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Mitochondria play a major role in the maintaining the cellular homeostasis, it is therefore to be expected that if this homeostasis is challenged, the mitochondria would be among the first to respond. Since homeostasis is essential to survival, the stress response achieves what is termed as "ALLOSTASIS" which can be defined as the process of maintaining homeostasis by active means, through the release of stress response mediators. Depression has been described as a representation of the activation of primary stress mediators. This thesis explores the effect of stress on neuronal energy metabolism. To achieve this, various experimental studies were designed to encompass a variety of stimuli; e.g. age, anxiety, inflammation and physiological stress i.e. elevated serum corticosterone. Mitochondria were prepared using differential centrifugation through 18% (v/v) percoll density gradient. To establish a sound base on which the study was to be based the mitochondrial preparation was validated using respiratory control index (RCI), marker enzymes and electron micrographs, and compared to various other studies using the same technique for preparation. The preparation used in this study was demonstrably superior in purity and function. Initial studies dealt with age and anxiety as sources of stress. It was demonstrated that older rats (24months+) have lower (20-25%) baseline mitochondrial function as compared to young animals (2-3 months). When exposed to anxiety, young animals demonstrated a transient decline in mitochondrial function seen as a fall in RCI, but older animals showed an inhibitory effect on Complex 11 of the mitochondrial respiratory chain which was seen as a significant (P<0.01) decrease in reactive oxygen species (ROS) production in the presence of 5 mM succinate but not 5 mM glutamate plus 5 mM malate (G+M). This could be attributable to the phenomenon of partial uncoupling, a protective mechanism that stems the production of large amounts of ROS in stressed mitochondria by lowering the membrane potential, and the consequent loss of reverse electron transfer that is responsible for the large amounts of ROS being produced at complex I in the presence of a complex 11 substrate. lnterleukin-tp (IL-1P) was found to block the improvement of brain mitochondrial function resulting from exposure to brain derived neurotropic factor (BDNF; 333 ng rnl"). This effect was found to be concentration-dependent. IL-1 p was seen to have no effect on the mitochondrial function in the absence of BDNF. An earlier study by this laboratory had shown the involvement of RAS/MAPK pathway in the mitochondrial propagation of the signal initiated by BDNF. IL-1 P was seen to interfere with this pathway at the site of scaffolding proteins that are essential for MAPK docking and therefore for signal propagation. Using an in vivo model of chronic stress involving oral corticosterone (0.5%) in drinking water for 14 days was developed. This model showed successfully that chronic exposure significantly reduced the RCI of treated animals from 8.70 ± 0.30 to 7.17 ± 0.17 for G+M and from 7.56 ± 0.60 to 5.50 ± 0.20 for succinate (P<0.05; n=5). To further validate the model, daily water consumption and weight gain charts were maintained and they showed that there was no significant variation between the treated and the untreated group. This model was then used to simulate chronic stress induced depression in subsequent studies. The prototypes of the three generations of antidepressants: imipramine for 1st generation, fluoxetine for 2nd generation and tianeptine for the novel agents or 3'd generation, were used in subsequent in vivo experiments designed to study the effect of stress and antidepressant therapy on mitochondrial function expressed in terms of RCI, ROS production and the effect on serum corticosterone through a quantitative enzyme immunoassay. The main findings of the in vivo studies were that imipramine acts primarily through a corticosterone- dependent pathway because it significantly lowered serum corticosterone concentration in corticosterone treated animals. However its effects on the mitochondrial function were not significant. Fluoxetine was found to have no effect on mitochondrial function, either in vitro or in vivo. Tianeptine was found to have an effect on the mitochondrial function in vitro but not in vivo. This study has identified a potential pivotal role for mitochondria in relation to the manifestation of stress responses, regardless of the origin of the stressor.

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Pronounced seasonal adaptations in physiology and behaviour are exhibited by mammals living in polar and temperate habitats. These include the development of a winter coat, altered fat reserves, reproductive quiescence and food hoarding. Maintaining constant body temperature (Tb) during winter is energetically very costly, and so many small mammals periodically abandon homeothermy in favour of heterothermy. The two principal heterothermic strategies are daily torpor and seasonal hibernation, in which bouts of profound hypothermia range from a few hours to several days (respectively). It is now clear that hypothalamic thyroid hormone (TH) regulation, and specifically the availability of the active metabolite triiodothyronine (T3), is a critical regulator of seasonal reproductive cycles in many species including birds and mammals. The impact of this signal as a switch for seasonal changes in physiology has been highlighted by the demonstration that blockade of this pathway prevents seasonal adaption in hamsters. Peripheral TH signalling is also a principle regulator of metabolic rate in mammals. Despite these findings nothing is yet known about the involvement of central (hypothalamic) and peripheral TH cycles in the expression of torpor and hibernation. Within this thesis, the role of TH dynamics both in the brain and peripheral circulation is examined within three models of heterothermia: the Siberian (Phodopus sungorus) and European (Cricetus cricetus) hamsters, which employ daily torpor and hibernation, respectively, and the laboratory mouse (Mus Musculus) which exhibits torpor in response to metabolic stress such as food restriction. To delineate TH regulation and signalling in the context of both seasonal and acute physiological responses, the expression of genes involved in thyroid hormones conversion (e.g. Deiodinase type II (Dio2) and type III (Dio3) and transport (e.g. Monocarboxylate transporter 8, Mct8) within the ependymal layer of the ventral 3rd ventricle have been detailed across seasonal (long (LD) and short day (SD)) photoperiods, and during normothermic and hypothermic conditions. Furthermore, TH concentrations have been directly measured within the hypothalami of P. sungorus and C. cricetus, and TH responsive genes (e.g. Hairless (Hr) and Thyrotropin releasing hormone (TRH) to determine the potential impact of regional T3 signalling. As expected, Dio2 and Dio3 expression in P. sungorus exhibited a strong seasonal cycle indicative of elevated T3 production during SD (reduced Dio2 and elevated Dio3). Unexpectedly, total T3 measures from hypothalamic extracts revealed no significant alteration either seasonally or during torpor/hibernation in hamsters. However, Hr expression in the ependymal layer and TRH expression in the paraventricular nucleus (PVN) suggests low T3 concentrations during SD are localised to specific regions and does not encompass the whole hypothalamus per se. In addition, altered serum TH concentrations implicate seasonal and torpor associated dynamics that may play a role in seasonal adaptation and hypothermia. Finally, data from transgenic mice strongly implicate the melatonin-related receptor (GPR50) in leptin signalling and aberrant thermogenesis in mice.

L'arthrose (OA) est une maladie articulaire chronique qui résulte de changements complexes dans le phénotype des chondrocytes. La présence de microcristaux contenant du phosphate dans les zones de cartilage lésées suggère que le métabolisme phosphocalcique contribue en partie aux modifications du phénotype chondrocytaire au cours de la maladie. De nombreuses études ont montré que des concentrations élevées en Phosphate Inorganique extracellulaire (ePi) ou en PyroPhosphate Inorganique (ePPi) ont respectivement un effet activateur ou répressif sur la minéralisation du cartilage articulaire. Comme le Fibroblast Growth Factor 23 (FGF23) régule les concentrations de Pi, FGF23 semble être un candidat aux modifications phénotypiques observées dans l'OA. De plus, il a récemment été mis en évidence que l’ePPi prévient la dédifférenciation in vitro des chondrocytes articulaires chez le rat, un effet provoqué par la production de PPi par la protéine Ank. Cela suggère que l’ePPi pourrait être un candidat pour prévenir les modifications du phénotype chondrocytaire. Premièrement, nous avons montré que l’expression de FGF23 est plus importante dans du cartilage lésé que dans du cartilage sain. Sous stimulation croissante de FGF23, les chondrocytes humains OA présentent une expression soutenue des marqueurs d’hypertrophie tels que COL10A1, VEGF et MMP13. Nous avons également démontré que l’expression de MMP13 est fortement dépendante de FGFR1 mais indépendante de Klotho et qu’elle est fortement régulée par la voie MEK/ERK et dans une moindre mesure par la voie PI3K/AKT. Deuxièmement, nous avons montré que FGF23 est produit de façon plus importante au cours de la différenciation des ATDC5 et qu’une stimulation par FGF23 augmente la minéralisation et l’expression des marqueurs d’hypertrophie, et ce, d’autant plus fortement en présence d’une stimulation par du Pi dans ces cellules. Dans la seconde partie, nous avons montré que des chondrocytes humains OA stimulés par du PPi présentent une expression diminuée des composants collagéniques de la matrice et une expression augmentée des MMPs, de la fibronectine et des intégrines. Une stimulation par le PPi active de façon importante la voie p38 et dans une moindre mesure la voie ERK pour réguler l’expression de ses gènes cibles et notamment MMP13 d’une manière Ank indépendante. Enfin, nous avons démontré qu’une stimulation par FGF23 entraine une augmentation de l’expression de Pit-1, ENPP1 et ANK ainsi que la production de PPi par les chondrocytes humains OA. Les résultats obtenus dans cette étude démontrent que le FGF23 permet localement une différenciation des chondrocytes OA vers un phénotype hypertrophique et peut potentiellement être considéré comme un facteur aggravant de l’OA. Contrairement aux données préliminaires chez le rat, le PPi permet un remodelage matriciel des chondrocytes humains OA et pourrait potentiellement contribuer aux effets pro-hypertrophiques du FGF23 / Osteoarthritis (OA) is the most common form of chronic joint disease, characterized by cartilage degeneration that results from complex changes in the chondrocyte phenotype. The presence of phosphate-containing microcrystals in the injured cartilage areas suggests the contribution of the phosphocalcic metabolism in the phenotype switch of chondrocytes during the disease. Numerous studies have shown that elevated concentrations of extracellular inorganic phosphate (ePi) or inorganic pyrophosphate (ePPi) have, respectively, activating or repressive mineralizing effects on articular cartilage. As Fibroblast Growth Factor 23 (FGF23) plays a major role in regulating concentrations of Pi, FGF23 is an attractive candidate to participate in the phenotype switch of the articular chondrocyte observed in OA. Moreover, we recently demonstrated that ePPi also prevents the in vitro dedifferentiation of articular chondrocyte in rats, an effect mostly triggered by Ank-induced release of PPi. This suggests that PPi may be an attractive candidate to prevent the phenotype switch of the articular chondrocyte. Firstly, we showed that FGF23 expression was higher in OA samples than in healthy one. When stimulated with increasing concentrations of FGF23, human OA chondrocytes displayed a sustained expression of markers of hypertrophy such as COL10A1, VEGF and MMP13. We demonstrated further, that MMP13 expression was mainly dependent on FGFR1 and independent of Klotho and was strongly regulated by the MEK/ERK cascade and to a lesser extent by the PI-3K/AKT pathway. Secondly, we showed that FGF23 and FGFRs were produced more importantly during ATDC5 differentiation and that FGF23 stimulation increased hypertrophic markers expression and mineralization in a synergic manner with Pi. In the second part, we showed that human OA chondrocytes stimulated with PPi displayed a decreased expression of collagen components of the matrix and sustained expression of MMPs, fibronectin and integrins. We demonstrated further that PPi stimulation mostly activates p38 pathway and to a lesser extent ERK pathway to regulate the expression of its target genes in an Ank-independent manner. Finally, we demonstrated that FGF23 stimulation increased Pit-1, ENPP1 and Ank expressions and PPi production by human OA chondrocyte. Altogether, the results obtained in this study demonstrate that FGF23 locally promotes differentiation of OA chondrocytes towards a hypertrophic phenotype and may therefore be considered as an aggravating factor for OA. In contrast to previous data obtained in rats, we demonstrated that PPi promotes matrix-remodeling of human OA chondrocytes and might contribute to FGF23 pro-hypertrophic effect