Добірка наукової літератури з теми "Mitochondrial respiration technique"

Mitochondrial dysfunction has been implicated in many neurological disorders that only develop or are much more severe in adults, yet no methodology exists that allows for medium-throughput functional mitochondrial analysis of brain sections from adult animals. We developed a technique for quantifying mitochondrial respiration in acutely isolated adult rat brain sections with the Seahorse XF Analyzer. Evaluating a range of conditions made quantifying mitochondrial function from acutely derived adult brain sections from the cortex, cerebellum, and trigeminal nucleus caudalis possible. Optimization of this technique demonstrated that the ideal section size was 1 mm wide. We found that sectioning brains at physiological temperatures was necessary for consistent metabolic analysis of trigeminal nucleus caudalis sections. Oxygen consumption in these sections was highly coupled to ATP synthesis, had robust spare respiratory capacities, and had limited nonmitochondrial respiration, all indicative of healthy tissue. We demonstrate the effectiveness of this technique by identifying a decreased spare respiratory capacity in the trigeminal nucleus caudalis of a rat model of chronic migraine, a neurological disorder that has been associated with mitochondrial dysfunction. This technique allows for 24 acutely isolated sections from multiple brain regions of a single adult rat to be analyzed simultaneously with four sequential drug treatments, greatly advancing the ability to study mitochondrial physiology in adult neurological disorders.

Mitochondria, that provide most of the ATP needed for cell work, and that play numerous specific functions in biosyntheses and degradations, as well as contributing to Ca2+; signaling, also play a key role in the pathway to cell death. Impairment of mitochondrial functions caused by mutations of mt-genome, and by acute processes, are responsible for numerous diseases. The involvement of impaired mitochondria in the pathogenesis of sepsis is discussed. By means of the skinned fiber technique and high resolution respirometry, we have detected significantly reduced rates of mitochondrial respiration in heart and skeletal muscle of endotoxaemic rabbits. Mitochondria from heart were more affected than those from skeletal muscle. Decreased respiration rates were accompanied by reduced activities of complex I+III of the respiratory chain. Endotoxin-caused impairment was also detectable at the level of the Langendorff perfused heart, where the coronary vascular resistance was significantly increased. For an investigation of the influence of bacteraemia on the mitochondrial respiratory chain, baboons were made septic by infusion of high and low amounts of E. coli. For complex I+III and II+III, a clear dose-dependent decrease was detectable and in animals which died in septic shock, a further decrease of enzyme activities in comparison to the controls were found. These results are discussed in the light of current knowledge on the role of mitochondria in cell pathology in respect to sepsis. In conclusion, we present evidence that mitochondrial function is disturbed during sepsis. Besides ischaemic and poison-induced disturbances of mitochondrial function, sepsis is a further example of an acute disease where impaired mitochondria have to be taken into account.

It has been long recognized that the impairment of platelet mitochondrial function occurs in a broad spectrum of diseases. Accordingly, the assessment of platelet respiratory dys/function has emerged as a putative approach allowing the characterization of the early impairment of human bioenergetic profile in several chronic pathologies. The aim of this study was to standardize the methodology for platelet isolation from peripheral blood and the measurement of mitochondrial oxygen consumption by means of high-resolution respirometry, respectively. The platelet isolation protocol consisted of two consecutive centrifugations of the whole blood collected from adult healthy females (n = 10) yielding a platelet-rich plasma sample. Respiration was measured at 370C using the Oxygraph-2k (Oroboros Instruments, Austria) according to a classic substrate-uncoupler-inhibitor-titration protocol. Platelets permeabilized with digitonin were allowed to respire in the presence of complex I (glutamate and malate) and complex II (succinate) substrates. We obtained a respiratory control ratio of 2.77 � 3.65 that indicates an accurate coupling efficiency of oxidative phosphorylation. The in vitro measurement of platelet respiration is a reliable method to evaluate the bioenergetic profile in humans. The standardized technique will be further used to assess the occurrence of mitochondrial dysfunction in peripheral blood in the setting of various chronic non-communicable diseases.

1. The yield of mitochondria isolated from perfused hearts subjected to 30 min ischaemia followed by 15 min reperfusion was significantly less than that for control hearts, and this was associated with a decrease in the rates of ADP-stimulated respiration. 2. The presence of 0.2 microM cyclosporin A (CsA) in the perfusion medium during ischaemia and reperfusion caused mitochondrial recovery to return to control values, but did not reverse the inhibition of respiration. 3. A technique has been devised to investigate whether the Ca(2+)-induced non-specific pore of the mitochondrial inner membrane opens during ischaemia and/or reperfusion of the isolated rat heart. The protocol involved loading the heart with 2-deoxy[3H]glucose ([3H]DOG), which will only enter mitochondria when the pore opens. Subsequent isolation of mitochondria demonstrated that [3H]DOG did not enter mitochondria during global isothermic ischaemia, but did enter during the reperfusion period. 4. The amount of [3H]DOG that entered mitochondria increased with the time of ischaemia, and reached a maximal value after 30-40 min of ischaemia. 5. CsA at 0.2 microM did not prevent [3H]DOG becoming associated with the mitochondria, but rather increased it; this was despite CsA having a protective effect on heart function similar to that shown previously [Griffiths and Halestrap (1993) J. Mol. Cell. Cardiol. 25, 1461-1469]. 6. The non-immunosuppressive CsA analogue [MeAla6]cyclosporin was shown to have a similar Ki to CsA on purified mitochondrial peptidyl-prolyl cis-trans-isomerase and mitochondrial pore opening, and also to have a similar protective effect against reperfusion injury. 7. Using isolated heart mitochondria, it was demonstrated that pore opening could become CsA-insensitive under conditions of adenine nucleotide depletion and high matrix [Ca2+] such as may occur during the initial phase of reperfusion. The apparent increase in mitochondrial [3H]DOG in the CsA-perfused hearts is explained by the ability of the drug to stabilize pore closure and so decrease the loss of [3H]DOG from the mitochondria during their preparation.

In the present study, we describe the existence of a large-conductance Ca2+-activated potassium (BKCa) channel in the mitochondria of the human endothelial cell line EA.hy926. A single-channel current was recorded from endothelial mitoplasts (i.e., inner mitochondrial membrane) using the patch-clamp technique in the mitoplast-attached mode. A potassium-selective current was recorded with a mean conductance equal to 270 ± 10 pS in a symmetrical 150/150 mM KCl isotonic solution. The channel activity, which was determined as the open probability, increased with the addition of calcium ions and the potassium channel opener NS1619. Conversely, the activity of the channel was irreversibly blocked by paxilline and iberiotoxin, BKCa channel inhibitors. The open-state probability was found to be voltage dependent. The substances known to modulate BKCa channel activity influenced the bioenergetics of mitochondria isolated from human endothelial EA.hy926 cells. In isolated mitochondria, 100 μM Ca2+, 10 μM NS1619, and 0.5 μM NS11021 depolarized the mitochondrial membrane potential and stimulated nonphosphorylating respiration. These effects were blocked by iberiotoxin and paxilline in a potassium-dependent manner. Under phosphorylating conditions, NS1619-induced, iberiotoxin-sensitive uncoupling diverted energy from ATP synthesis during the phosphorylating respiration of the endothelial mitochondria. Immunological analysis with antibodies raised against proteins of the plasma membrane BKCa channel identified a pore-forming α-subunit and an auxiliary β2-subunit of the channel in the endothelial mitochondrial inner membrane. In conclusion, we show for the first time that the inner mitochondrial membrane in human endothelial EA.hy926 cells contains a large-conductance calcium-dependent potassium channel with properties similar to those of the surface membrane BKCa channel.

The smolt stage of salmon has challenges in reaching adequate growth rates due to the changing environmental conditions at sea. Therefore, it is necessary to provide adequate diets to achieve sufficient growth. This study determined the impacts of glutamate and succinate (1% each) supplemented diet on the growth of Atlantic salmon smolts along with characterization of mitochondrial respiration using high-resolution respirometry technique. Results indicated that there was no significant difference in growth response between the treatment and control groups. Maximum oxidative phosphorylation (OXPHOS) was reached after addition of succinate. Analysis of heart homogenates revealed a significant difference in LEAK respiration state (P = 0.005). No significant difference was recorded between the diet groups for liver homogenates. Differences between heart and liver respiration revealed that mitochondrial activity is organ dependent.

Mechanisms responsible for limitation of exercise capacity in lung transplant recipients (LR) and benefits gained by exercise training were studied. Mitochondrial respiration parameters, energy transfer, and cell structure were assessed in vastus lateralis biopsies using the permeabilized fiber technique with histochemical and morphometric measurements. Twelve male controls (C) and 12 LR performed exercise training over 12 wk. Before exercise training, there were strong correlations between exercise capacity (maximal O2 consumption and endurance time at 70% maximal power output) and cellular events, as assessed by percentage of type I fibers and apparent Km for exogenous ADP. Anticalcineurins were not involved in LR exercise limitation, since there were no differences in maximal mitochondrial rate of respiration before exercise training and no abnormalities in respiratory chain complexes compared with C. Training resulted in a significant increase in physiological parameters both at the cellular (apparent Km for exogenous ADP and stimulating effect of creatine) and integrated (maximal O2 consumption, power output at ventilatory threshold, maximal power output, and endurance time at 70% maximal power output) levels in LR and C. After the training period, improvements in maximal O2 consumption and in maximal mitochondrial rate of respiration were noted, as well as changes in endurance time and percentage of type I fibers. Because there were no changes in diameters and fiber types, baseline alteration of apparent Km for exogenous ADP and its improvement after training might be related to changes within the intracellular energetic units. After the training period, intracellular energetic units exhibited a higher control of mitochondrial respiration by creatine linked to a more efficient functional coupling adenine nucleotide translocase-mitochondrial creatine kinase, resulting in better exercise performances in C and LR.

Background The authors previously reported that secondary carnitine deficiency may sensitize the heart to bupivacaine-induced arrhythmias. In this study, the authors tested whether bupivacaine inhibits carnitine metabolism in cardiac mitochondria. Methods Rat cardiac interfibrillar mitochondria were prepared using a differential centrifugation technique. Rates of adenosine diphosphate-stimulated (state III) and adenosine diphosphate-limited (state IV) oxygen consumption were measured using a Clark electrode, using lipid or nonlipid substrates with varying concentrations of a local anesthetic. Results State III respiration supported by the nonlipid substrate pyruvate (plus malate) is minimally affected by bupivacaine concentrations up to 2 mM. Lower concentrations of bupivacaine inhibited respiration when the available substrates were palmitoylcarnitine or acetylcarnitine; bupivacaine concentration causing 50% reduction in respiration (IC50 +/- SD) was 0.78+/-0.17 mM and 0.37+/-0.03 mM for palmitoylcarnitine and acetylcarnitine, respectively. Respiration was equally inhibited by bupivacaine when the substrates were palmitoylcarnitine alone, or palmitoyl-CoA plus carnitine. Bupivacaine (IC50 = 0.26+/-0.06 mM) and etidocaine (IC50 = 0.30+/-0.12 mM) inhibit carnitine-stimulated pyruvate oxidation similarly, whereas the lidocaine IC50 is greater by a factor of roughly 5, (IC50 = 1.4+/-0.26 mM), and ropivacaine is intermediate, IC50 = 0.5+/-0.28 mM. Conclusions Bupivacaine inhibits mitochondrial state III respiration when acylcarnitines are the available substrate. The substrate specificity of this effect rules out bupivacaine inhibition of carnitine palmitoyl transferases I and II, carnitine acetyltransferase, and fatty acid beta-oxidation. The authors hypothesize that differential inhibition of carnitine-stimulated pyruvate oxidation by various local anesthetics supports the clinical relevance of inhibition of carnitine-acylcarnitine translocase by local anesthetics with a cardiotoxic profile.

Changes in mitochondrial function are central to many forms of kidney disease, including acute injury, diabetic nephropathy, hypertension, and chronic kidney diseases. As such, there is an increasing need for reliable and fast methods for assessing mitochondrial respiratory function in renal cells. Despite being indispensable for many mechanistic studies, cultured cells or isolated mitochondria, however, often do not recapitulate in vivo or close to in vivo situations. Cultured and/or immortalized cells often change their bioenergetic profile and phenotype compared with in vivo or ex vivo situations, and isolated mitochondria are simply removed from their cellular milieu. This is especially important for extremely complex organs such as the kidney. Here, we report the development and validation of a new approach for the rapid assessment of mitochondrial oxygen consumption on freshly isolated glomeruli or proximal tubular fragments using Agilent SeaHorse XFe24 and XF96 Extracellular Flux Analyzers. We validated the technique in several healthy and diseased rodent models: the C57BL/6J mouse, the diabetic db/ db mouse and matching db/+ control mouse, and the Dahl salt-sensitive rat. We compared the data to respiration from isolated mitochondria. The method can be adapted and used for the rapid assessment of mitochondrial oxygen consumption from any rodent model of the investigator’s choice. The isolation methods presented here ensure viable and functional proximal tubular fragments and glomeruli, with a preserved cellular environment for studying mitochondrial function within the context of their surroundings and interactions.

Mitochondrial uncoupling is implicated in many patho(physiological) states. Using confocal live cell imaging and an optical O2 sensing technique, we show that moderate uncoupling of the mitochondria with plecomacrolide Baf (bafilomycin A1) causes partial depolarization of the mitochondria and deep sustained deoxygenation of human colon cancer HCT116 cells subjected to 6% atmospheric O2. A decrease in iO2 (intracellular O2) to 0–10 μM, induced by Baf, is sufficient for stabilization of HIFs (hypoxia inducible factors) HIF-1α and HIF-2α, coupled with an increased expression of target genes including GLUT1 (glucose transporter 1), HIF PHD2 (prolyl hydroxylase domain 2) and CAIX (carbonic anhydrase IX). Under the same hypoxic conditions, treatment with Baf causes neither decrease in iO2 nor HIF-α stabilization in the low-respiring HCT116 cells deficient in COX (cytochrome c-oxidase). Both cell types display equal capacities for HIF-α stabilization by hypoxia mimetics DMOG (dimethyloxalylglycine) and CoCl2, thus suggesting that the effect of Baf under hypoxia is driven mainly by mitochondrial respiration. Altogether, by activating HIF signalling under moderate hypoxia, mitochondrial uncoupling can play an important regulatory role in colon cancer metabolism and modulate adaptation of cancer cells to natural hypoxic environments.

La broncho-pneumopathie chronique obstructive (BPCO) est une pathologie qui affecte les muscles respiratoires mais aussi les muscles périphériques. Etant donné le rôle crucial du métabolisme énergétique dans la performance musculaire, nous avons émis l'hypothèse d'une participation des adaptations de cette composante énergétique dans les muscles respiratoires des patients porteurs d'une BPCO. Nous avons donc étudié la fonction mitochondriale et sa régulation, la fonction contractile (isoformes de chaînes lourdes de myosine), le système de transfert d'énergie comme la créatine kinase ainsi que certains enzymes des voies métaboliques aérobie et anaérobie dans le diaphragme costal et les muscles intercostaux externes chez le patient BPCO sévères ainsi que dans le diaphragme costal et les muscles périphériques chez le hamster emphysémateux. L'originalité de cette étude a été l'utilisation de la méthode de fibres perméabilisées à la saponine. Nous avons mis en évidence, dans le diaphragme et les muscles intercostaux externes chez l'homme, une augmentation de la capacité oxydative estimée par la respiration mitochondriale maximale ainsi qu'une amélioration du couplage phosphorylation oxydation. Ces adaptations sont plus importantes dans le diaphragme et sont corrélées aux indices fonctionnels que sont l'obstruction et la distension. Nous n'avons pas retrouvé ces résultats dans le diaphragme de hamster emphysémateux, ni aucune adaptation au niveau de deux muscles périphériques oxydatives et glycolytiques. L'ensemble de ces résultats montre chez les patients emphysémateux sévères une adaptation des muscles inspiratoires, similaire à celles d'un muscle qui serait entraîné en endurance sur une durée prolongée. Ces modifications joueraient un rôle important dans la prévention de la fatigue musculaire chez ces patients. L'absence de résultats obtenus chez le hamster emphysémateux suggère que ce modèle animal n'est pas entièrement superposable à l'emphysème chez l'homme
Chronic Obstructive Pulmonary Disease (COPD) is a pathology that affects respiratory and peripheral muscles. Given the crucial role of energy metabolism in muscle performance, we hypothesized that alterations in the energy metabolism would participate in the respiratory muscles of COPD patients. We thus investigated the mitochondrial function and its regulation, the contractile function (myofibrils) and the compartmentation of energy transfer systems from costal diaphragm and external intercostal muscles in COPD patients and costal diaphragm and two limb muscles in emphysematous hamsters. The originality of these studies was to use the in situ method of skinned fibers. We showed that costal diaphragm and external intercostal muscles in severe COPD patients exhibited adaptations in energy transfer systems and increase of their oxidative capacity. These adaptations were correlated in the diaphragm with indexes of obstruction and hyperinflation. We did not find any modification of diaphragm or peripheral muscles in emphysematous hamsters. Altogether, the results demonstrate in severe COPD patients, adaptations in respiratory muscles similar to the effects of long endurance training muscles. These modifications would play an important role in muscular fatigue prevention. The lack of results in emphysematous hamsters indicates that this model is not entirely superposable to the human emphysematous disease

Le complexe I de la chaîne respiratoire mitochondriale (ou NADH-ubiquinone oxydoréductase) est composé de 38 sous-unités codées par l’ADN nucléaire et de 7 sous-unités codées par l’ADN mitochondrial. Le déficit enzymatique du complexe I est une cause fréquente de cytopathie mitochondriale. Le grand nombre des mutations décrites dans les gènes nucléaires du complexe I, leur caractère le plus souvent privé, et l’absence de corrélation génotype – phénotype en cas de récurrence, compliquent l’identification des mutations pathogènes. Un dépistage systématique des mutations des gènes nucléaires du complexe I a été réalisé dans ce travail chez 19 patients atteints d’un déficit isolé en complexe I. Au total, 12 gènes nucléaires, codant pour 10 protéines de structure et 2 protéines chaperonnes, ont été étudiés par digestion enzymatique d’hétéroduplexes d’ADN complémentaires utilisant l’endonucléase Surveyor TM. Des mutations pathogènes ont été détectées chez 3 patients. Deux autres gènes d’assemblage, caractérisés par un polymorphisme important ou par la présence physiologique de variants de transcription, ont été étudiés par séquençage direct. Des anomalies de l’expression du complexe I ont été mises en évidence par la technique d’électrophorèse Blue Native couplée à une technique de Western Blot chez les patients étudiés. La technique de digestion enzymatique par l’enzyme Surveyor TM a montré son efficacité en limitant le nombre des séquençages effectués. Cette méthode s’inscrit dans le développement de nouvelles techniques de biologie moléculaire permettant un dépistage simultané des mutations dans un grand nombre de gènes
Mitochondrial complex I (or NADH-ubiquinone oxidoreductase) includes 38 nuclear subunits and 7 mitochondrial subunits. Complex I deficiency frequently accounts for mitochondrial disorders. Pathogenic mutations of complex I nuclear genes are often private. Moreover, the genotype – phenotype correlation is inconstant in the rare recurrent mutations. Identification of all mutations responsible for complex I deficiency is challenging but essential for providing accurate and helpful genetic counselling. An isolated complex I deficiency was biochemically detected in 19 patients. We screened for complex I nuclear genes mutations using the mismatch-specific DNA endonuclease Surveyor TM. Fragments of the complementary DNA of 12 complex I nuclear genes, encoding 10 structural proteins and 2 assembly proteins, were amplified for heteroduplexe formation followed by digestion. Pathogenic mutations were identified in 3 patients. Direct sequencing was performed for 2 other assembly genes, characterized by frequent polymorphisms or presence of several alternative transcripts. An abnormal complex I expression was detected by Blue Native electrophoresis followed by Western Blot in the studied patients. The Surveyor TM digestion – based method was efficient for limiting the number of sequence reactions. This method is useful and time – less consuming for the rapid mutation screening of a large number of genes

Exercise training provides health benefits to the general population, but there is considerable variability in the individual response to similar training. Some people have limited improvements following exercise (“low responders”), while others seem to improve considerably (“high responders”). To date, most exercise studies that have claimed to identify “low” or “high” responders assumed that if the participants were to repeat the same exercise training, they would show a similar response. However, within- subject variability has not been tested, which might lead to inaccurate classification of exercise responses at the individual level and the waste of precious research resources. Exposing individuals to a repeated or longer training intervention can assist in identifying the magnitude of responses to exercise training with better accuracy. Recent evidence also suggests that the response to exercise training may be influenced by epigenetic signatures. Epigenetics is a reversible process that affects how genes are expressed in cells, and it carries the memory of past cellular and environmental events. To date, no study has tested whether individual response is influenced by epigenetic marks. Thus, the overarching aim of this thesis is to identify the physiological, molecular, and epigenetic marks of exercise responses. Twenty young, healthy men from the Gene SMART (Skeletal Muscle Adaptive Response to Training) study completed a repeated and a longer exercise training intervention to measure within-subject variability and to obtain individual progress curves (See Figure 3.1 for study design). Participants underwent a four-week control period followed by four weeks of High-Intensity Interval Training (HIIT), had a washout period of > 1 year, and underwent another four weeks of HIIT followed by an additional 8 weeks of HIIT. The HIIT program was adjusted to individual fitness levels that were re-assessed every four weeks during the intervention to ensure improvements. Participants’ peak power output (Wpeak), lactate threshold (LT), and maximal oxygen uptake (VO2max) were assessed in duplicates at each time point. We used five known statistical methods to investigate changes in fitness and mixed models to estimate individual response. Muscle biopsies were collected at each time point to measure mitochondrial markers (i.e. mitochondrial respiration, citrate synthase, cytochrome C oxidase, succinate dehydrogenase, mitochondrial copy number, fibre typing, and myosin heavy chains PCRs), as well as genome-wide DNA methylation profiles in skeletal muscle using the Illumina HumanMethylation EPIC array. In Chapter 3, we show that at the group level, all physiological measures increased in a dose-response manner following HIIT (p<0.05). We found no changes in mitochondrial function and content or fibre type distributions. Baseline citrate synthase (CS) was associated with HIIT-induced changes in cytochrome-c oxidase (COX) and vice-versa (p < 0.05). At the individual level, we successfully identified trainability in physiological measurements using the repeated testing approach but failed to do so using the repeated intervention approach. We did not identify consistent individual response at the molecular level (mitochondrial function and content and fibre type distribution) using either approach, as measurements were highly variable within participants. We then investigated the reliability of the mitochondrial respiration technique (Chapter 4) by measuring the Technical Error of measurement (TEM) and the coefficient of variation (CV) for each mitochondrial complex. While the correlation between the two chambers was good for all complexes (R > 0.7 p < 0.001), the TEM was large (7.9 to 27 pmol·s-1·mg-1), and the CV was > 15% for all complexes. We performed statistical simulations to determine the sample size that would be required to detect a range of effect sizes at 80% power. We found that duplicate measurements on 75 participants are required to detect a 6% change in mitochondrial respiration after an intervention. Finally, Chapter 5 and 6 focus on the DNA methylation measures at the group and individual level respectively. For the first time at the group level, we have investigated DNA methylation patterns that are associated with fitness by combining three measurements of performance into a comprehensive z-score (Chapter 5). We found 12,107 DMPs that were associated with baseline fitness (z-score) (FDR < 0.005), 18.2% of which were hypomethylated and 81.8% hypermethylated with higher fitness levels. We identified 1,268 DMRs for baseline fitness, 15.3% of which hypomethylated and 85.7% hypermethylated. Hyper-DMRs were robustly over-represented in genic enhancers and flanking active TSS, and highly depleted in strong and weak candidate enhancers. Hypo and Hyper-DMRs had a moderate association with bivalent enhancers and promoters. Both hyper and hypo-DMRs presented a moderate representation in regions actively repressed by PolyComb proteins. Finally, significant DMRs were enriched for 26 GO terms, and these pathways were related to muscle system processes, actin cytoskeleton organization and regulation of actin filament and cytoskeleton processes. Next, we investigated the effects of exercise on the methylome, and surprisingly we observed an inverse pattern of DNA methylation profile after exercise. In summary, we found 568 DMPs that significantly changed after the 4 weeks (FDR < 0.005), and out of those only 1.4% were hypermethylated and 98.6% were hypomethylated. We identified 17 DMRs associated with changes in DNA methylation in response of 4 weeks of HIIT, and 100% of DMRs were hypomethylated. Lastly, we intersected DMPs that were significant for both baseline fitness z-score and after 4 weeks of HIIT. Five DMPs were significant, and they appeared to have inverse patterns for baseline z-score (more hypermethylation) and 4 weeks of HIIT (more hypomethylation). When we transitioned to the individual level study in Chapter 6, neither of our approaches (i.e. repeated intervention and repeated testing) yielded many significant results: only one DMP was significant (cg11260483, p-value: 3.22000e-10, adj.p-value: 0.00022) after the repeated intervention, and no DMPs significant after the repeated testing approach. The challenging experimental design of this thesis provided high resolution, longitudinal physiological and molecular profiles in skeletal muscle following repeated exercise training and testing. It yielded novel insights into the phenomenon of trainability in humans; young, healthy men displayed individual responses to HIIT at the physiological level, but not at the molecular level. This thesis also issued methodological considerations for protocols aimed at measuring individual response (Chapter 3). In particular, the high within-subject variability we observed led us to conclude that many repeated testings on the same individual at regular intervals during the training program, along with a moderate-to-large sample size, were necessary to estimate inter-individual variability in response to training. The mitochondrial respiration technique showed high technical variability (Chapter 4), making the measurement unreliable in our study with only n = 20 men and only two duplicates per individual. The typical sample sizes used in exercise training studies (n < 20) are likely insufficient to capture exercise-induced changes in mitochondrial respiration at the group level, let alone the individual level. Lastly, we observed a clear DNA methylation profile association with fitness levels (Chapter 5). However, when an exercise intervention was applied, we noticed a change in DNA methylation patterns that were inverse to those observed at baseline for the fitter participants. Such observations left us wondering on potential reasons to why this occurs. Thus, future research should also integrate the methylome with transcriptome and proteome to elucidate the mechanisms underlying adaptations to exercise training.

Breathing is essential for life in all of its stages. Cellular, mitochondrial respiration requires an adequate supply of oxygen, provided by the air we breathe, after airway conduction, treatment by the lungs, and transport to tissues. At different stages of life, pediatric dentists and orthodontists can intervene in the upper airway, expanding it, which helps with ventilation. The greater airway space, if used, contributes in different ways to the child’s development and the recovery of respiratory problems and should always be present as a weapon that physicians and the population should know. The value of the techniques becomes even more important when applied to children and young people with disabilities who can significantly improve their development and performance. Rapid Maxillary Expansion and Extraoral Traction Appliances are two important pediatric resources to treat these children. Clinical practice of the authors, is discussed, emphasizing the importance of early intervention and the need for multi and interdisciplinary collaboration in the follow-up of disabled people.