Functional magnetic resonance imaging (fMRI) based on blood oxygenation level dependent (BOLD) contrast has become a universal methodology in functional neuroimaging. However, the BOLD signal consists of a mix of physiological parameters and has relatively poor reproducibility. As fMRI becomes a prominent research tool for rehabilitation studies involving repeated measures of the human brain, more quantitative and stable fMRI contrasts are needed. This dissertation enhances quantitative measures to complement BOLD fMRI. These additional markers, cerebral blood flow (CBF) and cerebral blood volume (CBV) (and hence cerebral metabolic rate of oxygen (CMRO₂) modeling) are more specific imaging markers of neuronal activity than BOLD. The first aim of this dissertation assesses feasibility of complementing BOLD with quantitative fMRI measures in subjects with central visual impairment. Second, image acquisition and analysis are developed to enhance quantitative fMRI by quantifying CBV while simultaneously acquiring CBF and BOLD images. This aim seeks to relax assumptions related to existing methods that are not suitable for patient populations. Finally, CBF acquisition using a low-cost local labeling coil, which improves image quality, is combined with simultaneous acquisition of two types of traditional BOLD contrast. The demonstrated enhancement of CBF, CBV and CMRO₂measures can lead to better characterization of pathophysiology and treatment effects.

Insufficient sleep may be associated with obesity via increased energy intake and/or decreased energy expenditure. The present study therefore aimed to investigate effects of sleep restriction on energy balance in healthy young adults. Participants (14 men, 13 women) aged 35.3 ± 1.0 y with 23.6 ± 0.2 kg/m2 BMI completed a randomized, crossover study exposed to short and habitual sleep with 4 wk washout. Controlled diets were provided during the first 4 d, followed by 2 d of ad libitum eating. Ad libitum energy intake, energy expenditure and physical activity level were determined as well as energy balance and body weight. Results showed that ad libitum energy intake (p = 0.031), as well as total fat (p = 0.018) increased after short compared with habitual sleep, but physical activity level, energy expenditure, energy balance, and body weight remained unaffected by sleep duration. In conclusion, sleep deprivation elevates energy intake, which may lead to positive energy balance over time and increase the risk of weight gain and/or obesity.

Insufficient sleep may be associated with obesity via increased energy intake and/or decreased energy expenditure. The present study therefore aimed to investigate effects of sleep restriction on energy balance in healthy young adults. Participants (14 men, 13 women) aged 35.3 ± 1.0 y with 23.6 ± 0.2 kg/m2 BMI completed a randomized, crossover study exposed to short and habitual sleep with 4 wk washout. Controlled diets were provided during the first 4 d, followed by 2 d of ad libitum eating. Ad libitum energy intake, energy expenditure and physical activity level were determined as well as energy balance and body weight. Results showed that ad libitum energy intake (p = 0.031), as well as total fat (p = 0.018) increased after short compared with habitual sleep, but physical activity level, energy expenditure, energy balance, and body weight remained unaffected by sleep duration. In conclusion, sleep deprivation elevates energy intake, which may lead to positive energy balance over time and increase the risk of weight gain and/or obesity.

The primary objective of this PhD program was to investigate the impact of training at a constant-load moderate-intensity (FATmax) compared to work-matched high-intensity intervals (HIIT) on the metabolic, physiological and psychosocial health profiles of sedentary overweight and obese men. This study was unique in that it was the first time the effect of exercise intensity had been investigated to examine concurrently the components of whole-body energy metabolism and body composition as measured using gold standard techniques. Based upon the positive alterations in blood lipids, body composition, cardiorespiratory fitness and substrate oxidation, it appears that training at FATmax can positively impact health parameters as well as, or if not better than, high-intensity training. Furthermore, there are ramifications for public health messages and obesity management strategies arising from these findings, primarily attributable to the increased exercise adherence and the reduction in health risks stemming from the significant loss of abdominal visceral adipose tissue after FATmax training.

Immune activation is generally acknowledged to be costly. These costs are frequently assumed to result from trade-offs arising due to the reallocation of resources from other life-history traits to be invested in immunity. Here, I investigated the energetic basis of the costs associated with immune activation in Drosophila melanogaster. I found that immune activation significantly reduced fly fecundity (45%) and also caused a decline in metabolic rate (6%) but had no effect on body weight. To understand the factors behind reduced fecundity and metabolic rate I measured feeding and found that food intake was reduced by almost 31% in immune-challenged D. melanogaster. These findings suggest that fecundity costs of immune activation result not from the commonly accepted resource reallocation hypothesis but probably because resource acquisition is impaired during immune responses. The individuals of any animal population generally vary greatly in their ability to resist infectious disease. This variation arises due to both environmental heterogeneity and genetic diversity. Genetic variation in disease susceptibility has generally been considered to lie in the nuclear genome. Here, for the first time, I explored the influence of mitochondrial genetic (mtDNA) variation on disease susceptibility. I crossed 22 mitochondrial haplotypes onto a single nuclear genome and also studied epistasis interactions between mitochondrial and nuclear genomes (mitonuclear epistasis) by crossing five haplotypes onto five different genetic backgrounds. I found that fly susceptibility to Serratia marcescens was influenced significantly by mtDNA allelic variation. Furthermore, the effect of mitonuclear epistasis on fly susceptibility to S. marcescens was twice as great as the individual effects of either mitochondrial or nuclear genome. However, susceptibility to Beauveria bassiana was not affected by mtDNA allelic variation. These findings suggest the mitochondrial genome may play an important role in host-parasite coevolution. The Mother’s Curse hypothesis suggests that sex-specific selection due to maternal mitochondrial inheritance means that mitochondria are poorly adapted to function in males, resulting in impaired male fitness. Mother’s Curse effects have previously only been studied for two phenotypic traits (sperm-infertility and ageing) and their generality for broader life-history has not been explored. I investigated the impact of mtDNA allelic variation on 10 phenotypic traits and tested whether the patterns of phenotypic variation in males and females conformed to the expectations of the Mother’s Curse hypothesis. I found that seven of the 10 traits were significantly influenced by mtDNA allelic variation. However, there was no evidence that the effects of this variation differed between males and females. I therefore concluded that Mother’s Curse is unlikely to be a general phenomenon, nor to provide a general explanation for sexual dimorphism in life-history traits. Overall, this thesis explored the impacts of immunity costs, mitochondrial genetic variation, mitonuclear epistasis and sex-specific mitochondrial selection on D. melanogaster life-history.

Using a high throughput closed respirometry method to measure oxygen consumption, I determined metabolic rates in asexual and sexual Schmidtea mediterranea and Girardia dorotocephala, as a function of temperature, taxon, stressors, reproductive mode, age, regeneration, and specific dynamic action. This study has shown that oxygen consumption can reliably be measured in planaria using optode closed respirometry, and also provided a reliable method for measuring wet mass in planaria, which has been a challenge to researchers in the past. This research revealed that oxygen consumption in S. mediterranea is 1.5-2.1X greater in the sexual strain over the asexual strain at 13-18°C. Within the sexual strain, oxygen consumption is 1.5 -2.2X greater in sexually mature adults over the sexually immature groups (hatchlings, juveniles, and regenerating sexuals). Furthermore, I was able to quantify differences in sexual morphology between these groups exhibiting significant differences in oxygen consumption. The results of this research supports a theory of higher metabolic costs with sexual maturity in S. mediterranea. Therefore, this study has established sexual and asexual S. mediterranea as simple, yet attractive models for investigating energetic costs between sexual and asexual phenotypes. This research also provided quantitative values for specific dynamic action in planaria, with a maximum increase in oxygen consumption of 160% induced by feeding, as well as metabolic relationships in planaria involving temperature, age, and regeneration. These values establish planaria as one of the simplest animal models in which common metabolic patterns, such as SDA and poikilothermic temperature sensitivity, have been demonstrated. Therefore, this research has contributed to the overall knowledge of the basic physiology in this animal, providing the framework for future metabolic studies in planaria involving environmental factors, reproduction, regeneration, development, and aging. Information from this study may supplement interpretation and understanding of modern cellular, molecular, and genomic studies in planaria.

La spectroscopie par résonance magnétique nucléaire (RMN) est un outil puissant permettant d’acquérir des profils biochimiques du cerveau et de quantifier de nombreux paramètres cellulaires in vivo. Au cours de ce travail de thèse, nous nous sommes intéressés à trois techniques : (i) la spectroscopie RMN du 1H pondérée en diffusion, (ii) la spectroscopie RMN du carbone-13 (13C) et (iii) de l’oxygène-17 (17O) pour étudier la microstructure et la fonction cellulaire in vivo.Les métabolites cérébraux sont des traceurs endogènes spécifiques d’un type cellulaire (neurones et astrocytes) dont la diffusion dépend des nombreuses propriétés cellulaires (par exemple la viscosité du cytosol et la restriction intracellulaire). L’étude de la dépendance du coefficient de diffusion (ADC) aux temps de diffusion (td) permet de quantifier chacun de ces paramètres. En particulier, la mesure de l’ADC aux td longs permet d’évaluer la compartimentation des métabolites. Dans une première étude, nous avons mesuré l’ADC de plusieurs métabolites neuronaux et astrocytaires sur une large gamme de td (de ~80 ms à ~1 s) dans un large voxel dans le cerveau du macaque. Aucune dépendance de l’ADC de l’ensemble des métabolites au td n’a été observée suggérant que les métabolites diffusent majoritairement dans les prolongements neuronaux (axones, dendrites) et astrocytaires et ne sont pas confinés dans le corps cellulaire ou les organelles (mitochondries, noyau). La grande taille du voxel, liée à la sensibilité de détection limitée, ne nous a pas permis d’étudier la compartimentation des métabolites dans la substance blanche (SB) et la substance grise (SG). C’est pourquoi, une nouvelle étude a été réalisée dans le cerveau de l’Homme. Les résultats montrent que les métabolites diffusent dans des structures fibrillaires dans la SG et la SB. Enfin, une dernière étude, avec une gamme de td jusqu’à 2 s chez le macaque, nous a permis d’estimer, à l’aide de modèles analytiques simples mimant la structure cellulaire, la longueur des fibres neuronales (~110 μm) et astrocytaires (~70 μm). L’oxydation du glucose au sein des mitochondries permet de produire l’ATP (adénosine triphosphate), la principale source d’énergie de l’organisme. La spectroscopie du 13C permet de mesurer la vitesse de dégradation du glucose dans le cycle de Krebs (VTCA). Cette méthode est largement reconnue pour l’étude du métabolisme. Néanmoins, de nombreuses limitations, en termes de modélisation des données en détection indirecte ou de puissance émise dans le contexte du découplage hétéronucléaire en détection directe, ont été rencontrées sur notre scanner IRM. C’est pourquoi, la spectroscopie du 17O a ensuite été développée afin de quantifier la vitesse de consommation de l’oxygène pendant la phosphorylation oxydative (CMRO2). Des développements méthodologiques et technologiques ont été nécessaires et sont encore en cours pour mettre en place et valider cette technique qui n’a encore jamais été utilisée chez le macaque. / Magnetic Resonance Spectroscopy is a unique tool that allows acquiring brain biochemical profiles and quantifying many cellular parameters in vivo. During this thesis, three different techniques have been developed: (i) 1H diffusion-weighted, (ii) carbone-13 (13C) and (iii) oxygen-17 (17O) NMR spectroscopy to study brain structure and function in vivo. Brain metabolites are cell-specific endogeneous tracers of the intracellular space whose translational diffusion depends on many cellular properties (e.g.: cytosol vicosity and intracellular restriction). Studying the variation of the diffusion coefficient (ADC) as a function of diffusion time (td) allows untangling and quantifying those parameters. In particular, measuring metabolites ADC at long diffusion times gives information about the metabolites compartmentation in cells. In a first study, we measured neuronal and astrocytic metabolites ADC over a large time window (from ~80 ms to ~1 s) in a large voxel in the macaque brain. No dependence of all metabolites ADC on td was observed suggesting that metabolites primarily diffuse in neuronal (dendrites and axons) and astrocytic processes and are not confined inside the cell body and organelles (nucleus, mitochondria). The large size of the voxel, due to low detection sensitivity, did not allow us to study metabolites compartmentation in pure white (WM) and grey matters (GM). Therefore, we performed a new study in the human brain. Results showed that in both WM and GM metabolites diffuse in fiber-like cell structure. Finally, using an even larger time window (up to 2 s) in the macaque brain and analytical models mimicking the cell structure, we estimated the length of neuronal (~110 μm) and astrocytic (~70 μm) processes. ATP (adenosine triphosphate), the main source of energy in the organism, is produced thanks to glucose oxidation inside the mitochondria. 13C NMR spectroscopy is a well-known technique to study brain energy metabolism and can be used to estimate the rate of glucose degradation within the Krebs cycle (VTCA). However, many limitations, concerning data modeling when performing indirect detection or power deposition due to heteronuclear decoupling during direct detection, were encountered on our MRI scanner. Therefore, 17O NMR spectroscopy was developed to quantify the rate of oxygen consumption during oxidative phosphorylation (CMRO2). Methodological and technological developments were necessary and are still ongoing to validate this technique, which has never been used with macaque.

Most climate change predictions focus on the response of individual species to changing local conditions and ignore species interactions, largely due to the lack of a sound theoretical foundation for how interactions are expected to change with climate and how to incorporate them into climate change models. Much of the variability in species interaction strengths may be governed by fundamental constraints on physiological rates, possibly providing a framework for including species interactions into climate change models. Metabolic rates, ingestion rates and many other physiological rates are relatively predictable from body size and body temperature due to constraints imposed by the physical and chemical laws that govern fluid dynamics and the kinetics of biochemical reaction times. My dissertation assesses the usefulness of this framework by exploring the community-level consequences of physiological constraints. In Chapter 2, I incorporated temperature and body size scaling into the biological rate parameters of a series of realistically structured trophic network models. The relative magnitude of the temperature scaling parameters affecting consumer energetic costs (metabolic rates) and energetic gains (ingestion rates) determined how consumer energetic efficiency changed with temperature. I systematically changed consumer energetic efficiency and examined the sensitivity of network stability and species persistence to various temperatures. I found that a species' probability of extinction depended primarily on the effects of organismal physiology (body size and energetic efficiency with respect to temperature) and secondarily on the effects of local food web structure (trophic level and consumer generality). This suggests that physiology is highly influential on the structure and dynamics of ecological communities. If consumer energetic efficiency declined as temperature increased, that is, species did best at lower temperatures, then the simulated networks had greater stability at lower temperatures. The opposite scenario resulted in greater stability at higher temperatures. Thus, much of the community-level response depends on what species energetic efficiencies at the organismal-level really are, which formed the research question for Chapter 3: How does consumer energetic efficiency change with temperature? Existing evidence is scarce but suggestive of decreasing consumer energetic efficiency with increasing temperature. I tested this hypothesis on seven rocky intertidal invertebrate species by measuring the relative temperature scaling of their metabolic and ingestion rates as well as consumer interaction strength under lab conditions. Energetic efficiencies of these rocky intertidal invertebrates declined and species interaction strengths tended to increase with temperature. Thus, in the rocky intertidal, the mechanistic effect of temperature would be to lower community stability at higher temperatures. Chapter 4 tests if the mechanistic effects of temperature on ingestion rates and species interaction strengths seen in the lab are apparent under field conditions. Bruce Menge and I related bio-mimetic estimates of body temperatures to estimates of per capita mussel ingestion rates and species interaction strengths by the ochre sea star Pisaster ochraceus, a keystone predator of the rocky intertidal. We found a strong, positive effect of body temperature on both per capita ingestion rates and interaction strengths. However, the effects of season and the unique way in which P. ochraceus regulates body temperatures were also apparent, leaving room for adaptation and acclimation to partially compensate for the mechanistic constraint of body temperature. Community structure of the rocky intertidal is associated with environmental forcing due to upwelling, which delivers cold, nutrient rich water to the nearshore environment. As upwelling is driven by large-scale atmospheric pressure gradients, climate change has the potential to affect a wide range of significant ecological processes through changes in water temperature. In Chapter 5, my coauthors and I identified long-term trends in the phenology of upwelling events that are consistent with climate change predictions: upwelling events are becoming stronger and longer. As expected, longer upwelling events were related to lower average water temperatures in the rocky intertidal. Furthermore, recruitment rates of barnacles and mussels were associated with the phenology of upwelling events. Thus climate change is altering the mode and the tempo of environmental forcing in nearshore ecosystems, with ramifications for community structure and function. Ongoing, long-term changes in environmental forcing in rocky intertidal ecosystems provide an opportunity to understand how temperature shapes community structure and the ramifications of climate change. My dissertation research demonstrates that the effect of temperature on organismal performance is an important force structuring ecological communities and has potential as a tractable framework for predicting the community level effects of climate change. / Graduation date: 2013 / Access restricted to the OSU Community, at author's request, from Nov. 20, 2012 - Nov. 20, 2014

Consistently, reports in the literature have identified that a sedentary lifestyle contributes to the progression of a range of chronic degenerative diseases. The measurement of energy expenditure and physical activity pattern in children is a challenge for all professionals interested in paediatric health and from a broader perspective, the public health fraternity charged with considering longer term health consequences of physical inactivity. The primary objective of this thesis was to identify a suitable indirect and objective measurement technique for the assessment of energy expenditure and physical activity pattern in children. The ideal characteristics of such a technique are that it should be reproducible and have been validated against a criterion reference method. To achieve this goal, a series of methodological studies were undertaken (Chapters II and III). This work was essential to increase accuracy during the individualised laboratory calibration process and further minimise prediction errors when analysing data from 7 days of monitoring under free-living conditions in the second part of the study (Chapters IV and V). In the first study to verify the combined effect of body position, apparatus and distraction on children's resting metabolic rate (RMR), experiments were carried out on 14 children aged 8-12 (mean age = 10.1 years ± 1.4). Each participant underwent 2 test sessions, one week apart under three different situations: a) using mouthpiece and nose-clip (MN) or facemask (FM); b) sitting (SEAT) or lying (LY) and c) TV viewing (TV) or no TV viewing. In the first session, following 20 min rest and watching TV, the following protocol was used: LY: 20 min - stabilisation; 10 min using MN and 10 min using FM. Body position was then changed to seated: 20 min stabilisation; 10 min using FM; 10 min using MN. In the second session, FM and MN order was changed and participants did not watch TV. Data were analysed according to the eight combinations among the three studied parameters. Repeated measures ANOVA indicated statistically significant differences for &VO2 (p=0.01) and RMR (p=0.02), with TVMNSEAT showing higher values than TVFMLY. Bland-Altman analysis showed a bias for &VO2, &VCO2, RQ and RMR between TVFMLY and TVMNSEAT of -17.8±14.5 ml.min-1, -8.8±14.5 ml. min-1, 0.03±0.05 and -115.2±101.9 kcal.d-1, respectively. There were no differences in RMR measurements due to body position and apparatus when each variable was isolated. Analyses of distraction in three of four combinations indicated no difference between TV and no TV. In summary, different parameter combinations can result in increased bias and variability and thereby reported differences among children's RMR measurement. The second study dealt with treadmill adaptation and determination of self-selected (SS) walking speed. Assessment of individual and group differences in metabolic energy expenditure using oxygen uptake requires that individuals are comfortable with, and can accommodate to, the equipment being utilised. In this study, a detailed proposal for an adaptation protocol based on the SS was developed. Experiments were carried out on 27 children aged 8-12 (mean age = 10.3±1.2 yr). Results from three treadmill tests following the adaptation protocol showed similar results for step length with no significant differences among tests and lower and no statistically significant variability within- and between-days. Additionally, no statistically significant differences between SS determined over-ground and on a treadmill were verified. These results suggest that SS speed determined over-ground is reproducible on a treadmill and the 10 min familiarisation protocol based on this speed provided sufficient exposure to achieve accommodation to the treadmill. The purpose of the third study was to verify within- and between-day repeatability and variability in children's oxygen uptake ( &VO2), gross economy (GE) [ &VO2 divided by speed] and heart rate (HR) during treadmill walking based on SS. 14 children (mean age = 10.2±1.4 yr) undertook 3 testing sessions over 2 days in which four walking speeds, including SS, were tested. Within- and between-day repeatability was assessed using the Bland and Altman method and coefficients of variability (CV) were determined for each child across exercise bouts and averaged to obtain a mean group CV value for &VO2, GE and HR per speed. Repeated measures ANOVA showed no statistically significant differences in within- or between-day CV for &VO2, GE or HR at any speed. Repeatability within and between-day for &VO2, GE and HR for all speeds was verified. These results suggest that submaximal &V O2 during treadmill walking is stable and reproducible at a range of speeds based on children's SS. In the fourth study, the objective was to establish the effect of walking speed on substrate oxidation during a treadmill protocol based on SS. Experiments were carried out on 12 girls aged 8-12 (mean age = 9.9±1.4 yr). Each participant underwent 2 test sessions, one week apart. Workloads on the treadmill included 2 speeds slower than SS (1.6 [V1] and 0.8 km.h-1 [V2] slower than SS), SS (V3), and a speed 0.8 km.h-1 faster than SS (V4). Indirect calorimetry from respired gas measurements enabled total fat (FO) and carbohydrate (CHO) oxidation rates to be calculated according to the non-protein respiratory quotient (Peronnet and Massicote, 1991) and percentage of CHO and FO calculations using equations from McGilvery and Goldstein (1983). Repeated measures ANOVA followed by a Tukey Post Hoc test (p< 0.05) was used to verify differences in CHO and FO rates among speeds. Paired T-test was used to verify differences in CHO and FO rates between tests per velocity. The reliability between-day was assessed using intraclass correlation coefficient (ICC). Results showed significant differences for CHO among all speeds, as well as significant differences for FO between V1 and V2 against V3 and V4 in both tests. Analyses between trials per velocity showed no significant substrate use differences as well as acceptable reliability. At the self-selected speed (V3) there was an accentuation in FO reduction as well as an increase in CHO oxidation. The purpose of the fifth study was to determine whether there were differences in substrate oxidation between girls (G) and women (W) during a treadmill protocol based on SS. Experiments were carried out on 12 G aged 8-12 (mean age = 9.9±1.4 yr) and 12 W aged 25-38 (mean age = 32.3±3.8 yr). The treadmill protocol included 6 min workloads followed by 5 min rest periods. Workloads included 2 speeds slower than SS (1.6 (V1) and 0.8 km.h-1 (V2) slower than SS), SS (V3), and a speed 0.8 km.h-1 faster than SS (V4). Total fat and carbohydrate (CHO) oxidation rates were calculated from indirect calorimetry according to the non-protein respiratory quotient. Repeated measures ANOVA followed by a Tukey Post Hoc test was used to verify intra-test differences in CHO and fat oxidation rates among speeds. Intergroup differences were analysed using paired T-test. Fat utilisation in W achieved a plateau at a relative velocity 0.8 km.h-1 slower than SS, but for G, fat utilisation increased until SS, and then stabilised upon reaching the higher velocity. CHO oxidation curves rose abruptly above V2 for W, while for G the acute increase occurred after SS (V3). Collectively, these results indicate that as walking intensity increases G are able to meet the energy demands of the work by increasing fat oxidation together with the increased CHO oxidation up to SS. In contrast for W, increasing CHO oxidation is associated with an early decrease in fat utilisation at a velocity slower than the self-selected speed. The sixth study dealt with validation of indirect techniques for the measurement of energy expenditure in free-living conditions against the DLW technique. Experiments were carried out on 19 children aged 8-12 (mean age = 10.3±1.0 yr). To indirectly predict energy expenditure 12 different procedures were used. Only one procedure, combining activity and heart rate (AHbranched), was based on a group equation, the others were based on individualised regression. Three of the individually-based techniques were able to accurately predict energy expenditure in free-living conditions. These tecniques were HRPAnetRMR using HRnet [HR exercise minus sleep HR (SHR)] against PAnet (measured PA exercise minus measured RMR) and upper and lower body equations corrected by RMR; HRPAnet4act using the same procedure but corrected by the mean resting &VO2 for 4 resting activities [(4act) = supine watching TV, sitting watching TV, sitting playing computer games and standing], and HRPALBnet4act using only lower body activities and corrected by 4act. HRPAnetRMR was only slightly more accurate than HRPAnet4act and HRPALBnet4act, but this technique is only adjusted by RMR whereas the other two are heavily dependent on more complex laboratory calibration. Bland and Altman (1986) analyses showed no significant differences between AHbranched predicted and measured TEE using the DLW technique. A SEE of 79 kcal.d-1 and a mean difference of 72 kcal.d-1, with a 95% CI ranging from -238 to 93.9 kcal.d-1 was found. In addition, no significant differences between predicted HRPAnetRMR and measured TEE using DLW were found, showing an SEE of 99 kcal.d-1 and a mean difference of -67 kcal.d-1, and a 95% CI ranging from -276.6 to 141.9 kcal.d-1. AHbranched and HRPAnetRMR were both valid and similarly suitable for the prediction of energy expenditure in children under free-living conditions. Significant associations between DLWAEE and the after-school time window indicated that this time window as an important discretionary period representative of children physical activity. However, the duration of the after-school time windows should be more carefully considered. Accelerometer data showed a better association between the largest after-school time window (3.5 hr) and measured TEE. The final study, completed with 19 children aged 8-12 (10.3±1.0 yr) highlighted, under laboratory conditions across a range of walking and running speeds, the inadequacy of the use of the standard MET in children. This traditional approach overestimates energy expenditure with an increased difference linearly related to speed increments. Minute-by-minute analyses of 7 days of free-living monitoring showed an average overestimation of 64 minutes per day for moderate-to-vigorousphysical- activity (MVPA) using the standard MET compared with the individually measured MET. For all intensities, these differences were statistically significant (p< 0.001). The second part of this study showed a variability of 20% in the average time spent at MVPA when comparing HR I 140 bpm and HR > 50%P &VO2 (P &VO2 = the highest &VO2 observed during an exercise test to exhaustion). Results of the current study compared to observations in the literature showed that HR I 140 bpm consistently estimates lower MVPA time than HR > 50%P &VO2. When these two PA indices were compared with individual and standard MET measured minute-byminute, statistically significant differences were verified among all of them at MPA, but no differences were verified at VPA, except between individual and standard METs. However, whether each one of the PA indices used are under- or overestimating time at MVPA is still debatable due to the lack of a gold standard. Finally, each index used in this study classified different numbers of participants as achieving the PA target of 60 min.d-1. The wide variability between indices when attempting to classify children who are achieving the recommended target is cause for great concern because habitually these indices are utilised as screening tools in paediatric and public health settings and used to guide behavioural interventions.