specific for MCU among the molecular components of MCUC, with the exceptions of mitochondrial calcium uniporter b (MCUb) mRNA, which increased. We then investigated the miR-1/MCU axis, in murine and human heart models of physiologic and pathologic hypertrophy. Physiologic hypertrophy was obtained in mice with chronic exercise protocol, which caused enlargement in cardiac size, CM cross-sectional area, and a slight increase in contractility. As compared to sedentary littermates, miR-1 expression level decreased and, consistently, MCU protein content increased. Analysis of the uniporter complex biochemistry in hearts undergone pressure-overload through transverse aortic constriction (TAC) surgery demonstrated that during the initial, compensatory hypertrophy, characterized by modest CM growth with no contractile failure, changes in miR-1 and MCU were similar to those observed in hearts from exercised mice. Remarkably, the reciprocal miR-1 and MCU modulation occurred in a clinically relevant model of cardiac hypertrophy, as shown by the analysis of human heart biopsies obtained from healthy subjects and patients with aortic stenosis-induced hypertrophy. These results suggest that, regardless of the nature of the hypertrophic stimulus (physiologic or pathologic), the initial CM adaptation to increased heart work is characterized by similar enhancement in the availability of uniporter-forming MCU molecules. Given that similar changes in the miR1/MCU axis were detected both upon exercise and compensated pathologic hypertrophy, we made the hypothesis that a common regulatory mechanism may exist. We thus focused on the β-AR system, the primary physiologic mechanism engaged in response to increased heart load, a condition in common between exercise and TAC-induced pressure-overload. Activation of β-AR signalling leads to enhancement of cytosolic Ca2+ oscillations and mitochondrial Ca2+ uptake, and is involved in the parallel activation of hypertrophic pathways, such as the Akt-FOXO cascade. Interestingly, miR-1 expression has been shown to depend on FOXO3a, suggesting that in conditions of chronic β-AR activation, the blockade of FOXO3a nuclear translocation may inhibit miR-1 increase. In support of this hypothesis, treatment of mice undergone TAC with the β1-blocker metoprolol ablated miR-1 repression and prevented accordingly the increase in MCU protein content. Altogether, our data identifies miR-1 as a novel post-translational regulator of MCU, and supports that the miR-1/MCU axis is involved in physiologic and pathologic myocardial remodelling. Future experiments will be aimed at exploiting the mechanicism of miR-1 action on MCU, as well as understanding the complete signalling pathway involved in MCU modulation. Given that miRs are well-suited therapeutic targets, as they can easily be mimicked or antagonized pharmacologically (miR mimics or antagomiRs, respectively), even with target selectivity, our study of the miR-1/MCU axis may open to the refinement of the current therapeutic approaches to treat myocardial hypertrophy. 2. MCU participates to the myocardial adaptation to hypertrophic stimuli. The observation that MCU protein content drops during long-term TAC, in which maladaptation occurs, suggested that MCU protein content fluctuates during pathologic hypertrophy. This led us to investigate whether MCU may have a role in myocardial remodelling caused by chronic increase in cardiac workload. To test this hypothesis, we sought to characterize functionally, biochemically, and morphologically the effect of modulating MCU expression level prior to exposing hearts to pressure overload through TAC. To increase the insight on cellular signalling, we used adrenergic receptor agonists to study the effect of prolonged adrenergic stimulation in cultured CMs. To study the role of MCU in cardiac adaptation to hypertrophy in vivo, we efficiently overexpressed or downregulated MCU, via AAV9 injection. Altering MCU expression did not affect cardiac structure and performance at baseline. However, in mice undergone TAC, MCU overexpression resulted in enhanced hypertrophy, as demonstrated by higher increase in cardiac mass, as compared to TAC-operated WT TAC (injected with AAV9-Empty vector). Interestingly, hypertrophic remodelling had characteristics similar to that of physiologic hypertrophy (i.e. increased capillary density, reduced fibrotic remodelling, and preserved cardiac contractility) also in the advanced stages of hypertrophy (i.e. 8 weeks). On the contrary, silenced mice subjected to TAC displayed a dramatic phenotype caused by the rapid appearance of severe maladaptive remodelling with typical hallmarks of dilated cardiomyopathy, including reduced capillary density, massive replacement fibrosis and decreased cardiac function. Altogether, these processes result in HF and increased susceptibility to sudden cardiac death already four weeks after TAC. To gain insight on the molecular mechanism whereby changes in MCU impact on stress-induced CM growth, we used neonatal rat CMs in which MCU overexpression or downregulation were obtained with adenoviral vectors. Consistently, MCU overexpression and downregulation resulted in enhanced and reduced mitochondrial calcium uptake, respectively. Interestingly, while MCU overexpression did not affect CM size and morphology at baseline, MCU KD cells displayed a significant increased area and disarranged sarcomeres. To mimic the increased sympathetic tone that characterises both physiologic and pathologic hypertrophy, we treated CMs with the onset of both adrenergic agonist norepinephrine (NE). Interestingly, MCU OE cells had a significantly enhanced increase in cell size growth. Conversely, MCU KD cells had a remarkably divergent phenotype, characterized by sarcomere disarray and activation of apoptosis. These data were intriguingly similar to the phenotype observed in MCU KD hearts developing dilated cardiomyopathy after TAC. The following analyses regarded the activation state of several pro-hypertrophic pathways. Interestingly, MCU overexpression determined faster activation of calcineurin/NFAT pathway upon adrenergic stimulation. Our data point at the participation of Akt/GSK3axis in NFAT enhanced nuclear translocation, presumably downstream of CaMKII-mediated Akt phosphorylation. Indeed, inhibition of CaMKII in MCU OE cardiomyocytes resulted in hypertrophic growth comparable to control cells. To conclude, our studies show that increased heart workload, as achieved in vivo by TAC and mimicked in cells by NE treatment, is well tolerated when MCU levels are augmented by overexpression. Conversely, MCU downregulation leads, in the same conditions, to cell death and consistently faster maladaptive cellular and tissue remodelling. These data are well in accord with our preliminary observation that MCU content, increased in the compensated hypertrophy, decreases in the advanced remodelling associated to HF. Second, we have identified the AR/CaMKII/Akt cascade as a key signalling pathway involved in myocardial hypertrophy and dependent on MCU modulation. 3. In vitro maturation of cultured neonatal cardiomyocytes. Primary neonatal CMs are a widely used cellular model in molecular cardiology, which can be maintained in culture for several days and is easily amenable to genetic manipulation. However, this cell type has important functional and structural differences with the mature CMs. These differences range from the expression of different myosin isoforms to maximize contractile performance, to changes in metabolism allowing increased ATP production to sustain higher consumption. Importantly, postnatal cellular maturation involves structures that regulate Ca2+ dynamics. In particular, in neonatal cells contraction is mostly due to Ca2+ entering through the plasmalemmal L-type Ca2+ channels (LTCC), directly triggering the activation of the sarcomeres, with little contribution from intracellular Ca2+ release from the immature SR stores. In contrast, in adult cells the plasma membrane has fully developed invaginations known as T-tubules which face the terminal SR cisternae, so that LTCC are in close juxtaposition to the Ca2+ Release Units (CRU) formed by the intracellular Ca2+ release channel, ryanodine receptor (RyR). Such arrangement allows few Ca2+ ions entering the cell to trigger release of further Ca2+ from the SR, in a process known as Ca2+-Induced-Ca2+-Release (CICR), which drives contraction. In parallel with the development of SR, the mitochondrial population enriches and interfibrillary mitochondria tether to the SR, in proximity to the CRUs, a condition in which the organelle is found within the confines of a high Ca2+ microdomain, fundamental to drive the ion into the mitochondrial matrix. With these notions in mind, we sought to develop a protocol promoting maturation of neonatal CMs, thus obtaining a cellular model better suited to the study of subcellular Ca2+ handling in order to identify the mechanisms linking mitochondrial Ca2+ dynamics to hypertrophic remodelling. To induce maturation of neonatal CMs, we modified the composition of the media traditionally used to maintain cells in culture. By removing serum from the culture medium, we could avoid cell proliferation and de-differentiation. In addition, we reduced glucose content and added vitamin co-factors and trophic hormones, such as insulin, to compensate the absence of mammalian serum. Furthermore, we improved the preparation purity by eliminating contaminating cardiac fibroblasts, which secrete growth factors and matrix components, promoting cell de-differentiation and hyperplastic growth. With these changes in the isolation conditions, we obtained a pure population of CMs that can be maintained in culture for several weeks, and after few days already acquired a different morphology, compared to those obtained with the more commonly used protocol. Indeed, microscopy imaging showed that the cells were larger, rectangular-shaped, with a regular perimeter, lacking the typical ramifications of neonatal CMs, and a higher long/short axis ratio. Moreover, we observed an increased area occupied by the contractile apparatus, which appeared more regularly displaced. Mitochondria appeared longitudinally displaced along and between the sarcomeres, similarly to adult cells. In addition, immunostaining of RyRs revealed that the protein appeared in clusters more regularly distributed, thus mimicking the phenotype observed in fully differentiated CMs and suggesting increased maturation of the SR. In line with this, we observed shorter and smaller Ca2+ sparks, which are elementary Ca2+ signalling events depending on RyR opening, thus supporting that the more organized RyR clusters formed functionally active CRU, alike those of more mature cells. Interestingly, cells were more receptive to adrenergic agonists, displaying a more pronounced growth by hypertrophy as compared to traditional neonatal CMs. All the aforementioned aspects demonstrate that these cells may represent an in vitro model system well-suited to the study of Ca2+ dynamics and its relation with hypertrophic growth. Remarkably, these properties did not compromise the amenability for genetic manipulation, either via viral infection or transient plasmid transfection. Future experiment will aim at fully characterizing the Ca2+-related structures, such as T-Tubules, as well as formation of dyads.
Dal momento della nascita, per tutta la durata della vita, il miocardio è costituito da un numero pressoché fisso di cardiomiociti (CM). Infatti, la crescita postnatale del cuore è di tipo ipertrofico, per cui lo sviluppo del cardiomiocita, anch’esso di tipo ipertrofico, avviene attraverso un profondo rimodellamento strutturale, funzionale e metabolico. Una volta raggiunto un completo sviluppo, il cuore adatta continuamente la sua contrattilità e struttura in base alle richieste perfusionali dell’organismo, che variano in base a fattori intrinseci ed ambientali. Stimoli acuti determinano la modulazione della contrattilità, mentre stimoli cronici, che richiedono una performance elevata nel tempo, fanno sì che i cardiomiociti rimodellino la loro struttura, crescendo ulteriormente per sostenere l’aumentato carico meccanico. In base a tipo, intensità e durata dello stimolo ipertrofico, il rimodellamento cardiaco può portare ad ipertrofia fisiologica (come nel caso del “cuore d’atleta”) o patologica (ad esempio nel sovraccarico pressorio): in quest’ultimo caso, la crescita ipertrofica risulterà nel tempo in scompenso cardiaco, insufficienza cardiaca e morte. Nonostante i fenotipi cellulare e clinico siano distinti, il comune denominatore di queste condizioni è che, nelle fasi iniziali, i processi sono di tipo adattativo e comprendono la deposizione di nuovi sarcomeri nei cardiomiociti, per garantire una contrattilità migliorata o quanto meno preservata. Queste proprietà richiedono entrambe una maggiore produzione di ATP. Non sorprende quindi il fatto che la regolazione della funzione mitocondriale sia un processo critico per la produzione di ATP, per soddisfare il fabbisogno energetico durante la crescita ipertrofica. I mitocondri sono la “centrale energetica” della cellula e la concentrazione di Ca2+ opera come un regolatore dinamico primario della produzione di ATP. Nei cardiomiociti, l’influsso di Ca2+ nella matrice mitocondriale avviene durante l’aumento di Ca2+ sistolico ed è mediato dal complesso dell’uniporto mitocondriale per il calcio (MCUC), recentemente identificato. L’uptake di Ca2+ mitocondriale è un processo fondamentale nella modulazione acuta della contrattilità durante la risposta “fight-or-flight” attivata dall’attivazione dei recettori ß-adrenergici. A prova di ciò, la delezione di MCU nel modello murino diminuisce la capacità d’esercizio7, mentre la riduzione di MCU nelle cellule del nodo senoatriale o dei ventricoli riduce le risposte cronotropiche o contrattili, rispettivamente, indotte dalla stimolazione ß-adrenergica. Al momento non è noto se avvengano cambi nell’espressione delle proteine formanti MCUC in diverse situazioni fisiopatologiche, così come non è noto l’effetto della modulazione dell’uptake di Ca2+ mitocondriale durante il rimodellamento cardiaco. Su queste basi, gli obiettivi del mio progetto di dottorato sono: 1) Identificare i meccanismi molecolari coinvolti nella regolazione endogena di MCU; 2) Determinare se MCU ha un ruolo nel rimodellamento fisiologico e patologico del cuore; 3) Sviluppare un modello sperimentale di cardiomiociti isolati da cuori neonati per la caratterizzazione in vitro delle dinamiche del Ca2+mitocondriale su tempi prolungati. Risultati. 1. Il contenuto dell’uniporto mitocondriale per il calcio (MCU) nei cardiomiociti è dinamicamente regolato da miR-1 nell’ipertrofia fisiologica e patologica. In esperimenti preliminari condotti nel nostro laboratorio, abbiamo confrontato i livelli proteici di MCU in cuori normali ed ipertrofici, ed abbiamo osservato che le variazioni nel contenuto proteico di MCU non erano accompagnate da variazioni in parallelo del suo trascritto. Ciò ci ha portato ad investigare se, nel rimodellamento cardiaco, potesse avvenire una regolazione post-trascrizionale di MCU. Ci siamo così focalizzati sui microRNA (miR), piccole sequenze non codificanti di RNA (18-25 nucleotidi) capaci di modulare finemente l’espressione di svariati geni, grazie all’interferenza con la stabilità o la traduzione dell’mRNA target. Un numero crescente di evidenze rivela il ruolo fondamentale dei miRs nell’ipertrofia cardiaca e, in altri tessuti, è stato dimostrato come certi miR regolino il contenuto di MCU. Tramite ricerca bioinformatica, abbiamo identificato diversi microRNA che potrebbero appaiarsi alla regione 3’UTR di MCU. Tra questi, ci siamo focalizzati su miR-1 per la sua espressione muscolo-specifica, il suo ruolo critico nell’ipertrofia cardiaca, la sua omologia conservata tra diverse specie e la specificità per MCU tra i membri del complesso MCUC. Il saggio di luciferasi ha confermato quanto predetto dalla bioinformatica ed ha permesso di identificare specifiche sequenze complementari sul gene di MCU. Consistentemente, cardiomiociti over-esprimenti miR-1 hanno mostrato un diminuito contenuto proteico di MCU senza alterazioni nel suo mRNA, risultando in una riduzione significativa nella capacità di importare Ca2+ nella matrice mitocondriale. Quindi, abbiamo testato l’ipotesi che il contenuto di MCU fosse modulato in condizioni di ipertrofia associate a variazioni nell’espressione di miR-1, quali: i) lo sviluppo postnatale, ii) l’esercizio moderato, iii) il sovraccarico pressorio. Confrontando cuori neonati ed adulti abbiamo osservato che l’espressione di miR-1 aumenta, in linea col suo ruolo di repressore del programma genico fetale. Questo calo di miR-1 è accompagnato da un aumento nel contenuto proteico di MCU senza che ne aumentasse il trascritto. Inoltre, abbiamo osservato come solo il contenuto di MCU vari, tra i vari membri del complesso, eccezion fatta per l’mRNA di MCUb, che aumenta. Quindi, abbiamo analizzato l’asse miR-1/MCU in cuori ipertrofici murini e umani, con rimodellamenti sia fisiologici che patologici. Nei topi, l’ipertrofia fisiologica è stata indotta tramite protocollo di esercizio cronico, efficace nel determinare ingrandimento cardiaco, dei singoli cardiomiociti ed un aumento della contrattilità35. Il confronto coi cuori di topi sedentari ho dimostrato come il livello di miR-1 scenda nell’esercizio e, consistentemente, quello proteico di MCU salga. L’analisi del complesso in cuori sottoposti a costrizione aortica ha dimostrato come, durante l’iniziale fase compensata, caratterizzata da crescita dei cardiomiociti senza scompenso, le variazioni di miR-1 e MCU rispecchino quelle osservate nei topi esercitati. Inoltre, le reciproche variazioni di miR-1 e MCU accadono anche in un modello di ipertrofia di rilevanza clinica, come dimostrato dalle analisi di biopsie cardiache umane provenienti da donatori sani e pazienti con ipertrofia causata da stenosi aortica. Questi risultati indicano che, indipendentemente dalla natura dello stimolo ipertrofico (fisiologico o ipertrofico), l’iniziale adattamento cardiaco all’aumentata richiesta contrattile è caratterizzato da analoghi aumenti nella disponibilità cellulare di MCU. Viste le variazioni analoghe dell’asse miR-1/MCU riscontrate sia in ipertrofia indotta da esercizio che in quella compensata patologica, abbiamo ipotizzato che ci sia un meccanismo regolatorio comune. Ci siamo così focalizzati sul sistema ß-adrenergico, il primo meccanismo fisiologico coinvolto nella risposta all’aumentato carico di lavoro, condizione che accomuna sia ipertrofia da esercizio che da costrizione aortica. L’attivazione del signalling ß-adrenergico, infatti, determina aumento delle oscillazioni di Ca2+ citosolico e conseguentemente dell’uptake mitocondriale. In parallelo, l’attivazione di queste cascate di segnale è coinvolta nell’attivazione di vie di segnale di ipertrofia come Akt-FOXO. È interessante notare che l’espressione di miR-1, come è stato dimostrato, dipende da FOXO3a, indicando che, in condizioni di attivazione cronica dei recettori ß-adrenergici, il blocco della traslocazione nucleare di FOXO3a potrebbe inibire l’aumento di miR-1. Per supportare questa ipotesi, abbiamo trattato topi sottoposti a costrizione aortica col ß-bloccante metoprololo che, in linea con quanto ipotizzato, è stato in grado di abolire la repressione di miR-1 e di conseguenza l’aumento di MCU. Conclusioni e prospettive future. Complessivamente, i nostri dati identificano miR-1 come un nuovo regolatore post-trascrizionale di MCU e supportano l’idea che l’asse miR-1/MCU sia coinvolto nel rimodellamento ipertrofico fisiologico e patologico. Esperimenti futuri mireranno ad approfondire il ruolo causale di miR-1 nella modulazione di MCU, ed a identificare la via molecolare coinvolta nel processo. Attualmente esistono tools farmacologici (quali miR-mimics o antagomiRs) in grado di interagire coi miR endogeni, antagonizzandoli o sostituendoli, modulando con efficacia e selettività l’espressione degli mRNA target. Su queste basi, il nostro studio sull’asse miR-1/MCU può aprire a nuove prospettive terapeutiche per trattare l’ipertrofia cardiaca. 2. MCU partecipa all’adattamento cardiaco a stimoli ipertrofici. L’osservazione di come il contenuto di MCU cali durante la fase maladattativa dell’ipertrofia patologica, suggerisce che esso fluttui nelle varie fasi dell’ipertrofia. Questa osservazione ci ha indotto a cercare di determinare se MCU potesse avere un ruolo attivo nel rimodellamento cardiaco. Per testare quest’ipotesi, abbiamo modulato il livello di MCU in topi successivamente sottoposti a sovraccarico pressorio. Inoltre, per avere dettagli meccanicistici sul signalling cellulare, abbiamo modulato l’espressione di MCU in vitro, e abbiamo studiato l’effetto della sua overespressione o silenziamento nella risposta ad incubazione cronica con agonisti adrenergici. Per studiare il ruolo di MCU nell’adattamento cardiaco in vivo, abbiamo overespresso o silenziato l’uniporto mediante l’uso di vettori virali (AAV9). La modulazione di MCU, per sé, non ha alterato la struttura e la performance cardiaca. Tuttavia, quando abbiamo sottoposto gli animali a TAC, abbiamo osservato come l’overespressione di MCU comporti aumentata crescita ipertrofica, confrontando con animali WT allo stesso tempo dopo l’inizio della costrizione aortica. Inoltre, il rimodellamento nei topi overesprimenti ha caratteristiche simili a quello dell’ipertrofia fisiologica, quali aumentata densità capillare, scarsa fibrosi, funzionalità cardiaca preservata anche dopo 8 settimane di sovraccarico pressorio. Al contrario, il silenziamento di MCU ostacola l’adattamento cardiaco all’aumentata pressione, determinando un maladattamento prematuro, con caratteristiche tipiche della cardiomiopatia dilatativa, quali ridotta densità capillare, fibrosi diffusa ed inadeguata contrattilità. Queste caratteristiche hanno portato i topi MCU silenziati a sviluppare scompenso ed insufficienza cardiaca, ed a morire dopo solo 4 settimane dalla TAC. Per approfondire i meccanismi molecolari mediante i quali MCU impatta nella crescita ipertrofica dei cardiomiociti, abbiamo overespresso o silenziato MCU in cardiomiociti neonatali di ratto. Eseguendo esperimenti di live imaging delle dinamiche di Ca2+ mitocondriali con la sonda “mito-CaMeleon”, abbiamo appurato come la modulazione di MCU risulti in aumentato o diminuito uptake di Ca2+ mitocondriale. Se da un lato l’over-espressione di MCU non determina alterazioni morfologiche in condizioni basali, cellule silenziate dimostrano dimensioni maggiori rispetto a cellule di controllo, con evidente alterazioni nella struttura sarcomerica. Per mimare l’iperattivazione del sistema nervoso simpatico che si riscontra nell’ipertrofia sia fisiologica che patologica, abbiamo incubato le cellule con norepinefrina. Anche in questo caso, l’overespressione di MCU aumenta la crescita ipertrofica, mentre il suo silenziamento ha un effetto opposto, contraddistinto da compromissione dei sarcomeri ad attivazione di apoptosi, in evidente analogia ai dati ottenuti in vivo. Le successive analisi sono state mirate per approfondire lo stato di attivazione di divere vie di segnale medianti ipertrofia. Abbiamo rilevato come l’overespressione di MCU, in cardiomiociti sottoposti a stimolazione adrenergica, acceleri l’attivazione dell’asse calcineurina/NFAT. Inoltre, i nostri dati suggeriscono la partecipazione dell’asse Akt/ GSK3ß all’aumentata attivazione di NFAT, in una cascata presumibilmente a valle di CaMKII che fosforila Akt. Infatti, l’inibizione di CaMKII in cardiomiociti MCU overesprimenti determina una crescita ipertrofica comparabile a cellule di controllo. Per concludere, i nostri risultati dimostrano come l’aumento del carico cardiaco, indotto in vivo da TAC ed in vitro da trattamento con noradrenalina, sia ben tollerato quando i livelli di MCU sono aumentati dall’overespressione. Al contrario, il silenziamento di MCU induce, nelle stesse condizioni, morte cellulare e prematuro rimodellamento maladattativo. Questi dati sono in accordo con le nostre osservazioni preliminari che indicano come il contenuto proteico di MCU, che aumenta nell’ipertrofia compensata, diminuisca nel successivo rimodellamento patologico che determina scompenso cardiaco. Inoltre, abbiamo identificato l’asse ß-AR/CaMKII/Akt come cruciale nell’ipertrofia cardiaca e dipendente dalla modulazione di MCU. 3. Sviluppo di un protocollo di coltura che induca la maturazione di cardiomiociti neonatali in vitro Le colture primarie di cardiomiociti neonatali sono un modello cellulare ampiamente utilizzato nella cardiologia molecolare, in quanto possono esser mantenuti in coltura per più giorni e sono facilmente manipolabili geneticamente28. Tuttavia, questo tipo cellulare ha importanti differenze funzionali e strutturali rispetto ai cardiomiociti adulti. Queste differenze vanno dall’espressione di diverse isoforme di miosina (nel topo, dalla ß alla α), necessario per ottimizzare la performance contrattile, a cambi nel metabolismo (che passa da glucidico ad ossidativo), in modo da garantire maggior apporto di ATP in vista di un maggior consumo29. Inoltre, il processo di maturazione postnatale delle cellule comprende alterazioni nelle strutture coinvolte nelle dinamiche di Ca2+ 30. In particolare, nelle cellule neonatali, la contrazione avviene principalmente grazie al Ca2+ che entra dai canali del Ca2+ di tipo L situati nella membrana citoplasmatica. Il Ca2+ che entra attiva direttamente i sarcomeri, con un minimo contributo del Ca2+ contenuto nelle vescicole che costituiscono un immaturo reticolo sarcoplasmatico. Al contrario, nelle cellule adulte la membrana plasmatica ha sviluppato una serie di invaginazioni note come tubuli T che penetrano nella cellula e giungono all’estremità del reticolo sarcoplasmatico, ora costituito dal tipico sistema di cisterne, cosicché i canali del Ca2+ di tipo L siano a stretto contatto coi RyR2, formando cosi le Unità deputate al Rilascio del Ca2+ (CRUs). Questa sofisticata struttura fa sì che le poche molecole di Ca2+ che entrano dai canali nei tubuli T possano scatenare il Rilascio di Ca2+ indotto dal Ca2+ (CICR), determinando l’uscita di un’ingente quantità di ione dal reticolo sarcoplasmatico. Un altro importante cambiamento interessa i mitocondri che, se nel cardiomiocita neonatale occupano principalmente la zona perinucleare, in quello adulto si dispongono anche negli spazi sub-sarcolemmali ed inter-miofibrillari. In questi distretti, i mitocondri sono in prossimità del reticolo sarcoplasmatico, al quale possono ancorarsi fisicamente, trovandosi così in distretti cellulari caratterizzati da elevate concentrazioni di Ca2+. Tenendo a mente questi fattori, il nostro obiettivo è stato quello di sviluppare un protocollo che promuovesse la maturazione di cardiomiociti neonatali verso un fenotipo adulto, ottenendo così un modello sperimentale ottimale per lo studio delle dinamiche del Ca2+ cellulare, ed identificare così i meccanismi che connettono il Ca2+ mitocondriale al rimodellamento ipertrofico. Per indurre la maturazione dei cardiomiociti neonatali abbiamo modificato la composizione dei terreni di coltura tradizionalmente usati. Per mantenere le cellule ad una concentrazione di glucosio simile a quella fisiologica, abbiamo cambiato il costituente principale del terreno, passando da DMEM (Dulbecco’s modified eagle medium) a MEM (minimum essential medium) e riducendo così la concentrazione da 25 mM a 5 mM, valore, quest’ultimo, paragonabile alla concentrazione fisiologica in vivo. Per ridurre la proliferazione dei fibroblasti, che tramite secrezione di fattori di crescita e componenti della matrice extracellulare determinerebbero de-differenziamento dei cardiomiociti, abbiamo fortemente ridotto il quantitativo di siero ed aggiunto un agente proliferativo (BrdU). Per compensare la rimozione del siero, abbiamo aggiunto co-fattori vitaminici ed ormoni trofici, come l’insulina. In tal modo abbiamo ottenuto una popolazione pura di cardiomiociti che può essere tenuta in coltura per più settimane, e che già dopo pochi giorni mostrano una morfologia diversa dalle cellule ottenute col protocollo tradizionale. Analisi alla microscopia hanno evidenziato come queste cellule siano più grandi, rettangolari con un asse maggiore ben distinto da un asse minore, ed un perimetro regolare senza le tipiche ramificazioni dei cardiomiociti immaturi neonatali. A livello subcellulare, abbiamo osservato una maggiore estensione dell’apparato contrattile, rivelatosi disposto in maniera più regolare. I mitocondri appaiono disposti longitudinalmente accanto e tra i sarcomeri, come nelle cellule adulte. Inoltre, l’immunofluorescenza per il recettore rianodinico ne ha evidenziato la presenza in clusters, distribuiti in maniera regolare, in analogia alla loro distribuzione in cellule mature, suggerendo così la presenza di un reticolo sarcoplasmatico maggiorente formato. Consistentemente con ciò, abbiamo osservato minori e più rapidi Ca2+ sparks, eventi elementari di dinamiche di calcio, determinati dall’apertura transiente di RyR. La minore frequenza ed entità di questi sparks suggerisce che i RyR disposti in maniera regolare in clusters determini la formazione di vere e proprie unità deputate al rilascio di calcio (Calcium Release Units, CRUs), strutture fondamentali nei cardiomiociti adulti. Infine, queste cellule han risposto maggiormente al trattamento con agonisti adrenergici, riportando una crescita ipertrofica maggiore rispetto a cellule neonatali tradizionali sottoposte allo stesso trattamento. Tutte queste caratteristiche sopracitate indicano come queste cellule possano rappresentare un modello in vitro adatto allo studio delle dinamiche di Ca2+ intracellulare, specialmente nel rimodellamento ipertrofico. È importante sottolineare come questo maggior grado di maturazione dei cardiomiociti neonatali non sia a discapito della capacità di manipolarli geneticamente, con tecniche di trasfezione od infezione. Esperimenti futuri cercheranno di caratterizzare a fondo le strutture coinvolte nelle dinamiche di calcio intracellulari, come ad esempio la formazione di Tubuli T ed il rapporto di questi con il reticolo sarcoplasmatico ed i mitocondri.

Endurance training results in profound skeletal muscle adaptations that improve fatigue resistance and enhance exercise capacity. To maximise these adaptations, athletes often engage in extensive training regimes, involving 10 to 16 training sessions∙wk-1. The use of cold water immersion (CWI) as a recovery intervention has emerged as a strategy to maintain training performances between sessions. However, its concurrent influence on muscle aerobic adaptations to training is unclear. Thus, the overall purpose of the four research studies contained within this thesis was to determine the influence of post-exercise CWI on muscle metabolic activity, acute and long-term adaptations to exercise and exercise training, respectively. The first two studies of this thesis were designed to determine the reliability and between limb differences of the near infrared spectroscopy (NIRS)-derived indices describing muscle oxygenation and metabolic activity. Such information was necessary to substantiate the use of NIRS to monitor muscle aerobic adaptations to training/cooling as well as the onelegged cooling model utilised throughout this thesis. In study 1, it was found that there were considerable differences in reliability levels with regards to the analytical technique chosen. However the variables demonstrated CVs ranging from 3 to 35%, which is lower than currently reported changes in training-induced adaptations and/or group differences between athletes and sedentary controls (23% - 450%). In study 2, it was shown that there were no between limb differences in NIRS-derived variables. As such, studies 1 and 2 indicate that changes in NIRS-derived variables are suitable indices to monitor the influence of cooling on training-induced adaptations in the muscle, as well as substantiate that the exercise/training protocols induced similar physiological stimulus in both the intervention and control limbs. In study 2, it was also shown that cooling one leg (15 min at 10°C) from the gluteal fold downwards resulted in significant decreases in post-exercise vastus lateralis skin temperature (35.1 ± 0.6 vs. 16.9 ± 1.7°C, p < 0.001), microvascular perfusion (20 ± 4%, p < 0.01) and muscle metabolic activity (p < 0.05) while not resulting in shivering thermogenesis. While these responses may improve local muscle recovery, its simultaneous effect is on muscle aerobic adaptations are unclear. Indeed, reduced muscle metabolism might attenuate mitochondrial biogenesis via inhibiting AMPK activation or via a decrease in the Q10 effect. Conversely, cooling in cell and rodent models has been shown to up-regulate the expression of the transcriptional coactivator PGC-1α, which is implicated in the regulation of non=shivering thermogenesis. As such, studies 3 and 4 investigated the acute and chronic influence of post-exercise cooling on muscle aerobic adaptations to exercise and exercise training, respectively. In study 3, it was shown that cooling resulted in significantly lowered intramuscular temperatures (28.9 ± 2.3°C vs. 37.0 ± 0.8°C, p < 0.001). This change was associated with a significant increase in the mRNA content of PGC-1α in the cooled limb compared with control. However, associated PGC-1α targets related to vascular and metabolic adaptation, namely VEGF and nNOS, only demonstrated significant changes from baseline (i.e. time effects) with no significant differences between conditions evident. These data indicate that an acute post-exercise cooling intervention enhances the gene expression of PGC-1α and therefore may provide a valuable strategy to enhance exercise-induced mitochondrial biogenesis. However its influence on VEGF and nNOS expression and associated functional adaptations warrants further research. In study 4, we investigated the effect of regular post-exercise CWI on training induced AMPK activity and mitochondrial biogenesis. Ten males performed 3 sessions∙wk-1of endurance training for 4 wks, where following each session subjects immersed one leg in a cold water bath (10°C) to the level of their gluteal fold for 15 min, while the contra-lateral leg served as control. Subjects’ maximal oxygen consumption and maximal aerobic running speed were improved by 5.4% and 6.4% following training (p < 0.05). Additionally, regular post-exercise cooling enhanced exercise-induced increases in basal AMPK activity. Despite an increase in AMPK activity, a concomitant increase in downstream targets PGC-1α and most mitochondrial electron chain subunits was not observed. However, a significant increase in COX3 protein content was evident and hence indicates that mitochondrial biogenesis may be enhanced. Regardless, we advocate caution with regards to regular use of this intervention as cold-induced mitochondrial biogenesis may concomitantly decrease mitochondrial efficiency. Further research should focus on muscle aerobic function following regular CWI also verify if increases in AMPK activity observed in this study translates to improved glucose disposal and fatty acid oxidation.

Abstract The impact of hand-rearing on the morphology and physiology of captive and wild grey partridges (Perdix perdix) and capercaillies (Tetrao urogallus) was studied in three feeding trials conducted under laboratory conditions, and two comparative studies between wild and captive birds. Finally, wild and hand-reared grey partridges from several localities in Europe were sampled and the control region 1 of mitochondrial DNA was sequenced to reveal genetic variation between populations, as well as to compare wild and captive stocks. Wild capercaillies had heavier pectoral muscles, hearts, livers and gizzards, longer small intestines than hand-reared ones, and a higher cytochrome-c oxidase activity in muscle and heart. Invertebrates were essential to the growth, primary and temperature regulation development in grey partridge chicks. Fish was not sufficient to replace invertebrates in the diet. A change in diet from commercial to natural decreased the assimilation efficiency in the grey partridge. It also increased the mass of gizzard reflecting the need for greater grinding ability. Of hepatic P450 enzymes used in this study 7-ethoxyresorufin-0-deethylase and 7-pentoxyresorufin-0-deethylase differed between wild and hand-reared birds. Coumarin-7-hydroxylase activity was higher in grey partridges than capercaillies. Diet differences may have caused these differences. Quebracho tannin added to the diet lowered nitrogen concentration in caecal feces, and elevated the level of excreted tannin. Otherwise its effects were slight. Mitochondrial control region revealed 14 variable sites between two main lineages detected. Nucleotide and haplotype diversities varied greatly between populations. The markedly deep divergence between the two lineages indicated most probably post-glacial recolonisations from geographically isolated refuges. In Finland, wild birds represented the eastern lineage, while the farmstock represented the western lineage. Surprisingly little trace, contrary to expectations, from the large-scale releasing of imported partridges could be seen in the European populations.

The focus of this dissertation is centered on the mass spectral analysis of lipids and changes occurring in keeping with the concept of homeoviscous adaptation [1]. Homeoviscous adaptation is the process of modification of membrane lipids in response to environmental stimuli [1]. Dissertation investigations applied this concept to prokaryotic and eukaryotic organisms, and expanded the perception of environmental factors from exogenous organic solvents to intracellular environment. The field of lipidomics deals with the analysis of phospholipid and fatty acid components of membranes the changes that occur due to environmental stimuli and their biological significance [2-6]. The high sensitivity of mass spectrometry (MS) is an ideal tool for lipidomics allowing detection, quantification and structural elucidation [6]. Coupling of a mass spectrometer to a chromatographic system, such as gas chromatograph (GC), allows the separation of fatty acid methyl esters analytes prior to analysis [7]. The research investigations that comprise this dissertation are divided into three interrelated projects. The first project involved the analysis of composition and structure of Clostridium thermocellum membranes from wild-type and ethanol-adapted strains in response to adaptation of cultures to growth in ethanol. The hypothesis being that adaptation of cultures to growth in ethanol would result in compensatory change to the membrane composition. Rat mitochondrial fatty acid profiles isolated from brain, liver, kidney and heart tissues were compared. The hypothesis being that differences in cellular environments found among various tissues would be reflected in the mitochondrial membrane composition. These data support the concept that variations to the lipid content of neurological mitochondria may increase susceptibility to the products of oxidative stress. Lastly, changes in neurological mitochondria as a function of Alzheimer’s disease progression were studied. The hypothesis being that changes to the mitochondrial lipidome would be significantly reflected during advanced stages of AD, in addition to being more prevalent in regions displaying greater pathology. The three interrelated projects increased our understanding of the boundaries established by the concept of homeoviscous adaptation. Project specific hypotheses were supported by data obtained from these investigations.

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

Ce travail etait destine a etudier les effets de l'hypoxie chronique (fo2 = 10% ; pendant 3 semaines), sur le metabolisme energetique et la fonction du muscle cardiaque de rat. - une etude in situ de la fonction contractile des ventricules droit (vd) et gauche (vg), grace a l'introduction d'un microcapteur de pression millar dans les cavites ventriculaires, a revele une legere depression de la fonction du vg, alors que l'ensemble des variables fonctionnelles enregistrees au niveau du vd (pression developpee, dp/dt max et min) sont fortement augmentees. Ces variations sont associees a une augmentation de la masse du ventricule droit. L'evaluation de la fonction ventriculaire gauche sur un modele de cur isole perfuse ne revele aucune limitation des capacites fonctionnelles dans le groupe hypoxique, la fonction apparaissant meme augmentee lors de stimulations inotropes par le calcium et l'isoprenaline. - les principales observations sur le metabolisme energetique des curs isoles de rats hypoxiques sont les suivantes: (1) la balance energetique myocardique est preservee en hypoxie chronique et le taux d'atp intracellulaire est identique a celui des curs temoins. (2) la consommation d'oxygene myocardique du cur isole est globalement reduite, pour une activite contractile egale ou superieure a celle des curs temoins. (3) la production de lactate des curs isoles, perfuses en presence de glucose 11 mm, est augmentee. (4) la variation des concentrations des composes phosphoryles (phosphate inorganique pi, phosphocreatine pcr, et atp), detectee par la spectroscopie rmn du p-31, lors de situations de perturbations de la balance energetique (stimulations inotropes ou hypoxie aigue), est attenuee. De plus, apres -stimulation (isoprenaline 10#-#6 m), on observe une augmentation de la vitesse de resynthese de la pcr dans les curs d'animaux adaptes a l'hypoxie. (5) l'addition d'hexanoate dans le milieu de perfusion modifie profondement le comportement metabolique des curs de rats temoins, alors qu'elle est sans effet dans le cas du groupe hypoxique. - l'etude des caracteristiques de la respiration mitochondriale, sur une preparation de fibres cardiaques permeabilisees, a permis de montrer, pour les curs de rats exposes a l'hypoxie, une reduction de l'effet stimulant de la creatine sur la vitesse de respiration. Cette donnee traduit une modification des systemes de canalisation de l'energie dans la cellule myocardique, entre les sites d'utilisation (atpases myofibrillaires) et de production (mitochondries) = la navette creatine-phosphocreatine. Les resultats obtenus evoquent une plus grande sollicitation de la voie glycolytique, associee a une efficacite amelioree des processus oxydatifs apres acclimatation a l'hypoxie. Ces donnees conduisent a l'hypothese d'une reorganisation des systemes cellulaires responsables du transfert de l'energie dans le cardiomyocyte

Collembola (=springtails) is one of the most abundant, widespread and ancient lineages of basal hexapods. During their long evolutionary history, springtails have adapted to most damp environments on Earth, including those of South Pole. Antarctic springtails are endemic to the frozen Continent and among the few invertebrate taxa adapted to its strictly terrestrial ecosystem. These species have evolved when Antarctica was still linked to the Gondwanaland at lower latitudes and have adapted and survived to the cooling, isolation and southwards migration of the landmass. Antarctic springtails’ habitats are restricted to the few coastal areas, seasonally ice-free and accounting for less than the 0.5% of the entire continental area and off-shore islands. The niche fragmentation, together with springtails poor dispersal capability (due to the primary absence of wings), entail a severe degree of isolation among populations, with very low levels of gene flow. The Antarctic springtail species composition is limited without overlap among the two main Antarctic bioregions (i.e., the maritime and the continental Antarctica), with Friesea antarctica being the only species found both in the Antarctic Peninsula and Victoria Land (continental Antarctica). The high levels of endemism and fragmentation among populations, as well as the low invertebrate biodiversity and the complex and delicate array of physiological adaptation these species evolved, make Antarctic taxa particularly susceptible to anthropogenic climate changes, that we are all experiencing since the second industrial revolution in the XIXth century. In this respect, studying the molecular mechanisms underlying springtail adaptation to such a harsh environment, as well as the genetic structure of the populations and the way in which specimens may have been and can be influenced by the Antarctic terrestrial environment, may greatly assist the development of adequate and biogeographically-specific (thus, effective) conservational plans. In order to address these issues, different studies have been carried out during the current PhD project. A genetics of population study was performed to investigate the genetic structure of the Antarctic springtail species Cryptopygus terranovus. As previously observed in other Antarctic species (i.e, F. antarctica), high levels of genetic divergence were detected, with very few haplotypes shared among populations nearly suggesting the absence of gene flow, as well as the presence of cryptic species. One way to address this issue is the integration of morphological, molecular and biogeographic data to assess whether the detection of cryptic species is due to an ongoing phylogenetic niche conservatism process or to overlooked morphological differences. In this respect, an integrative taxonomic analysis has been carried out on the Antarctic springtail F. antarctica. Nuclear and mitochondrial markers were used in bioinformatic analyses of species delimitation. Although applied tools rely on different algorithms and biological assumptions, and the chosen molecular markers are generally subject to different evolutionary pressures, the results obtained in this study would suggest that at least two more species may be hidden within the F. antarctica complex. Specimens from the same localities were also morphologically re-described so that new species could be possibly established. These analyses would suggest an even higher species richness of the Antarctic terrestrial ecosystem, that should be taken into account when developing conservational plans. Our ability to safeguard Antarctica relies also on our in-depth understanding of the terrestrial ecosystem functioning and dynamics. In this perspective, an initial descriptive analysis of the microbial communities associated to four Antarctic springtail species was performed (specifically on: Cryptopygus antarcticus antarcticus and F. antarctica from the Antarctic Peninsula and C. terranovus and F. antarctica collected along Victoria Land). The results obtained are in line with previous studies on Collembola microbiomes. In addition, the occurrence of particularly interesting OTUs, such as those of the genera Streptomyces and Bacillus, was, to my knowledge, firstly detected among springtails and may break new grounds for biotechnology development, especially starting from such an unspoiled ecosystem, like the Antarctic one. Finally, the mitochondrial genomes from 13 springtail species, both living at low and high latitudes, have been applied to maximum likelihood analyses of positive selection in order to investigate whether or not the organelle chromosome may have been involved in Antarctic springtails adaptation to such an extreme environment. The results pointed out that some mitochondrial genes involved in the oxidative phosphorylation process may have been under positive selection, thus suggesting the development of additional thermoregulatory mechanism within the mitochondrion, complementary to the well-known cold hardiness strategies.

L’anorexie mentale (AN), un trouble du comportement alimentaire multifactoriel, se traduit par une perte de poids. La sévère dénutrition retrouvée dans l’AN est associée à des altérations métaboliques induisant une dérégulation de l’axe intestin cerveau. Les mécanismes physiopathologiques sont encore mal connus. Le travail de cette thèse était de mieux appréhender les dysfonctions de l’axe intestin cerveau en évaluant le métabolisme protéique de divers tissus (hypothalamus, côlon et estomac) dans un modèle murin d’anorexie par une approche protéomique. Le premier travail a permis de mieux caractériser le modèle d’anorexie nommé activity-based anorexia (ABA) en fonction du sexe. Puis les différentes analyses protéomiques ont permis de constater une adaptation tissu dépendant des mécanismes régulant l’équilibre énergétique, avec une activité cérébrale potentiellement augmentée au détriment des fonctions digestives. Chez les souris femelles ABA, il a été constaté une augmentation d’expression de protéines mitochondriales au niveau de l’hypothalamus et à l’inverse, une diminution du métabolisme protéino-énergétique au niveau colique avec un rôle de la voie de signalisation mTOR. L’autophagie était augmentée dans ces deux tissus. Ensuite, nous avons démontré un ralentissement de la vidange gastrique secondaire à la dénutrition, et l’analyse protéomique a permis de constater une augmentation du stress oxydant au niveau de l’antre des souris ABA femelles. Ces altérations peuvent contribuer aux troubles fonctionnels gastro intestinaux. En conclusion, nos études soulignent des mécanismes d’adaptation tissu dépendants dans l’anorexie, qui devront être ultérieurement approfondis
Anorexia nervosa, a multifactorial eating disorder, is a major public health problem and results in a severe body weight loss. The severe malnutrition observed in anorectic patients is associated with metabolic alterations inducing disturbance of the gut-brain axis. However, involved mechanisms remained poorly understood. The aim of the present thesis was to better understand the alterations of the gut-brain axis in the activity-based anorexia (ABA) model by evaluating the protein metabolism of various tissues (hypothalamus, colon and stomach) by proteomic approach. Firstly, we have better characterized the response to ABA model according to sex. Then, different proteomic analyses were performed using female C57BL/6 mice. Our results revealed a tissue-dependent adaptation of protein and energy metabolism with an increased hypothalamic activity and a decrease in the gastrointestinal tract. Indeed, ABA mice exhibited an increased expression of proteins involved in mitochondrial metabolism at the level of the hypothalamus, and conversely a decrease of proteins involved in protein and energy metabolism in colonic mucosa with a key role of the mTOR signaling pathway. Both in hypothalamus and colon, autophagy was increased. We were also able to show that gastric emptying was delayed in ABA mice that is mainly due to malnutrition. In addition, proteomic analysis revealed an increase in gastric oxidative stress in female ABA mice. These alterations may contribute to the gastrointestinal functional disorders frequently described in anorexia nervosa. In conclusions, our study underlined tissue-dependent adaptive metabolic process during anorexia that should be further explored

Exercise intensity and its distribution is probably the most important and most heavily debated variable of endurance training. Training induces adaptation but also induces stress responses. Controlling the training-intensity distribution may provide a mechanism for balancing these two effects. It has been reported that elite endurance athletes train with a high volume and load, relative to the sport. These athletes spend the vast majority (>80%) of training time at relatively low intensities (lower than the lactate threshold, zone one), and therefore <20% of training time above the lactate threshold (zones two and three). Experimental studies support the beneficial effects of a high training volume in zone one, and show detrimental effects of replacing zone one training with training in zone two. This is likely due to enhanced recovery from training in zone one compared with training in zone two. The acute recovery following training sessions in zones two and three has been reported to not be different, but the recovery following training in zone one has been reported to be faster. Improvements in physiological adaptation and endurance performance have been reported to be greater following training programmes with higher exercise intensities. Therefore, it has been suggested that a polarised training model, which includes ~80% of training in zone one with ~20% of training in zone three is more beneficial than a threshold training model, with the majority of training in zone two. However, research into an optimal training-intensity distribution is limited. Therefore, the aims of this thesis were to assess the effectiveness of training-intensity distribution on the improvements in physiological adaptation, endurance performance and assess if manipulating training-intensity distribution had an effect on immune function. Study one revealed that the lactate threshold, the lactate turnpoint and maximal performance measures in swimming, cycling and running, assessed using the methods outlined in the study, are reproducible in trained endurance athletes. These tests can therefore be used by trained endurance athletes as part of a physiological testing programme to assess not only endurance performance, but also to demarcate training intensity zones for exercise intensity prescription and monitor moderate to large adaptations to training. Practitioners should take care when deciding on the duration between tests to test for adaptations from training, as adaptations need to be greater than these detected test-retest variations to be considered physiologically meaningful. To the best of the author’s knowledge, study two was the first study to have assessed training-intensity distribution in a group of multisport athletes. Training was monitored over a 6-month period, and testing took place every two months to assess the effect of the training on physiological adaptation. Although speculative due to the number of variables involved, the results suggest that a greater proportion of training time spent in zone one and a lower proportion of training time spent in zone two is beneficial to physiological adaptation. However, given the number of variables associated with assessing the training-intensity distribution in multisport athletes, it is not easy to draw conclusions as to the effectiveness of the training in the different disciplines on the key measures of adaptation in the different disciplines. Study two highlighted the need for future research to focus on experimental manipulation of training-intensity distribution and thus improve our understanding of its impact on the training-induced adaptations in endurance athletes. Study three manipulated the training-intensity distribution in trained endurance athletes in just one discipline, to reduce the number of variables involved. A polarised training model was compared to a threshold training model on the effectiveness to improve physiological adaptation and endurance performance. Results revealed that a polarised training model is recommended for trained cyclists wishing to maximally improve performance and physiological adaptation over a short-term (six week) training period. The first part of study four assessed the effect of a polarised and a threshold training model on immune function markers in trained cyclists. Both endurance training programmes had similar volume, and were sufficient to induce improvements in performance and physiological adaptation. However, despite likely differences in recovery, both training programmes had no effect on the proportion of low or high differentiated or senescent CD8+ or CD4+ T-cells in blood. Therefore, training adaptation was achieved at no cost to this particular aspect of immune function. From these results and evidence from previous studies, it seems likely that athletes need to be overreached to induce any change in immune function following a period of intensified training. The second part of study four assessed the impact of an ironman triathlon race on Epstein-Barr virus (EBV) and Varicella-Zoster virus (VZV) antibody titres and the frequency of low and high differentiated and senescent blood T-cells in trained endurance athletes. Previous work has revealed that an ironman triathlon race increases the proportion of senescent CD4+ T cells and decreases the proportion of naive CD4+ T cells, and thus induces changes the immune space which could leave an individual at a greater risk of infection. This study however, did not find any changes in the proportions of these T cell subsets following an ironman triathlon race. The mean results of this study suggest that there is no relationship between EBV and VZV-specific antibody concentrations and the proportion of senescent, low and highly differientiated T cells. However, on analysis of individual subject data, it seems possible that subjects with a high antibody titre for EBV or VZV 3 wks before a competition might be more at risk of infection post race. A greater subject number would be needed in order to make a more conclusive statement about this relationship. The results of this thesis suggest that future research is required in the area of training-intensity distribution. Firstly, our understanding of the physiological mechanisms responsible for the effectiveness of a polarised training model in trained endurance athletes is limited, and thus studies should attempt to address this issue. Our current knowledge on the mechanisms underlying a blunted T cell response following strenous exercise is also limited. A change in the immune space to a greater proportion of senescent T cells and a lower proportion of naive T cells might contribute to this blunted response. In the current thesis however, the proportions of these T cell markers were unchanged following the training/racing interventions. It is possible that with a higher training load, there could be changes in these markers, and thus this is an exciting area that could have potential implications on athlete health. Finally, testing for antibody titres in endurance athletes is possibly an avenue to detect individuals at the greatest risk of infection if subjected to a large physical and/or mental stress. This could have implications on maintaining athlete health and therefore, allowing athletes to train consistently.

Le stress thermique résulte d'une exposition à une température située au-delà de la plage optimale pour un organisme. L’impact du stress thermique est variable selon son intensité, allant d’un effet bénéfique à la mort de l’organisme. Mon travail de thèse a établi un modèle de stress thermique aigu (aHS pour acute Heat Stress) chez C. elegans et a étudié ses effets sur l'homéostasie cellulaire, le développement des vers et la réponse autophagique. Un aHS au cours du 4ème stade larvaire induit un retard de développement, mais aucune létalité ni stérilité. Ce stress de développement entraîne la fragmentation massive mais transitoire des mitochondries, la formation d'agrégats dans la matrice et la diminution de la respiration mitochondriale. En outre, l’aHS déclenche un flux autophagique associé à des événements de mitophagie dans de nombreux tissus et en particulier dans l'épiderme. Nous avons montré que la réponse autophagique à l’aHS était protectrice pour les animaux. De plus, nous avons découvert que dans l’épiderme, les mitochondries sont les principaux sites de biogenèse des autophagosomes, en conditions physiologique et en aHS. Nous avons également constaté que la protéine DRP-1 (dynamin related protein 1) est impliquée dans le processus de mitophagie induite par l'aHS. Chez les animaux mutants drp-1 soumis au aHS, la fission mitochondriale est impossible, l’autophagie est induite mais les autophagosomes sont anormaux et agrégés sur la mitochondrie. À partir de ces données, nous proposons que DRP-1 participe au contrôle de la qualité des mitochondries stressées en coordonnant la fission mitochondriale et la biogenèse des autophagosomes. J'ai également étudié plusieurs protéines pouvant être impliquées dans les zones de contact entre le réticulum endoplasmique et les mitochondries, ainsi que leurs rôles sur la morphologie mitochondriale et l'autophagie, dans des conditions physiologiques ou d’aHS. De plus, nous avons développé de nouveaux outils pour analyser les sites de contact ER-mitochondries
Heat stress results from an exposure to a temperature beyond the optimum range of an organism. The impact of heat stress can range from beneficial to lethal due to the severity of stress. My thesis work established an acute heat stress (aHS) model in C. elegans and studied its effects on cell homeostasis, worm development and autophagy response. aHS during the 4th larval stage induces a developmental delay but no lethality or sterility. This developmental stress results in the massive but transitory fragmentation of mitochondria, the formation of aggregates in the matrix and the decrease of mitochondrial respiration. In addition, aHS triggers an active autophagy flux associated to mitophagy events in many tissues and particularly in epidermis. We showed that the autophagy response upon aHS is protective for the animals. Moreover, we discovered that in the epidermis, the mitochondria are the major sites for autophagosome biogenesis in both standard and aHS. We also found that the dynamin related protein DRP-1 is involved in aHS-induced mitophagy process. In drp-1 animals submitted to aHS, mitochondrial fission is unable to achieve, and despite autophagy induction the autophagosomes cluster and elongate abnormally on mitochondria. From these data, we propose that DRP-1 is involved in the quality control of stressed mitochondria by coordinating mitochondrial fission and autophagosomes biogenesis. I also studied several proteins which may be involved in contact zones between endoplasmic reticulum and mitochondria, and their roles on mitochondrial morphology and autophagy, in physiological or aHS conditions. Furthermore, we have developed new tools for further studying the ER-mitochondria contact sites

Les pathologies chroniques sont la toute première cause de décès dans le monde. En plus de diminuer considérablement les capacités fonctionnelles des patients et de dégrader leur qualité de vie, elles constituent un poste de dépenses majeures en termes de santé publique. Une partie de la prise en charge de ces pathologies passe par l’activité physique adaptée et la mise en place d’un réentrainement à l’effort. Celui-ci vise à améliorer les capacités des sujets, notamment les capacités d’endurance et de force musculaire, afin de gagner en autonomie et diminuer les risques de morbi-mortalité. Classiquement, les exercices d’endurance de pédalage ou de course sur tapis roulant sont réalisés à une intensité métabolique sous maximale (~60 %) et en mode de contraction musculaire concentrique. Ces conditions de réentrainement et les adaptations associées sont cependant limitées par la capacité des patients à atteindre ou à maintenir dans le temps de telles sollicitations. Il est donc nécessaire de mettre au point des stratégies d’entrainement alternatives prenant en considération la problématique de limitations cardiaque, respiratoire et/ou musculaire liées à la pathologie tout en permettant la mise en place de réponses adaptatives optimales. L’une des pistes est celle de l’entrainement excentrique dynamique au travers d’exercices de pédalage en résistance ou de course en descente. Comparativement à la modalité concentrique, l’excentrique à la capacité de générer des charges mécaniques importantes pour une sollicitation cardio-respiratoire moindre. Depuis plusieurs années, la faisabilité de ce type d’entrainement a été observée, y compris auprès de patients atteints de maladies chroniques. Son efficacité dans la prise de masse musculaire et le gain de force a aussi largement été démontrée. De récents travaux montrent également son intérêt dans la gestion du surpoids et de l’obésité grâce à ses effets sur la composition corporelle via de la réduction de masse grasse. Toutefois, les adaptations aérobies suite à un entrainement excentrique restent incomplètes aux regards des attentes initiales d’amélioration de la consommation d’oxygène et de la fonctionnalité mitochondriale, pour lesquelles l’intensité métabolique de l’exercice semble être le facteur déterminant. Ainsi, des approches mixtes peuvent être envisagées afin de développer les meilleures combinaisons qui permettront d’optimiser les résultats globaux de l’entrainement en améliorant à la fois la force musculaire et l’endurance
Chronic pathologies are the world leading cause of death. In addition to reducing functional capacities and degrading patients' life quality, they constitute a major public health expenditure. Part of the management involves appropriate physical activity and exercise training. This aims to improve subjects' capacities, in particular endurance and muscle strength, in order to increase their autonomy and reduce the risk of morbidity and mortality. Classically, endurance cycling or treadmill running exercises are performed at a sub-maximal metabolic intensity (~60%) and in a classic concentric muscle contraction mode. These training conditions, and the associated adaptations, are however limited by the patients' ability to achieve or maintain such stresses over time. It is therefore necessary to develop alternative strategies that take into account cardiac, respiratory and/or muscular limitations linked to the pathology while allowing optimal adaptive responses. One approach is that of dynamic eccentric training through resistance pedaling or downhill running exercises. Compared to concentric mode, eccentric has the ability to generate significant mechanical loads for less cardio-respiratory stress. For several years, the feasibility of this type of training has been observed, including in patients with chronic diseases. Its effectiveness in increasing muscle mass and strength has also been widely demonstrated. Recent work also shows its interest in overweight and obesity management through its effects on body composition and fat reduction. However, aerobic adaptations following eccentric training remain incomplete with regard to initial expectations of improved oxygen uptake and mitochondrial function, for which exercise metabolic intensity appears to be the determining factor. Thus, mixed approaches can be considered in order to develop the best combination that will optimize the overall physical training outcomes by enhancing both muscle strength and endurance

An important goal of biology is to understand the key mechanisms of evolution underlying the diversity of living organisms on Earth. In that respect, the recent innovations in the field of new generation sequencing technologies (NGS) are bringing new and exciting opportunities. This thesis presents results obtained with these tools in the specific context of the study of two sister species of cold-adapted leaf beetles, Gonioctena intermedia and G. quinquepunctata. More specifically, this work is focused around four research directions: the two first explore methods of statistical inference using a spatially explicit model of coalescence, by (1) evaluating the potential of various summary statistics to discriminate phylogeographic hypotheses, and (2) investigating the dispersal abilities of a montane leaf beetle, G. quinquepunctata, using an original method that avoids using summary statistics. The third research direction focuses on the adaptation to cold conditions in this montane leaf beetle, by testing the association between genetic polymorphism across tens of thousands of genetic markers and altitude in samples collected at various elevation levels in the Vosges (France). Finally, the fourth, and last, research axis presents the discovery of mitochondrial heteroplasmy, i.e. the presence in an individual of multiple copies of the mitochondrial genome, in natural populations of G. intermedia. We illustrate, here, how NGS technologies could help identify this phenomenon, probably underestimated in animals, on a large scale.
Doctorat en Sciences
info:eu-repo/semantics/nonPublished

La formation du glutaminyl (Q)-tRNAQ cytoplasmique (c) permettant l’insertion de Q dans les protéines lors de la traduction ribosomale se fait généralement par aminoacylation directe de l’ARNt par une Q-ARNt synthétase (QRS). Cependant les mécanismes de synthèse du QtRNAQ mitochondrial (m) requis pour le bon fonctionnement de la machinerie traductionnelle organellaire ne sont toujours pas bien caractérisés. En effet, aucune mQRS n’a été retrouvée dans les génomes eucaryotiques séquencés jusqu’à ce jour. Ainsi, il est impossible de prédire quelle voie génère cet aminoacyl (aa)-ARNt dans un eucaryote donné. Les eucaryotes ont à priori deux possibilités pour générer du Q-mtRNAQ: soit utiliser la voie directe via l’import de cQRS, ou alors utiliser une voie ARNt-dépendante de transamidation, ce qui requiert dans ce cas la présence d’une ERS non discriminante (nd) et d’une amidotransférase ARNt-dépendante (AdT) dans la mitochondrie. Nous avons montré que la protéine Pet112 fait partie d’une amidotransférase, mais également que la ndERS essentielle à la voie est l’ERS du cytoplasme qui est capable de se relocaliser dans la mitochondrie. La double localisation de la nd-cERS est contrôlée par Arc1p, son partenaire cytoplasmique. Ces résultats représentent une avancée intéressante dans le domaine de l’aminoacylation et de l’import mitochondrial. En effet nous décrivons une nouvelle stratégie : l’utilisation d’une plateforme d’ancrage cytoplasmique afin de réguler la capacité de double-localisation d’un seul et même produit traductionnel, suggérant que toute protéine dans un complexe pourrait être capable d’atteindre d’autres compartiments une fois relâchées. Nous avons alors montré que la transcription d’ARC1 est contrôlée par la voie de signalisation Snf1/4 qui induit la diminution de la quantité d’Arc1p lors de l’adaptation à la respiration. Cependant ses deux partenaires, la cERS et la cMRS, restent exprimées de manière stable ce qui induit une augmentation de la quantité de leur forme libre. Les cMRS et cERS libres sont alors capables d’être importées dans le noyau et la mitochondrie respectivement, dans le but de synchroniser l’expression des partenaires de la chaîne respiratoire (RC). En effet, les partenaires de la RC sont encodés de manière séparée dans le noyau et la mitochondrie, la cMRS promeut la transcription d’une partie des gènes de la RC qui sont encodés dans le noyau, alors que la cERS augmente le taux de traduction des partenaires de la RC produits dans la mitochondrie. En prouvant qu’Arc1p est un relai essentiel pour la voie Snf1/4 et l’adaptation à la respiration, nous décrivons pour la première fois un mécanisme de synchronisation de la chaine respiratoire. C’est ce concept qui semble être le point le plus important de mon travail, nous montrons les avantages que représente l’utilisation d’un complexe protéique en tant qu’élément synchroniseur de l’expression de gènes présents dans différents compartiments. Nous pouvons en effet penser que ce mécanisme est utilisé pour beaucoup d’autres fonctions où la synchronisation des différents acteurs est essentielle
The formation of cytoplasmic (c) glutaminyl (Q)-tRNAQ allowing insertion of Q into proteins during ribosome-mediated translation proceeds via direct tRNA aminoacylation by a specific Q-tRNA synthetase (QRS). However, the synthesis of mitochondrial (m) Q-tRNAQ required for the specific organellar translation system is still matter of debate. In fact, no mQRS can be found in any eukaryotic genomes sequenced so far. Thus, it is almost impossible to predict which pathway, direct or indirect, generates this organellar aminoacyl (aa)-tRNA species in a given eukaryote. Eukaryotes have, a priori, two possibilities to generate a Q-mtRNAQ: either they use the direct pathway via the import the cQRS or they use an indirect tRNA-dependent transamidation pathway which implies the presence of a non discriminating (nd) ERS and of a tRNA-dependent amidotransferase (AdT) in the organelle. We have shown that Pet112 is a part of a yeast mitochondrial amidotransferase, but also that the necessary ndERS is the cytoplasmic form of ERS (nd-cERS) which is able to be localized both in the cytoplasm and the mitochondrion. The dual localization of the nd-cERS is controlled by Arc1p, the cytoplasmic partner of the nd-cERS. This project represents an important breakthrough in the fields of aminoacylation and mitochondrial import. We describe a new strategy: the use of a cytosolic anchoring platform, for the dual localization of a single translational product, suggesting that any protein in a complex, even if well characterized in a specific subcellular compartment, might be able to reach other compartments upon release from the complex. We then show that ARC1 transcription is controlled by the Snf1/4 pathway that decreases Arc1p upon adaptation to respiration. However, its two partners, cERS and cMRS, stay stably expressed leading to an increase of the free cMRS and cERS pools. These released forms are then imported in the nucleus and the mitochondria respectively, in order to synchronize expression of respiratory chain (RC) partners. RC partners are encoded in a split manner in the nucleus and the mitochondrion, cMRS promotes transcription of a subset of the RC genes encoded by the nucleus, whereas cERS increase the translation rate of mitochondrial-encoded partners of the RC. By proving that Arc1p is an essential relay for the Snf1/4 pathway we propose for the first time a mechanism explaining how synchronization of the RC gene expression is achieved. This represents the most important conceptual change we made, in which we show the advantages of the dynamic control of a protein complex as a strategy to synchronize gene expression of genomes located in different compartments

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Com aumento global da temperatura, compreender como o metabolismo de bovinos se adapta ao estresse térmico, permitirá selecionar animais melhor adaptados ao aumento da temperatura ambiente. Uma das possibilidades é compreender como cada raça responde a esse efeito ambiente. O trabalho objetivou expor ao sol duas raças distintas de bovinos com diferente resistência ao calor e estudar as alterações no metabolismo mitocondrial e comportamental após a exposição ao sol ou não por 60 dias. Foram analisados 23 animais da raça Angus – susceptível ao calor (12 ao sol e 11 sob sombra) e 25 Nelores – tolerantes ao calor (13 ao sol e 12 sob sombra). Após todos os animais passarem por período de adaptação de 60 dias, foram divididos entre os grupos e analisados os parâmetros ambientais (THI, HLI e AHLU), movimentação (por radiotelemetria), concentrações séricas de testosterona e LH por radioimunoensaio, espessura de tecido adiposo subcutâneo por ultrassonografia e atividade enzimática mitocondrial de músculo esquelético do complexo I (NADH oxidoredutase). A atividade mitocondrial foi avaliada em amostras da musculatura do tríceps braquial colhidas por biópsia periodicamente ao longo do experimento. Os parâmetros ambientais caracterizam o desconforto térmico que os animais foram submetidos, principalmente Angus sol com índice de AHLU muito acima de estresse extremo. Os Angus se movimentaram mais que os Nelore durante o período experimental (p<0,05) e os Nelore se movimentaram mais à noite em comparação ao dia (p<0,05). As concentrações de testosterona aumentaram do período de adaptação para o experimento para ambas as raças e tratamentos. Os animais da raça Nelore apresentaram maior depósito de gordura subcutânea que os Angus. A atividade mitocondrial aumentou significativamente do período de adaptação (P0) para o experimento (P1) e não diferiu durante o experimento entre as raça e os tratamentos. Animais endotérmicos mantidos em altas temperaturas respondem com adaptações metabólicas e comportamentais mediante o estresse exposto. Os Nelore se movimentam mais à noite quando a temperatura é mais amena e os Angus durante o dia à procura de conforto térmico. A atividade mitocondrial é aumentada devido a alta demanda de ATP provavelmente em repercussão da necessidade de sinalizar biogênese mitocondrial, reparação celular, movimentação e níveis aumentados de testosterona.
Cattle farmers are suffering challenges in the animal breeding, consequence of the global increase of the temperature. Understanding how the homeothermic metabolism adapts to the thermal stress, allows the selection of better adapted animals to the of the environmental temperature increase. One of those possibilities is to understand each breed reacts to this ambient effect. The work aimed to expose in the sun two different bovine breeds that have different heat resistance and evaluate the mitochondrial metabolism and behavioral changes during sun exposure. Twenty three Angus - susceptible to heat (12 in the sun and 11 in shadow) and 25 Nellore - heat tolerant (13 in the sun and 12 in shadow) animals were analyzed. After all the animals went through a 60 days adaptation period, the animals were sorted composing the groups and analyzed the environmental parameters, movement (by radiotelemetry) subcutaneous fat thickeness and mitochondrial enzymatic activity from squelectical muscle samples.The activity mitochondria were evaluated by the enzymatic reaction of the compound I (NADH oxidoreductase) in muscular tissue samples that were collected periodically during the experiment. The environmental parameters characterize the thermal discomfort that the animals were subjected to, mainly Angus sun with AHLU index well above extreme stress. The Angus moved more than the Nellore during the experimental period (p <0.05) and the Nellore moved more at night compared to the day (p <0.05). Testosterone concentrations increased from the adaptation period to the experimental for both breeds and treatments. The animals of the Nelore breed presented greater subcutaneous fat deposition than the Angus. Enzymatic mitochondrial activity increased significantly from the adaptation period (P0) to the experiment (P1) and did not differ during the experiment between breed and treatments. Homeothermic animals kept at high temperatures respond with metabolic and behavioral adaptations through exposed stress. The Nelore move more at night when the temperature is milder and the Angus during the day looking for thermal comfort. Mitochondrial activity is increased due to high ATP demand probably in repercussion of the need to signal mitochondrial biogenesis, cell repair, movement and increased levels of testosterone.

Exposure to real or simulated microgravity is sensed as a stress by mammalian cells, which activate a complex adaptive response. Culture of human endothelial cells for 10 days in real microgravity onboard the ISS resulted in the modulation of more than a thousand genes, some of which involved in stress response. We cultured human endothelial cells for 4 and 10 days in the Rotating Wall Vessel, a NASA developed surrogate system for bench-top microgravity research on Earth. We highlight the crucial role of the early increase of HSP70, since its silencing markedly impairs cell survival. Once HSP70 upregulation fades away after 4 days of simulated microgravity, a complex and articulated increase of various stress proteins - SIRT2, PON2, SOD2, p21, HSP27, P-HSP27 all endowed with cytoprotective properties – occurs and counterbalances the upregulation of the pro-oxidant TXNIP. Interestingly, TXNIP was the most overexpressed transcript in endothelial cells after space flight. We conclude that HSP70 upregulation sustains the initial adaptive response of endothelial cells to mechanical unloading and drives them towards the acquisition of a novel phenotype that maintains cell viability and function through the involvement of different stress proteins. We also demonstrated that mitophagy contributes to endothelial adaptation to gravitational unloading. After 4 and 10 days of exposure to simulated microgravity in the Rotating Wall Vessel, the amount of BNIP3, a marker of mitophagy, was increased and, in parallel, mitochondrial content and oxygen consumption were reduced, suggesting that HUVEC acquire a thrifty phenotype to meet the novel metabolic challenges generated by gravitational unloading. Moreover, we suggested that microgravity induced-disorganization of the actin cytoskeleton triggers stress adaptation and mitophagy, thus creating a connection between cytoskeletal dynamics and mitochondrial content upon gravitational unloading. We also found that Mg homeostasis was modulated in microgravity, since a reduction of total intracellular magnesium and modulation of its transporters was found in EC exposed to simulated microgravity. We also investigated a new 3D cell culture method, a microfluidic system where EC are cultured in 3D and in presence of fluid laminar flow, in perspective of using these systems for experiments in microgravity.

Le but de ce travail a été d'étudier l'adaptation β-cellulaire au stress oxydant (SO) dans un modèle animal de diabète de type 2, le rat GK/Par. Dans le premier volet de cette thèse, nous avons évalué le stress oxydant insulaire et l'impact des espèces réactives de l'oxygène (ERO) sur la sécrétion de l'insuline. Les îlots de rats diabétiques GK/Par apparaissent protégés contre le SO, car (i) les marqueurs de SO ciblent essentiellement la périphérie insulaire (aires vasculaires et inflammatoires) ; (ii) l'accumulation d"ERO est plus faible comparativement aux îlots témoins (Wistar) ; (iii) leur sécrétion de l'insuline n'est pas altérée après exposition aux ERO ; et (iv) ces adaptations sont associées à une augmentation des défenses antioxydantes insulaires. Dans un deuxième volet, nous avons vérifié si ces mécanismes adaptatifs pouvaient expliquer la protection des cellules β contre l'apoptose chez le rat GK/Par diabétique. Nous avons montré que le phénotype particulier de l'îlot GK/Par diabétique consiste, ex vivo, en une augmentation des gènes anti-apoptotiques, associée in vitro à une résistance contre l'apoptose induite par les ERO. Nous avons démontré que l'AMPc joue un rôle dans cette résistance à l'apoptose. Compte tenu de l'existence de sources d'ERO proches des cellules endocrines insulaires chez le rat GK/Par, nous avons, dans un dernier volet, étudié le rôle d'une dysfonction endothéliale et du processus inflammatoire associé (phénomène au potentiel pro-oxydant) dans l'adaptation antioxydante des îlots. Le blocage de la voie IL-1 via un traitement in vivo par IL-1Ra a permis, au niveau insulaire, de diminuer l'activation endothéliale, le stress inflammatoire et de normaliser l'expression des gènes antioxydants. Enfin, puisqu'il existe une production d'ERO insulaire précoce chez le rat GK/Par (avant l'installation du diabète), nous suggérons qu'elle soit à l'origine de l'augmentation des défenses antioxydantes/anti-apoptotiques. Cette adaptation est en place après installation du diabète et permet à l'îlot GK/Par de contrer l'environnement diabétogène pro-oxydant (glucolipotoxicité, inflammation). Enfin, nous proposons que la dysfonction β-cellulaire engendrée par un excès d'antioxydants endogènes soit le prix à payer pour protéger la cellule β du rat GK/Par de la mort cellulaire.

Les complexes multisynthétasiques (MSC) sont des complexes multi-protéiques identifiés dans un grand nombre d’organismes pro- et eucaryotes. Ils impliquent des protéines d’assemblages et des aminoacyl-ARNt synthétases (aaRSs), responsables de l’aminoacylation de leurs ARNts homologues au cours de la traduction. La taille et la composition des MSC varient selon les organismes, et le rôle de ces complexes n’est pas encore totalement compris. Il semblerait néanmoins que chez les eucaryotes, l’accrétion en complexe soit une stratégie mise en oeuvre par les cellules pour empêcher les aaRSs d’assurer des fonctions additionnelles. Chez S.cerevisiae,nous montrons que la dynamique du complexe AME, composé de la méthionyl- et de la glutamyl-ARNt synthétase (MRS et ERS) ainsi que de la protéine d’ancrage Arc1p, est dépendante du métabolisme de la levure. En respiration la MRS joue le rôle de facteur de transcription et régule l’expression des gènes nucléaires du complexe III et V de la chaîne respiratoire, tandis que l’ERS active la traduction mitochondriale. Cette étude montre que la relocalisation synchrone est primordiale pour l’adaptation des cellules au métabolisme respiratoire
Multisynthetase complexes (MSC) are complexes made of several proteins and were identified in a wide variety of organisms from pro- to eukaryotes. They are usually made of assembly factors and aminoacyl-tRNA synthetases (aaRSs), which are responsible for the aminoacylation of their corresponding tRNAs during translation. Depending on the organisms, size and composition of these complexes differ greatly and their role is not fully understood yet. Although it seems that in eukaryotes, accretions of aaRSs into MSC prevent aaRSs to perform their additional functions. In the yeast Saccharomyces cerevisiae, we show that the dynamic of the AME complex, made of the méthionyl- and glutamyl-tRNA synthetases (MRS and ERS) and the assembly protein Arc1p is linkedto yeast metabolism. In respiration, MRS is imported in the nucleus to act as a transcription factor and regulates the expression of nuclear genes belonging to complex III and V of the respiratory chain, while ERS is imported in mitochondria to activate translation. This study shows that synchronous relocation of both aaRSs is crucial for yeast cells to adapt to respiratory metabolism

Plutella xylostella (L. ) (Lepidoptera : Plutellidae) est un ravageur important des cultures de Brassicacées dans le monde. Les problèmes pour lutter contre les populations de ce ravageur sont essentiellement liés à des résistances aux pesticides et à des échecs de la lutte biologique. Des différences morphologiques, biologiques et génétiques ont été étudiées entre des populations mondiales d'origines géographiques différentes. Pour l'étude des différences morphologiques et biologiques, deux paramètres ont été mesurés : la longueur des nymphes et l'activité de ponte des femelles. La longueur des nymphes et la fécondité sont variables au sein des populations et entre elles. La répartition des œufs au cours de la période de ponte permet de différencier les populations en deux groupes, liés aux régions tropicales et sub-tropicales et aux régions tempérées. L'adaptation locale des populations est discutée. Pour l'étude des différences génétiques, deux marqueurs ont été utilisés : les allozymes et les séquences nucléotidiques d'un fragment du gène mitochondrial du cytochrome oxydase I. La variabilité génétique est relativement importante au sein de P. Xylostella. Les fréquences allèliques des enzymes montrent des déficits d'hétérozygotes dans de nombreuses populations. Les populations les plus différenciées sont celles du Japon et d'Australie. L'analyse des haplotypes mitochondriaux montre que des groupes de populations se différencient d'un groupe principal. Certains groupes correspondent à une zone géographique, ce sont les populations d'Australie ou d'Amérique du Sud. Un troisième groupe est constitué de populations de différentes régions. Les processus de différenciation sont discutés
Plutella xylostella (L. ) (Lepidoptera : Plutellidae) is a major pest of brassica crops worldwide. Problems to control pest's populations are mainly related to pesticides resistance and failure of biological control methods. Morphological, biological and genetic differences were assessed among world populations with different geographic origins. In the study of the morphological and biological differences, two parameters were measured: pupae size and females oviposition activity. The size of pupae and the fecundity were variable within and among populations. The distribution of the eggs laid during the laying period allowed differentiating populations in two groups, related to tropical or sub-tropical areas and to temperate areas. The local adaptation of the populations was discussed. In the study of genetic differences, two markers were used: allozymes and nucleotidic sequences of a part of cytochrome oxydase I mitochondrial gene. The genetic variability was relatively high in P. Ylostella species. The enzymes allelic frequencies exhibited heterozygotes deficits in a majority of the populations. Populations the most different were from Japan and Australia. Analysis of mitochondrial haplotypes exhibited the differentiation of groups of populations from a major group. Two groups were related to geographic areas: populations from Australia or South America. A third group consist of populations from different regions. The mechanisms of differentiation were discussed

La compréhension des mécanismes physiologiques mis en jeu en réponse à l’hypoxie aigüe et chronique ainsi que leur retentissement global sur l’énergétique cardiaque nécessite l’application d’approches globales. L’approche intégrative développée par notre équipe, l’Analyse Modulaire du Contrôle (MoCA) permet une description quantitative de l’ensemble des interactions au sein d’un système biologique complexe décomposé en différents modules liés par des intermédiaires communs, ce qui en fait un outil puissant pour l’étude des interactions au sein de l’énergétique cardiaque, normale ou pathologique. Dans cette thèse, MoCA a été appliquée sur des coeurs isolés de souris contrôles et de souris soumises à une hypoxie chronique de 21 jours, perfusés en conditions d’oxygénation normale ou avec un milieu dont la concentration en oxygène dans le milieu a été réduite. Après exposition à l’hypoxie chronique, les coeurs sont caractérisés par une réponse (élasticité) plus importante des processus d’apport en énergie face à une variation des intermédiaires énergétiques (PCr, ATP, Pi), et ceci malgré une diminution de la masse mitochondriale. De plus, contrairement aux coeurs sains, aucune modi?cation de l’activité contractile et de la concentration d’intermédiaires énergétiques n’a été mesurée sur les coeurs hypoxiques après diminution de la concentration en oxygène dans le milieu de perfusion. Ces résultats suggèrent une adaptation fonctionnelle de l’ensemble de la bioénergétique cardiaque après exposition à l’hypoxie chronique, l’augmentation de l’élasticité des processus d’apport en énergie permettant de compenser en partie les altérations énergétiques induites par un dé?cit en oxygène
An important issue in the comprehension of the link between molecular events developed in pathologies such as chronic hypoxia adaptation, is the development of new experimental strategies aimed at the study of the integrated organ physiology. Our Modular Control Analysis (MoCA), gives quantitative information on the internal control and regulation of integrated heart energetics on the basis of a supply-demand system and is therefore of particular interest to better understand the overall effect as well as the relative importance of the various modi?cations developed during pathologies. In this thesis, MoCA was applied on isolated hearts of control and chronic hypoxic mice perfused with high or low oxygen in the medium. Despite a severe mitochondrial alteration after chronic hypoxia exposure, a surprizing higher response of energy supply (elasticity) to energetic intermediates changes (PCr, ATP, Pi) was detected in chronic hypoxic hearts. Moreover, chronic hypoxic hearts energetics was unchanged by oxygen reduction while a strong concomitant decrease in heart contractile activity and in PCr concentration, was measured in control hearts. As suggested by these results, this increase in energy-supply elasticity could be considered as an adaptive mechanism developed after chronic hypoxia counteracting hypoxia-induced altered cardiac energetics

L'objectif de cette these est l'etude des interactions genetiques entre differentes formes de truite commune (salmo trutta, l. ) dans les pyrenees occidentales francaises. Dans cette region, deux formes de truites sauvages sont presentes et cohabitent avec une forme domestique. Une etude de genetique des populations a ete realisee a l'aide de 6 locus microsatellites. En dehors des parametres genetiques classiques, l'interpretation a fait appel a des techniques d'analyses multivariees (analyse factorielle des correspondances) ou d'assignations individuelles (reseaux de neurones artificiels, methodes probabilistes). Par ailleurs, la variabilite de la region de controle de l'adn mitochondrial a ete etudiee par pcr-sscp puis sequencage. Ces marqueurs ont permis de mettre en evidence une forte structuration genetique entre populations et entre groupes de populations. Des deficits en heterozygotes importants ont ete observes dans certaines populations. Par ailleurs, l'impact des repeuplements a ete estime dans cette zone et indique une situation contrastee, avec des populations tres introgressees et d'autres peu influencees par les repeuplements. L'efficacite et l'interet de ces pratiques sont discutes. Par ailleurs, la sequence des differents haplotypes mitochondriaux a permis de les situer par rapport aux grands types europeens pour une etude phylogeographique. La plupart faisaient partie du groupe atlantique et n'avaient pas ete identifies auparavant. Des hypotheses concernant l'evolution et l'origine des populations de cette region sont proposees.

Les zones d'hybridation sont des laboratoires naturels pour l'etude des phenomenes selectifs qui preservent l'integrite des especes biologiques. Deux cyprinides, le barbeau commun et le barbeau meridional ont donne naissance a la zone d'hybridation de la lergue (affluent de l'herault, france, chapitre i) que nous avons etudiee a la fois par une approche classique de genetique des populations (allozymes) et par la methode de capture/recapture en milieu naturel. Cette zone d'hybridation presente des clines allozymiques concordants mais non coincidents (chapitre ii). Les genotypes hybrides analyses sont recombines (tres peu de f1). Les differents echantillons etudies ne presentent pas d'ecart significatifs forts par rapport aux proportions d'hardy-weinberg. Le desequilibre de liaison mis en evidence est important. L'analyse genetique d'un retrocroisement male b. Barbus x femelle f1 (male b. Meridionalis x femelle b. Barbus) a demontre l'existence d'une distorsion de segregation en faveur des alleles de barbus meridionalis a la f1 et d'un phenomene d'affinite meiotique (chapitre iii). La liaison entre les marqueurs genetiques (allozymes) et les marqueurs morphologiques (diverses mesures) est forte. L'analyse de la survie in natura a permis de montrer que les hybrides subissaient une faible contreselection (chapitre iv). La dispersion ecologique et la dispersion efficace semblent fortes (chapitre v). Nous proposons un scenario de fonctionnement de la zone d'hybridation base sur l'ensemble de ces considerations (chapitre vi)

Les mitochondries possèdent leur propre matériel génétique (ADN mitochondrial ou ADNmt) qui code pour des peptides interagissant avec ceux codés par l'ADN nucléaire pour former les complexes du système de transport des électrons (ETS) ainsi que l'ATP synthase qui participent au processus de phosphorylation oxydative (OXPHOS). Il a été suggéré que la sélection sur l'ADNmt peut mener à des haplotypes adaptés à différents environnements. Dans cette thèse, Drosophila simulans a été choisie pour examiner le potentiel adaptatif des divergences de l'ADN mitochondrial. Cette espèce présente trois haplogroupes (siII, siII et siIII) subdivisés avec approximativement 3% de divergences inter-haplogroupes mais n'ayant aucune subdivision nucléaire observée au niveau des loci codés par l'ADN nucléaire. Le principal objectif de ce travail était d'examiner le rôle de l'ADN mitochondrial sur l'établissement de caractères phénotypiques tel que le métabolisme mitochondrial des haplotypes siII et silll de Drosophila simulans et de déterminer le potentiel adaptatif des divergences du génome mitochondrial sur les propriétés fonctionnelles des mitochondries en fonction des variations de température. Le premier objectif était d'identifier les différences au niveau des performances mitochondriales et de la thermosensibilité associées à la divergence des mitotypes sill et silll de Drosophila simulans en évaluant l'activité des différentes enzymes de l'ETS à quatre températures différentes grâce à une approche in vitro (isolations mitochondriales). Nous avons montré que les différentes enzymes de l'ETS ont différentes thermosensibilités, ce qui peut mener à une distribution différente du contrôle de la respiration par les composantes de l'ETS et par les déshydrogénases en amont de l'ETS à différentes températures. Par exemple, nous avons détecté un excès apparent au niveau du complexe IV d'environ 604% et 613% pour sill et silll respectivement, mais seulement à basse température (12°C), ce qui nous a amené à penser que cela était dû à un dysfonctionnement des déshydrogénases à basse température. Le second volet de cette thèse reprenait les mêmes objectifs que le premier. Cependant, pour ce chapitre, une nouvelle méthode (approche in situ) a été développée sur des fibres musculaires perméabilisées en utilisant un protocole en respirométrie à haute résolution. Nous avons montré, et ce pour la première fois, que l'approche in situ est très appropriée pour évaluer les performances mitochondriales chez des invertébrés et serait même plus pertinente que l'approche in vitro. De plus hautes capacités catalytiques des complexes de l'ETS ont été détectées à 24°C pour le mitotype siII. Cette capacité catalytique plus élevée pour siII peut lui donner un avantage en termes d'intensité du métabolisme aérobie, d'endurance, ou des deux si l'intensité de l'exercice qui peut être effectué au niveau aérobique est dictée par la capacité aérobique du tissu. De plus, les résultats obtenus sur la thermosensibilité ont montré que même si la température affecte les capacités catalytiques des différentes enzymes de l'ETS, les mitotypes sill et silll ont une grande tolérance aux variations de température. Le troisième volet de cette thèse se concentrait sur l'évaluation du potentiel adaptatif des divergences de l'ADNmt aux quatre températures déjà testées dans les chapitres précédents en utilisant des introgressions. Les performances mitochondriales des haplotypes ainsi créés (sill-introgressé et siIII-contrôle) ont ensuite été mesurée avec l'approche in situ. Nos résultats ont montré que les capacités catalytiques des différentes enzymes de l'ETS dans les organismes introgressés (sill-introgressé) étaient quasiment similaires à celles détectées dans le mitotype sill, du moins à 24°C. De plus, les différences entre sill et silll détectées à 24°C dans le second volet se retrouvent aussi entre sill-introgressé et siIII-contrôle, dénotant que les propriétés fonctionnelles des mitochondries sont principalement conférées par l'ADN mitochondrial. Cependant, l'impact de la température divergeait entre sill-introgressé et silIl-contrôle, principalement au niveau de l'excès apparent de COX à 12°C (excès d'environ 193% pour silIl-contrôle, mais pas d'excès pour sill-introgressé) et au niveau des coefficients de température (Q10) mesurés entre 12 et 18°C. Il est donc possible que les interactions entre ADN nucléaire et ADN mitochondrial soient nécessaires pour permettre aux organismes de faire face aux variations de température. C'est, selon nos connaissances, l'une des premières démonstrations claires du potentiel adaptatif de différents ADNmt sur les propriétés fonctionnelles des mitochondries. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : respiration mitochondriale, système de transport des électrons, interactions mitonucléaires, ADNmt, température.

Changes in intracellular Ca2+ ([Ca2+]f) and high-energy phosphates are known to induce adaptive changes in skeletal muscle during endurance exercising training, including mitochondrial biogenesis. Levels of [Ca2+]f are regulated by sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs) which are further regulated by sarcolipin (SLN), through a reduction in the apparent affinity of SERCAs for Ca2+. Furthermore, SLN reduces the efficiency of Ca2+ transport by SERCAs supporting a thermogenic role for SLN in skeletal muscle. Thus, it is possible SLN ablation could reduce Ca2+ and metabolic signaling during exercise training and attenuate increases in mitochondrial content. To investigate the potential role of SLN in the exercise-induced adaptive response of skeletal muscle, mice devoid of SLN (SLNKO) underwent endurance training for 8 weeks and were compared to WT controls. Maximal oxygen uptake (V̇O2 max) was measured with an exercise stress test while mitochondrial content was assessed through measurement of protein expression and maximal enzyme activities of several mitochondrial enzymes in soleus and extensor digitorum longus (EDL) muscles, which express high and low levels of SLN, respectively. All data were analyzed using a two-way analysis of variance (ANOVA) and student t-tests were conducted on enzyme data. V̇O2 max was found to not be significantly altered with exercise training in either genotype. Exercise training significantly increased the contents of adenine nucleotide translocase (ANT), cytochrome-c (cyt-c) and cytochrome-c oxidase subunit IV (COXIV) in soleus independent of genotype. Likewise, exercise training significantly increased cyt-c and COXIV expression (P<0.04), while increases in ANT expression were not significant (P=0.13) in the EDL. Two-way ANOVAs of mitochondrial enzymes in soleus revealed an interaction existed for succinate dehydrogenase (SDH) where its activity was increased only in the SLNKO mice (P<0.02). In comparison, exercise training significantly elevated activities of cytochrome c oxidase (COX) and citrate synthase (CS) activities (P<0.02) but not β-hydroxyacyl-CoA dehydrogenase (β-HAD; P=0.08), independent of genotype. Upon closer examination using student t-tests, it was determined that exercise training induced greater increases in COX and CS activity in SLNKO compared to WT controls (P<0.02), similar to and consistent with SDH data. In EDL, only SDH activity increased following exercise training, an effect that was independent of genotype. In conclusion, these data suggest that SLN ablation does not attenuate exercise-induced mitochondrial adaptations and may increase mitochondrial enzyme adaptations to exercise training in slow-twitch muscle. Further examination of the effects of SLN on Ca2+ and metabolic signaling may provide mechanisms explaining the results of this thesis.

Mitochondria are key components of skeletal muscles as they provide the energy required for almost all cellular activities, and play an important role in ageing and cell pathology. Different forms of exercise training have been associated with mitochondrial adaptations, such as increased mitochondrial content and function, and enhanced mitochondrial biogenesis, as well as improved endurance performance. However, the role of training intensity and training volume, in determining these changes remains elusive. Therefore, the aim of this thesis was to investigate the role of training intensity and volume on changes in mitochondrial content and function (as measured by mitochondrial respiration in permeabilised muscle fibres), in the skeletal muscle of healthy humans, and to study the molecular mechanisms underlying these changes. It was demonstrated that training intensity is a key factor regulating changes in mitochondrial respiration, but not mitochondrial content, and that an apparent dissociation exists between changes in these two parameters. Training consisting of repeated 30-s “all-out” sprints lead to improved mitochondrial (mt)-specific respiration (indicative of improved mitochondrial quality). Conversely, training volume was shown to be a key factor regulating mitochondrial content, with the associated increase in mitochondrial respiration being likely driven by the increase in mitochondrial content (i.e., unchanged mt-specific respiration). A training volume reduction resulted in a rapid decrease in most mitochondrial parameters, underlining the importance of maintaining the training stimulus to preserve training-induced mitochondrial adaptations. The protein content of PGC-1α, p53 and PHF20 was shown to be regulated in a training intensity-dependent manner, and was more strongly associated with changes in mitochondrial respiration rather than content, whereas changes in the protein content of TFAM were primarily associated with changes in mitochondrial content. Moreover, it was demonstrated that exercise intensity induced an increase in nuclear PGC-1α protein content and nuclear p53 phosphorylation, two events that may represent the initial phase of different pathways of the exercise-induced adaptive response. Collectively, this research provides novel information regarding mitochondrial adaptations to different training stimuli, and could have important implications for the design of exercise programs in conditions of compromised mitochondrial function.

Dissertação de Mestrado em Biologia Celular e Molecular apresentada à Faculdade de Ciências e Tecnologia
A hipóxia tecidual resulta do desequilíbrio entre a necessidade e o fornecimento de oxigénio. A insuficiente disponibilidade de oxigênio perturba a homeostasia redox e metabólica. Assim, múltiplas alterações metabólicas e transcripcionais ocorrem para prevenir danos nos tecidos ou até mesmo a morte celular. O HIF-1α é um fator de transcrição crucial na indução destas respostas adaptativas à hipóxia. No entanto, vários fatores podem exacerbar essa resposta, como é o caso da hiperglicemia. Como as mitocôndrias são um dos primeiros organelos afetados, tanto pela hipoxia como pela hiperglicemia, estas também estão envolvidas nas primeiras etapas da resposta adaptativa. Sesn2, uma proteína antioxidante ativada pela hipóxia, tem vindo a ser associada a biogênese mitocondrial e mitofagia, demonstrando a sua capacidade de melhorar a saúde mitocondrial. Assim, a nossa hipótese é que a Sesn2 possa estar envolvida na resposta adaptativa à hipoxia, através da modulação da morfologia e função mitocondrial.No presente estudo, usámos um modelo de privação de oxigênio e glicose (OGD/R) para estudar in vitro os mecanismos envolvidos na resposta adaptativa à hipoxia e como esta pode ser influenciada pela hiperglicemia e também explorar um possível papel da Sesn2 nesta adaptação. Os nossos resultados parecem indicar que a exposição a condições de hiperglicémica durante um mês altera a resposta adaptativa à hipóxia, que se refletiu numa aparente diminuição dos níveis de LC3-II/LC3-I, sugerindo a existência de prejuízos na ativação da autofagia. Além disso, a expressão genética de FIS1 e PGC-1α foi parcialmente aumentada durante OGD1h e OGD/R1h ao passo que esta expressão diminuiu às 6h de recuperação quando comparada às células C2C12 na euglicemia. Estes resultados sugerem que a hiperglicemia pode afetar a resposta adaptativa principalmente a nível mitocondrial. Ainda acerca do envolvimento da Sesn2 nestas alterações, analisámos o conteúdo proteico e a sua expressão genética. Verificámos que os níveis de Sesn2 estavam significativamente aumentados nas células em euglicemia 6h após a exposição OGD1h em comparação com as células em hiperglicemia. Esta diferença parece ser observada na expressão genética de SIRT1 e PGC-1α, sugerindo que a Sesn2 pode promover a biogênese mitocondrial através da via SIRT1/PGC-1α durante OGD e OGD/R. Além disso, também suspeitamos que a indução da expressão da Sesn2 tenha sido mediada por HIF-1α, e que a Sesn2 também pode promover biogênese mitocondrial através da diminuição da acumulação de HIF-1α e, consequentemente, induzir o aumento da expressão de PGC-1α.
Tissue hypoxia results from the mismatch between oxygen demand and oxygen delivery. The insufficient availability of oxygen perturbs redox and metabolic homeostasis. Thus, multiple transcription and metabolic alterations occur to prevent tissue damage or even cell death. HIF-1α is a crucial transcription factor involved in the induction of these adaptive responses to hypoxia. However, several factors can exacerbate this response, namely, hyperglycemia. As mitochondria are one of the primary organelles affected by both hypoxia and hyperglycemia, they are also involved in the first steps in the adaptive response. Sesn2, an antioxidant protein activated by hypoxia, has been implicated in mitochondrial biogenesis and mitophagy, demonstrating its ability to improve mitochondrial health. Thus, we hypothesized that Sesn2 can be involved in the adaptive response, through the modulation of mitochondrial morphology and function during hypoxia.In the current study, we used an oxygen-glicose deprivation (OGD/R) model to study in vitro the mechanisms involved in the adaptive response to hypoxia and how hyperglycemia influences it and also to explore a possible role of Sesn2 in that adaptation. We found that hyperglycemia exposure for one month seems to alter the adaptive response to hypoxia that was reflected in an apparent decrease in LC3-II/LC3-I protein, suggesting impairment in autophagy activation. Furthermore, the gene expression of FIS1 and PGC-1α was partially increased during OGD1h and OGD/R1h whereas it decreased at 6h recovery when compared to C2C12 cells in euglycemia. This demonstrates that hyperglycemia affects the adaptive response mainly at the mitochondrial level. To conclude about the involvement of Sesn2 in that alterations, we analyzed protein content and gene expression of Sesn2. We verified that the levels of Sesn2 were significantly increased in cells in euglycemia 6h after OGD1h exposure in comparison to cells in hyperglycemia. This difference seems to be observed in the gene expression of SIRT1 and PGC-1α. So, we suspect that Sesn2 might promote mitochondrial biogenesis through SIRT1/PGC-1α pathway during OGD and OGD/R. Furthermore, we also suspect that the induction of Sesn2 expression was induced by HIF-1α and that Sesn2 may also promote mitochondrial biogenesis by decreasing HIF-1α accumulation and, consequently, increasing PGC-1α expression.

Tese de mestrado, Oncobiologia, Universidade de Lisboa, Faculdade de Medicina, 2019
Durante o desenvolvimento e progressão do tumor, as células cancerígenas adquirem um conjunto de caraterísticas – the hallmarks of cancer – que contribuem para o crescimento, proliferação e invasão do tumor. Uma dessas características reside na capacidade das células malignas reprogramarem o seu metabolismo celular. Em 1926, Otto Warburg constatou que, as células tumorais adotavam preferencialmente a glicólise em relação à fosforilação oxidativa como forma de obtenção de energia mesmo na presença de oxigénio, dando particular relevância a esta característica das células tumorais. Este fenómeno, denominado “efeito de Warburg” é também conhecido por glicólise aeróbica e é uma característica transversal à maioria dos tumores. Desde então, múltiplas adaptações metabólicas têm sido descritas, incluindo, por exemplo, a elevada síntese de ácidos gordos e a sobreexpressão de enzimas específicos de determinadas vias metabólicas. Além disso, estudos recentes têm demonstrado que o “efeito de Warburg” não é uma condição sine qua non de todos os tumores e a fosforilação oxidativa assume um papel relevante na agressividade do cancro, nomeadamente num contexto de migração e invasão das células tumorais. Atualmente, o cancro da mama é o tipo de cancro mais frequente e o segundo mais mortífero entre mulheres. Em parte, este problema resulta da enorme complexidade do tumor do ponto de vista molecular e morfológico, o que se traduz numa ampla diversidade de situações clínicas que, por sua vez, são afetadas pelas caraterísticas biológicas do hospedeiro, contribuindo ainda mais para a heterogeneidade tumoral. Existem diversos tipos de cancro da mama, sendo estes classificados de acordo com a presença ou ausência de expressão de recetores hormonais de estrogénio (ER) e progesterona (PR) e do recetor do fator de crescimento epidérmico 2 (HER2). Devido às caraterísticas intrínsecas de maior agressividade e à falta de terapêutica dirigida, o cancro da mama triplo-negativo apresenta pior prognóstico comparativamente aos outros subtipos. O microambiente do tumor assume um papel crucial na progressão do cancro, uma vez que as células não-malignas que constituem o estroma tumoral produzem e libertam moléculas que podem ter uma função pro-tumoral. A par dos macrófagos e dos fibroblastos, os adipócitos são um dos componentes principais do estroma do cancro da mama e existe um importante crosstalk entre estes e as células tumorais, através da libertação recíproca de moléculas sinalizadoras, metabolitos e citocinas, que favorecem o desenvolvimento da doença. Além disso, os adipócitos podem fornecer lípidos às células tumorais que, por sua vez, podem ser utilizados como “combustível” para o crescimento e proliferação do tumor. Adicionalmente, a maioria dos tumores apresenta desregulação do metabolismo lipídico, fator que potencializa os efeitos anteriormente mencionados, contribuindo para um fenótipo mais agressivo. Os estudos epidemiológicos não oferecem dados muito consensuais acerca da associação entre elevados níveis de LDL e elevado risco de cancro da mama. Contudo, um estudo recente mostrou que determinadas variações genéticas associadas a elevados níveis de colesterol correlacionam-se positivamente com risco de cancro da mama. Adicionalmente, o nosso Laboratório mostrou que, durante o diagnóstico do cancro da mama, mulheres com elevados níveis de LDL desenvolvem tumores maiores e com maior índice proliferativo e, in vivo, ratos sujeitos a dieta rica em colesterol também apresentam estas caraterísticas: tumores maiores e com maior índice proliferativo. Observou-se também em estudos in vitro, em ambientes ricos em LDL, o aumento da capacidade migratória e proliferativa, bem como a perda de expressão de proteínas envolvidas nos mecanismos de adesão celular (caderina, claudina 7 e ocludina, por exemplo) e a sobreexpressão de intermediários das vias Akt e ERK, ambas responsáveis por uma resposta de sobrevivência. Contudo, os mecanismos moleculares subjacentes à maior agressividade tumoral induzida pela exposição a LDL permanecem pouco esclarecidos. Além da reprogramação do metabolismo celular, as células tumorais também apresentam frequentemente disfunções da dinâmica mitocondrial. As mitocôndrias são organelos versáteis, capazes de modelar a sua forma e distribuição na célula, consoante as funções que estão a desempenhar e as necessidades da mesma. Relativamente à dinâmica, as mitocôndrias podem apresentar-se com uma forma mais oval e alongada, designando-se por “mitocôndrias elongadas” ou mais redondas e pequenas denominando-se por “mitocôndrias fragmentadas”. O balanço entre mitocôndrias elongadas e fragmentadas é controlado por um processo de fissão-fusão, bastante regulado do ponto de vista fisiológico e que se encontra alterado nas células tumorais. Por exemplo, as células tumorais apresentam essencialmente uma rede mitocondrial mais fragmentada e tal está associado a um fenótipo mais agressivo. Em relação à distribuição celular, a rede mitocondrial pode localizar-se preferencialmente em torno do núcleo – rede perinuclear - ou distribuir-se ao longo de toda a célula, sob a forma de longos filamentos – rede filamentosa. Neste processo, o citosqueleto com o auxílio de proteínas motoras desempenham um importante papel na mobilização da rede mitocondrial ao longo da célula. Tendo em conta todos estes aspetos, não é surpreendente que as células tumorais consigam reprogramar o metabolismo, a morfologia, a dinâmica e a própria distribuição da rede mitocondrial na célula, em função do microambiente que as rodeia e do que é mais favorável para o crescimento e proliferação do tumor. A reprogramação do metabolismo lipídico pode estar associada à disfunção mitocondrial, no entanto os mecanismos envolvidos na regulação destes dois fatores permanecem pouco esclarecidos, nomeadamente no contexto de migração e invasão das células tumorais. Outro problema responsável pela elevada taxa de recidiva e de morte em pacientes com cancro, é a aquisição de resistência às terapêuticas. Devido à falta de terapêutica dirigida, em pacientes com cancro da mama triplo-negativo este fator encontra-se agravado, uma vez que os pacientes estão sujeitos às terapêuticas mais convencionais, nomeadamente à quimioterapia. Estudos recentes têm reportado a disfunção mitocondrial e a reprogramação do metabolismo lipídico como mecanismos de resistência à quimioterapia em cancro da mama. Deste modo, a caraterização dos aspetos morfológicos e moleculares subjacentes à reprogramação metabólica e à disfunção mitocondrial envolvidos na resistência à quimioterapia são importantes, pois podem contribuir para o desenvolvimento de eventuais terapias dirigidas e desse modo reverter a resistência aquirida. O presente estudo focou-se na resposta a duas questões principais: por um lado, pretendeu-se compreender de que modo as mitocôndrias se adaptam à exposição a LDL e como é que isso de algum modo contribui para um aumento da capacidade migratória em cancro da mama triplo-negativo; por outro lado, também decidiu-se explorar a influência do papel da exposição crónica a um agente quimioterapêutico, nomeadamente o Taxol, no fenótipo de células tumorais de cancro da mama triplo-negativo (mais especificamente, na capacidade migratória e proliferativa e na massa mitocondrial), bem como se a exposição a LDL também influencia estes parâmetros nestas mesmas células. No geral, os nossos resultados sugerem que o fenótipo agressivo provocado pela exposição a LDL, em células de cancro de mama triplo-negativo, é acompanhado por alterações em diversos parâmetros relacionados com a morfologia e rede mitocondrial. Adicionalmente, os nossos resultados mostram que este facto também poderá ser dependente de interações específicas entre mitocôndria e citosqueleto. Por sua vez, no contexto de resistência à quimioterapia, os dados mostraram que os efeitos produzidos pelo LDL são particularmente pronunciados após uma exposição prolongada ao Taxol e não durante o tratamento. Assim, este estudo fornece novos conceitos acerca do papel do LDL na agressividade do cancro da mama triplo-negativo, incluindo no contexto de resistência à terapia, um assunto ainda muito pouco explorado, providenciando, deste modo, potenciais ideias que poderão ser relevantes para realização de estudos posteriores e as quais poderão ter interesse do ponto de vista clínico.
Breast cancer remains the most common cancer and the second cause of cancer related death among women despite the significant advances in diagnostic and therapy. In one hand, tumors display great molecular and morphological heterogeneity; on the other hand, host biological characteristics contribute to the wide diversity of clinical situations. During cancer progression, tumor cells acquire specific features, designated as the hallmarks of cancer that enable tumor growth, proliferation and dissemination, which globally may contribute to escape apoptosis and therapeutic intervention. Deregulated cellular energetics is nowadays considered an emerging hallmark of cancer. In particular, abnormal lipid metabolism has been reported as one of the metabolic adaptations exhibited by tumors, including breast cancer, which allows the acquisition of aggressiveness and favors tumor invasion and metastasis. Besides presenting deregulated cellular energetics, tumor cells often exhibit mitochondrial dysfunction. Reprogramming of lipid metabolism has been associated with mitochondrial dysfunction, but the exact nature of the mechanisms underlying this regulation remains poorly understood. Resistance to therapy, including chemotherapy, is a major cause of cancer related death. More recently, it was described that reprogramming lipid metabolism, as well as mitochondrial dysfunction are both involved in the acquisition of resistance to conventional cancer therapy. In this study, we aim to unravel how mitochondria adapt in high hypercholesterolemic environments (LDL) and if mitochondrial adaptations are required for the acquisition of increased migratory and proliferative phenotype of triple-negative breast cancer (TNBC) under these conditions. Also, we aim at understanding how chronic exposure to a chemotherapeutic agent, Taxol, influences TNBC cells mitochondrial mass and phenotype (migration and proliferation capacity) and if LDL exposure could modulate these parameters. Our results suggest that LDL-induced aggressiveness is accompanied by adaptations in mitochondrial morphology and network arrangement, which could be dependent on specific mitochondria-cytoskeleton interactions. Regarding response to chemotherapy, our data show that the effects produced by LDL exposure are particularly pronounced after prolonged exposure to Taxol and not during acute treatment. Overall, this study provides novel mechanistic concepts about the impact of LDL exposure in the acquisition of TNBC cells aggressive phenotype, including response to chemotherapy, which offer important insights for the development of future studies.

博士
國立陽明大學
生化暨分子生物研究所
100
Myoclonic epilepsy and ragged-red fibers (MERRF) syndrome is a maternally inherited mitochondrial encephalomyopathy characterized by various clinical presentations involving both muscular and nervous systems. It has been documented that 80-90% of the patients with MERRF syndrome are caused by the A8344G mutation in the tRNALys gene of mitochondrial DNA (mtDNA) that leads to severe defects in mitochondrial protein synthesis. Mitochondrial dysfunction caused by mtDNA mutation results in not only inefficient generation of ATP but also increased production of reactive oxygen species (ROS). Previously, primary cultures of skin fibroblasts from several patients with MERRF syndrome were established in our laboratory. In the present study, I first investigated the energy metabolism shifts from mitochondrial oxidative phosphorylation (OXPHOS) to glycolysis as a bioenergetic adaptation in skin fibroblasts from MERRF patients as compared to those from age-matched normal subjects. I contended that the bioenergetic metabolism in skin fibroblasts would be perturbed by oxidative stress. In order to unravel the molecular mechanism involved in the alteration of energy metabolism in response to oxidative stress, I treated normal human skin fibroblasts (CCD-966SK cells) with sub-lethal doses of H2O2 to study the response of bioenergetic adaptation. The results showed that several glycolytic enzymes including hexokinase type II (HK II), lactate dehydrogenase A (LDHA) and glucose transporter 1 (GLUT1) were up-regulated in H2O2-treated normal skin fibroblasts. In addition, the glycolytic flux of skin fibroblasts was increased by H2O2 in a dose-dependent manner through the activation of AMP-activated protein kinase (AMPK) and phosphorylation of its downstream target, phosphofructokinase 2 (PFK2). Moreover, I found that the AMPK-mediated increase of glycolytic flux was accompanied by an increase of intracellular NADPH content in response to H2O2–induced oxidative stress via the action of glucose-6-phosphate dehydrogenase (G6PD). By treatment of the CCD-966SK cells with glycolytic inhibitors, an AMPK inhibitor, and genetic knockdown of AMPK, respectively, I found that the H2O2-induced increase of NADPH was abrogated and resulted in the overproduction of intracellular ROS and cell death. Significantly, I showed that the phosphorylation level of AMPK and glycolytic flux were also increased to confer an advantage of survival for MERRF skin fibroblasts. Taken together, I suggest that the increased production of NADPH by AMPK- mediated increase of the glycolytic flux contributes to the oxidative stress adaptation of MERRF skin fibroblasts and H2O2-treated normal skin fibroblasts, respectively. The findings of this study have provided new information for us to better understand the bioenergetic response to oxidative stress of human skin fibroblasts and shed new light in the unraveling of the molecular basis of the pathophysiology of mitochondrial diseases such as MERRF syndrome.

Tese de mestrado, Ciências Biofarmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2017
Cancer cells can acquire biological capabilities that enable tumor growth and dissemination, including the reprograming of their metabolism in order to obtain the necessary energy and metabolic intermediates to sustain tumor proliferation. The energetic reprogramming in cancer was first proposed by Otto Warburg, who suggested an anomalous energetic metabolism in cancer cells. This metabolic adaptation is characterized by an increased dependence on mitochondrial respiration and fatty acid boxidation (FAO), which has been recently correlated with increased tumor progression and aggressiveness and is recognized as a critical metabolic pathway in triple negative breast cancer (TNBC) cells, an aggressive subtype of breast cancer which has poor prognosis due to its largely resistance to conventional chemotherapy. Exposure of triple negative breast cancer cells to lipid-enriched environments, namely LDL, leads to the acquisition of an aggressive phenotype, characterized by increased proliferative and migratory capacities of the cells. Currently, the metabolic pathways responsible for the acquisition of the LDL-induced phenotype in TNBC cells remain unknown. The aim of this project is to characterize the metabolic program adopted by MDA-MB-231 TNBC cells exposed to LDL and determine its implication in the acquisition of the aggressive phenotype conferred by LDL, which is characterized by increased proliferative and migratory capacities. We investigated the expression of key regulators of mitochondrial biogenesis, glycolysis, oxidative phosphorylation (OXPHOS) and FAO to assess the deregulated metabolic pathways adopted by MDA-MB-231 cells and also investigated whether such modulations of metabolic pathways were conserved in other TNBC cell line, MDA-MB-436 and in the MCF-7 Luminal A breast cancer subtype. By inhibiting key enzymes of glycolysis, OXPHOS and FAO we have identified the metabolic pathways preferentially adopted by MDA-MB-231 cells to sustain their proliferation and migration capacities. We next investigated the impact of LDL exposure in the mitochondrial mass, mitochondrial membrane potential and mitochondrial dynamics of MDA-MB-231 cells and its implications in the aggressive phenotype induced by LDL. In conclusion, we identified metabolic adaptations of MDA-MB-231 cells exposed to LDL. We observed that MDA-MB-231 cells can reprogram their metabolism in presence of high amounts of extracellular lipids relying on glycolysis and OXPHOS to proliferate and migrate. The presence of exogenous LDL leads to adoption of a glycolytic phenotype related with PGC-1a modulation. Mitochondrial metabolism play a role in the aggressiveness of TNBC, characterized by increased mitochondria number and modulation of mitochondrial dynamics. We also identified PA as one of the components of the LDL molecule responsible for the modulation of the aggressive phenotype of TNBC.
Durante o desenvolvimento do tumor as células cancerígenas adquirem um conjunto de adaptações biológicas conhecidas como “the cancer hallmarks” que lhes permitem tornar-se malignas. Uma destas adaptações consiste na alteração do metabolismo energético, primeiramente observada por Otto Warburg. Em condições de oxigénio suficiente, as células normais metabolizam a glucose através da glicólise para sintetizar piruvato, que é transportado para a mitocôndria onde entra no ciclo dos ácidos tricarboxílicos para gerar ATP, pela chamada respiração aeróbia. Pelo contrário, as células cancerígenas, mesmo em condições de oxigénio suficiente para suportar a respiração aeróbia metabolizam o piruvato resultante da glicólise a lactato, através da fermentação, que acontece em células normais apenas em condições de insuficiência de oxigénio, uma vez que a quantidade de energia resultante deste processo é muito menor que a proveniente da oxidação do piruvato na mitocôndria. Warburg afirmou que seria uma deficiência ao nível das mitocôndrias a causa desta reprogramação metabólica levada a cabo pelas células cancerígenas, no entanto atualmente sabe-se que este fenómeno é devido a uma reprogramação metabólica para melhor satisfazer as necessidades do tumor a nível de intermediários metabólicos que suportam o seu rápido crescimento. Esta reprogramação do metabolismo baseia-se no aumento da utilização da respiração mitocondrial e da b-oxidação dos ácidos gordos, cuja importância e potencialidade como alvo terapêutico não estão ainda exploradas, devido à incidência do estudo do metabolismo tumoral ao nível da glicólise, glutaminólise e da síntese de ácidos gordos, embora tenha sido recentemente associada ao aumento da capacidade proliferativa do tumor e consequentemente à sua agressividade. As mitocôndrias são responsáveis simultaneamente por funções vitais e pela morte da célula, tanto em condições normais, como em condições patológicas como o cancro. Estes organelos possuem uma dinâmica contínua ao nível do seu número e morfologia, que determina o balanço entre a produção da energia celular e a apoptose. Assim, a desregulação ao nível da mitocôndria está implicada na tumorigénese a na progressão tumoral e a sua regulação metabólica é fundamental. A dinâmica da mitocôndria varia continuamente dependendo da atividade metabólica da célula e de condições de stress a que está sujeita. Nestas condições e na ausência de nutrientes, as mitocôndrias tornam-se alongadas e estabelecem redes através de fenómenos de fusão, aumentando a capacidade oxidativa. Em condições de baixa de concentração de oxigénio ou apoptose, fenómenos de fissão tornam as mitocôndrias mais fragmentadas. Apesar de serem conhecidos os processos que levam à alteração da morfologia da mitocôndria, e de se saber estarem alterados em vários tipos de cancro, a sua relação com a agressividade do tumor não está ainda estabelecida. O cancro da mama é tipo mais comum de cancro em mulheres e exibe diferentes características biológicas e fenótipos clínicos. O subtipo mais agressivo de cancro da mama é o subtipo triplo negativo que está associado a um prognóstico clínico pouco favorável, devido à sua heterogeneidade e desconhecimento sobre as vias metabólicas mais favorecidas e ainda por não possuir à superfície das células os recetores que são atualmente os alvos da terapêutica convencional. A obesidade é reconhecida como um fator de risco para o desenvolvimento de cancro da mama, embora a relação entre níveis elevados de lípidos no sangue e a incidência de cancro da mama não esteja ainda estabelecida. O nosso laboratório reportou recentemente que níveis elevados de colesterol (LDL) em doentes com cancro da mama estão associados a tumores maiores e mais metastáticos. Sabe-se também que as células cancerígenas têm uma necessidade acrescida de lípidos relativamente às células normais, pelo que aumentam o seu importe e síntese, e que a exposição de linhas do subtipo triplo negativo de cancro da mama a ambientes ricos em LDL resulta na aquisição de propriedades agressivas pelo tumor que levam à resistência à terapia. No entanto as alterações metabólicas requeridas em condições de elevada concentração de lípidos para este aumento de agressividade não estão ainda estabelecidas. Com este trabalho, propusemo-nos a caracterizar o fenótipo metabólico de células de cancro da mama triplo negativo, MDA-MB-231, expostas a LDL e determinar a importância deste lípido para o fenótipo agressivo adotado por esta linha celular, caracterizado pelo aumento da sua capacidade proliferativa e migratória. Para determinar as vias metabólicas preferencialmente adotadas por estas células quando expostas a LDL fizemos uma análise à expressão génica de enzimas da glicólise, fosforilação oxidativa e b-oxidação dos ácidos gordos e inibimos enzimas-chave destas vias. Para determinar a contribuição da mitocôndria para o fenótipo agressivo verificado investigámos a expressão génica de fatores de biogénese mitocondrial e a expressão das proteínas responsáveis pela dinâmica mitocondrial. Verificámos ainda o número de mitocôndrias e a quantidade de DNA mitocondrial em células expostas a LDL em comparação com a condição controlo. Estudámos também a importância do fator de biogénese mitocondrial PGC-1a para a adoção preferencial da fosforilação oxidativa e da b-oxidação dos ácidos gordos na presença de colesterol através da verificação dos níveis de expressão génica de enzimas destas vias, e ainda a importância deste fator de biogénese mitocondrial para a capacidade de proliferação e migração das células de cancro da mama triplo negativo. Verificámos que a modulação PGC-1a está relacionada a adoção da glicólise por parte das células MDA-MB-231 para manter a sua atividade proliferativa. Verificámos também a importância dos ácidos gordos presentes na molécula de LDL para o aumento da proliferação e migração das células de cancro da mama triplo negativo, através da exposição destas células a ácido palmítico, o ácido gordo saturado mais abundante na dieta que é simultaneamente um dos ácidos gordos com maior concentração no sangue. A exposição da linha celular MDA-MB-231 a ácido palmítico aumentou tanto a capacidade proliferativa como a capacidade migratória das células, o que sugere que este será um dos componentes da molécula de colesterol responsáveis pela agressividade do cancro da mama triplo negativo. Comparámos a capacidade proliferativa e migratória das células MDA-MB-231 com outra linha de cancro da mama triplo negativo, MDA-MB-436 e com a linha celular MCF-7, do subtipo Luminal A, positiva para o recetor de estrogénio. Verificámos que a exposição a LDL é responsável por um aumento das capacidades proliferativa e migratória de ambas as linhas celulares, o que nos permite concluir que este não é um efeito isolado apenas numa linha celular de cancro da mama triplo negativo e inferir que a exposição a LDL poderá ser responsável pelo aumento de agressividade deste subtipo de cancro da mama. Também a capacidade proliferativa da linha celular MCF-7 aumentou na presença de LDL, o que permite inferir que a exposição a LDL contribui para a aquisição de um fenótipo agressivo tanto nos subtipos de cancro da mama responsivos a hormonas com nos subtipos que não possuem recetores hormonais. Ao contrário do verificado nas duas linhas de cancro da mama triplo negativo estudadas, a quantidade de DNA mitocondrial nas células da linha celular MCF-7 não aumentou com a exposição a LDL, o que pode ser indicativo da aquisição de vias metabólicas diferentes para a agressividade dos dois subtipos de cancro da mama. Com este trabalho verificámos a adoção preferencial de vias metabólicas em detrimento de outras por parte de células do subtipo triplo negativo do cancro da mama e abrimos novas perspetivas à importância da mitocôndria e da sua reprogramação a nível metabólico e da sua dinâmica para a agressividade deste subtipo agressivo de cancro da mama. São necessários mais estudos para determinar possíveis inibições metabólicas que possam em cooperação com as terapias convencionais contribuir para o tratamento de doentes com cancro da mama triplo negativo.