The goal of this project has been to develop therapeutic strategies for protein misfolding diseases caused by excessive degradation of misfolded proteins and loss of protein function. The focus for this work is lysosomal storage disorders (LSDs), a group of more than 50 known inherited metabolic diseases characterized by deficiency in hydrolytic enzymes and consequent buildup of lysosomal macromolecules. Gaucher’s Disease (GD) is used as a representative of the family of LSDs in this study. GD is caused by mutations in the gene encoding lysosomal glucocerebrosidase (GC) and consequent accumulation of the GC substrate, glucocerebroside. The most prevalent mutations among GD patients are single amino acid substitutions that do not directly impair GC activity, but rather destabilize its native folding. GC normally folds in the ER and trafficks through the secretory pathway to the lysosomes. GC variants containing destabilizing mutations misfold and are retrotranslocated to the cytoplasm for ER-associated degradation (ERAD). However, evidence shows that if misfolding-prone, mutated GC variants are forced to fold into their 3D native structure, they retain catalytic activity. This study describes strategies to remodel the network of cellular pathways that maintain protein homeostasis and to create a folding environment favorable to the folding of unstable, degradation-prone lysosomal enzyme variants. We demonstrated that folding and trafficking of mutated GC variants can be achieved by modulating the protein folding network in fibroblasts derived from patients with GD to i) upregulate the expression of ER luminal chaperones, ii) inhibit the ERAD pathway, and iii) enhance the pool of mutated GC in the ER amenable to folding rescue. We also demonstrated that the same cell engineering strategies that proved successful in rescuing the folding and activity of mutated GC enable rescue of mutated enzyme variants in fibroblasts derived from patients with Tay-Sachs disease, a LSD caused by deficiency of lysosomal hexosaminidase A activity. As a result, the current study provides insights for the development of therapeutic strategies for GD based on the modulation of general cellular pathways that maintain protein homeostasis that could in principle be applied to the treatment of multiple LSDs.

Sensor array calibration is a major problem in engineering, to which a biological approach may provide alternative solutions. For animals, perception is relative. The aim of this thesis is to show that the relativity of perception in the animal kingdom could also be applied to robotics with promising results. This thesis explores through various behaviours and environments the properties of homeostatic mechanisms in sensory cells. It shows not only that the phenomenon can solve partial failure of sensors but also that it can be used by robots to adapt to their (changing) environment. Moreover the system shows emergent properties as well as adaptation to the robot body or its behaviour. The homeostatic mechanisms in biological neurons maintain fi ring activity between predefi ned ranges. Our model is designed to correct out of range neuron activity over a relatively long period of time (seconds or minutes). The system is implemented in a robot’s sensory neurons and is the only form of adaptability used in the central network. The robot was fi rst tested extensively with a mechanism implemented for obstacle avoidance and wall following behaviours. The robot was not only able to deal with sensor manufacture defects, but to adapt to changing environments (e.g. adapting to a narrow environment when it was originally in an open world). Emergence of non-implemented behaviours has also been observed. For example, during wall following behaviour, the robot seemed, at some point, bored. It changed the direction it was following the wall. Or we also noticed during obstacle avoidance an emerging exploratory behaviour. The model has also been tested on more complex behaviours such as skototaxis, an escape response, and phonotaxis. Again, especially with skototaxis, emergent behaviours appeared such as unpredictability on where and when the robot will be hiding. It appears that the adaptation is not only driven by the environment but by the behaviour of the robot too. It is by the complex feedback between these two things that non-implemented behaviours emerge. We showed that homeostasis can be used to improve sensory signal processing in robotics and we also found evidence that the phenomenon can be a necessary step towards better behavioural adaptation to the environment.

The redox properties of Copper (Cu) make it both an ideal cofactor for many enzymes, and, in its free form, a highly toxic molecule capable of stimulating production of reactive oxygen species or binding to protein thiol groups. Therefore, living organisms have evolved homeostatic systems to “handle” Cu avoiding dangerous and wasteful aspecific interactions. These systems comprise uptake, carrier, storage and excretion proteins. The importance of Cu-homeostatic systems was initially discovered in humans where alterations of Cu-excretory proteins were shown to be responsible for two lethal genetic disorders; the Wilson and Menkes diseases. The levels of bioavailable Cu in the aquatic environment is important because concentrations in oceanic waters tend to be minute, whilst in some fresh and coastal waters, particularly around areas of mineral extraction, viniculture and farming operations, concentrations can be excessive. In contrast to terrestrial vertebrates, fish are not only exposed to dietary sources of copper but are also exposed to dissolved ionic copper that may enter via the skin and gills. Indeed, the latter route is important in fish and it has been demonstrated in physiological studies that under conditions of dietary deficiency, fish can satisfy their own body requirements by uptake from water. Therefore, fish must have systems relating to both gill and gut to enable maintenance of body homeostasis of this essential, yet toxic, metal. In an attempt to understand the mechanisms of Cu homeostasis in fish, whether under conditions of deficiency, adequacy or excess, it is essential to consider the expression of known Cu-homeostasis proteins. Thus, cDNAs for sea bream (Sparus aurata) homologues of copper transporter 1 (Ctr1), antioxidant protein 1 (Atox1), Menkes protein (ATP7A), Wilson protein (ATP7B), and metallothionein (MT), which are responsible for the uptake, delivery to the secretory pathway and scavenging of intracellular Cu, were cloned and their mRNA tissue expression levels measured. To investigate the molecular basis of the different homeostatic and toxic responses to waterborne or dietary Cu, sea bream were exposed to sub-toxic levels of Cu in the diet (130 mg/Kg of dry diet) or water (0.3 mg/L) and tissue mRNA and Cu levels were measured. Moreover, to discriminate between the effect of different metals on the transcriptional regulation of Cu homeostasis genes in fish, Sparus aurata fibroblast (SAF1) cells were exposed to sub-toxic levels of Cu (25 μM), Zn (100 μM) and Cd (10 μM). In addition, a microarray was used to gain a broader overview of the transcriptional response of SAF1 cells to Cu (25 μM). Waterborne or dietary Cu resulted in distinct expression profiles of Cu-homeostasis genes and markers of oxidative stress. After dietary exposure, Cu increased in intestine and liver, whilst after waterborne exposure Cu increased in gill and liver. Exposure to dietary Cu resulted in decreases in Ctr1 and ATP7A mRNA in both liver and intestine. Renal Ctr1 levels remained unchanged, whilst ATP7A mRNA decreased. In contrast, waterborne Cu exposure increased intestinal Ctr1 and ATP7A mRNA, and increased renal Ctr1 and decreased renal ATP7A mRNA. Both dietary and waterborne Cu increased ATP7B mRNA in liver. Metallothionein (MT) mRNA increased in liver and gill after waterborne Cu. Glutathione reductase (GR), a marker of oxidative stress, increased expression in liver and gill after waterborne Cu exposure, but decreased in intestine. Thus, exposure to Cu via water or diet has different, often opposite effects on Cu-homeostasis genes. The decrease in expression of both Cu-transport genes in intestine after dietary exposure may indicate a defensive mechanism to limit uptake of Cu. The opposite effects in intestine after waterborne exposure are more difficult to explain, but again may reflect a defence mechanism against excess bloodborne Cu coming from the gill. Since both dietary and waterborne Cu increased Cu levels in liver and increased hepatic ATP7B it is likely that well-characterised mammalian route of Cu excretion to bile is active in sea bream. However, only hepatic Cu derived from gill increased the expression of the stress markers MT and GR. This suggests that Cu is delivered to liver in a different form from gill as that from intestine, the intestinally derived pool being less toxic. Thus the increase in copper transport gene expression in intestine after gill exposure might be a mechanism to enable incorporation of excess bloodborne Cu into the intestinal pathway of Cu delivery to liver, thus minimizing toxicity. The in vitro exposure of SAF1 cells to Cu showed a similar response to liver of fish exposed to waterborne Cu indicating similar Cu availability and complexation. ATP7A mRNA levels were induced by Cu but not by Zn or Cd suggesting Cu-specific regulation. Conversely, MT and GR were induced by all metals tested. The transcriptomic analysis highlighted that the biological processes most significantly affected by Cu were secretion, protein trafficking and stress. Overall, these results show that in fish copper has distinct effects on tissue Cu transporter genes and oxidative stress depending on whether it is taken up via the gill or gut and that intestinal absorption may be required for normal uptake and metabolism of Cu, regardless of the route of uptake. Moreover, changes in mRNA levels indicate that Cu homeostasis genes, at least in fish, may be regulated at the transcriptional level. Although more work needs to be done to identify genes that are robust predictors of Cu toxicity, the microarray results presented here show a clear transcriptional fingerprint which may characterize Cu toxicity in fish.

The subfornical organ (SFO) is a sensory circumventricular organ, lacking a proper blood-brain barrier. Neurons of the SFO are exposed directly to the ionic environment and circulating signaling molecules in the plasma, providing a unique window for communication of physiological status from the periphery to the central nervous system (CNS). The SFO is recognized as a key site for hydromineral balance, cardiovascular regulation and energy homeostasis. Neuropeptide Y (NPY) is a potent stimulator of food intake when released centrally, and has well-documented pressor effects when released peripherally. It has been demonstrated that the SFO expresses NPY receptors, however the effects of NPY on SFO neurons has never been investigated. The aim of this study was to determine the effects of NPY on the electrophysiological properties of SFO neurons dissociated from Sprague Dawley rats. Using whole cell patch clamp techniques in the current-clamp configuration, we report that 300 nM NPY caused 16% of SFO neurons to depolarize and 26% to hyperpolarize. The remaining neurons were insensitive to NPY. These effects were dose-dependent with a combined EC50 of 3.7 nM. Specific NPY receptor antagonists were applied, suggesting that the Y5 receptor predominately elicited a hyperpolarizing effect, while the Y1 receptor had a mixed response that was predominately hyperpolarizing, and the Y2 receptor had a mixed response that was predominately depolarizing. Using the voltage-clamp configuration, it was also observed that NPY caused an increase in the voltage-gated K+ current density as well as a shift in membrane activation of the persistent Na+ current, mediating the hyperpolarizing and depolarizing effects, respectively. These findings indicate that NPY elicits electrophysiological changes on SFO neurons, suggesting that the SFO is a key site of action for NPY in mediating energy regulation and/or cardiovascular output. / February 2017

No description available.

No description available.

Spike-timing dependent plasticity is a learning mechanism used extensively within neural modelling. The learning rule has been shown to allow a neuron to find the onset of a spatio-temporal pattern repeated among its afferents. In this thesis, the first question addressed is ‘what does this neuron learn?’ With a spiking neuron model and linear prediction, evidence is adduced that the neuron learns two components: (1) the level of average background activity and (2) specific spike times of a pattern. Taking advantage of these findings, a network is developed that can train recognisers for longer spatio-temporal input signals using spike-timing dependent plasticity. Using a number of neurons that are mutually connected by plastic synapses and subject to a global winner-takes-all mechanism, chains of neurons can form where each neuron is selective to a different segment of a repeating input pattern, and the neurons are feedforwardly connected in such a way that both the correct stimulus and the firing of the previous neurons are required in order to activate the next neuron in the chain. This is akin to a simple class of finite state automata. Following this, a novel resource-based STDP learning rule is introduced. The learning rule has several advantages over typical implementations of STDP and results in synaptic statistics which match favourably with those observed experimentally. For example, synaptic weight distributions and the presence of silent synapses match experimental data.

Mast cells are potent effectors of the inflammatory response, playing an important role in atopy, bacterial immunity, and animal models of arthritis, multiple sclerosis, and heart disease. Hence controlling mast cell numbers and responsiveness is essential for preventing inflammatory disease. This work demonstrated that the cytokine TGF-β1 is a potent inducer of mast cell apoptosis, a finding that was consistent for cultured mouse bone marrow-derived mast cells, peritoneal mast cells, and human mast cells. Cell death appeared to be the result of TGF-mediated repression of IL-3 receptor expression and function, leading to mitochondria1 damage and activation of an apoptotic cascade acting via p53 and caspases. While IL-3 receptor expression was reduced within one day of TGF-βl stimulation, apoptosis required at least 3 days to occur. This delay in onset is postulated to allow for protective mast cell effector functions, protecting the host from infection while preventing the establishment of chronic inflammation. These studies support the theory that TGF- β1 is an inhibitor of mast cell survival. Because of the widespread expression of TGF-β1, this cytokine may be an ideal candidate for control of mast cell homeostasis.

Anthropogenic activities are fundamentally altering the chemistry of the world’s oceans. Many of these modifications could have a significant impact on the health of marine organisms. Yet, despite being proposed as one of the most significant threats that marine ecosystems face, to date very little is known about the impact of anthropogenic climate change, and ocean acidification in particular, on host defence. The aims of this thesis are to investigate the impact of environmental stressors on the invertebrate immune response, providing empirical data on how anthropogenically induced stressors will impact the invertebrate immune system and how this will impact organism condition and subsequent physiological trade-offs. Exposure to reduced seawater pH and increased temperature significantly reduced the immune response in the blue mussel, Mytilus edulis. This reduction in immune response could indicate stress-induced immune dysfunction. However, the immune system protects an organism from infectious disease, ensuring survival, and should therefore be evaluated functionally rather than immunologically. By subsequently exposing mussels to a bacterial challenge this study demonstrated that an earlier study which measured a reduction in host defence represented a trade-off of immune system maintenance costs, with mussels maintaining a capacity to up-regulate immune defence when required. However, whilst this immune plasticity ensures mussels are able to survive a pathogen exposure, such a strategy appears to be physiologically costly. This cost is seen as a reduction in reproductive investment, an altered energy metabolism and an altered fatty acid composition in organisms exposed to low pH. Therefore the overarching picture that emerges is, without measuring physiological processes functionally, and in neglecting any physiological trade-offs, it is possible that many studies may misinterpret the complex physiological responses of marine organisms to ocean acidification.

Tout au long de la vie d’un individu, il existe un nombre optimal de cellules à produire et de progéniteurs à conserver en réserve. On parle de maintien de l’homéostasie tissulaire. De façon générale, l’organisme a cinq possibilités pour réguler l’homéostasie : l’autorenouvellement et la quiescence, souvent utilisés pour maintenir un ‘pool’ fonctionnel de progéniteurs, la différenciation qui permet de produire des cellules effectrices, l’apoptose et la sénescence, qui permettent de limiter la production de cellules ou encore d’en faire diminuer le nombre quand elles sont en excès. La régulation de ces quatre mécanismes peut se faire de façon extrinsèque en passant par différentes voies de signalisation combinées à l’action intrinsèque de facteurs de transcription comme Ikaros et GATA1. Le facteur de transcription Ikaros joue un rôle critique dans le devenir des cellules progénitrices et la différenciation des lignages hématopoïétiques. Cependant, il demeure surtout connu pour son influence sur la voie Notch dans les cellules lymphoïdes, notamment les lymphocytes T. Les cellules érythroïdes sont hautement sensibles à l’environnement et donc, particulièrement adaptées à l’étude des régulations de l’homéostasie. Les résultats de différentes études ont permis de démontrer qu’Ikaros et la voie Notch influencent l’érythropoïèse. Cependant le détail de leurs actions demeure en grande partie inconnu à ce jour. Au cours de notre étude nous avons voulu déterminer l’action d’Ikaros dans le maintien de l’homéostasie des cellules érythroïdes et si son rôle passe par un dialogue avec la voie Notch. Nous avons voulu décrypter les mécanismes de régulation transcriptionnelle utilisés par Ikaros et par Notch au cours de l’érythropoïèse et leurs effets. Notre étude montre qu’Ikaros réprime à l’aide de GATA1 le gène Hes1, une cible importante de la voie Notch, en recrutant un complexe de la famille Polycomb, le PRC2 ii (Polycomb Repressive Complex 2). Cette répression permet la promotion de la différenciation des cellules érythroïdes. Au niveau du maintien de l’homéostasie par régulation de l’apoptose, Ikaros est connu pour cibler l’anti-apoptotique Bcl2l1 dans les lymphocytes. Puisque Gata-1, partenaire préférentiel d’Ikaros cible Bcl2l1 dans les cellules érythroïdes, nous avons caractérisé leur effet sur l’expression de Bcl2l1. Nous avons découvert qu’Ikaros active de façon directe Bcl2l1 et qu’il recrute sur le gène deux complexes partenaires d’élongation : un de la famille SET1/MLL, et le complexe P-TEFb-NuRD. En l’absence d’Ikaros, le fragment intracellulaire de Notch (NICD) et son cofacteur RBP-J remplacent Ikaros et favorisent l’hyper-activation de l’expression de Bcl2l1. Ceci est associé à la modification du complexe d’élongation recruté, ainsi qu’à la mise en place de modifications épigénétiques distinctes de celles observées avec Ikaros ce qui modifie l’élongation transcriptionnelle du gène. Ikaros et Notch sont fréquemment mutés ou présentent des fonctions altérées dans les leucémies. Notre étude montre un dialogue Ikaros/Notch influençant aussi bien la différenciation que l’apoptose et met en évidence l’existence d’un circuit génétique dont le dérèglement pourrait favoriser l’apparition d’une hématopoïèse maligne. / Throughout the life of an individual, there is an optimal count of cells to produce and progenitors to conserve in stock. This is the tissue homeostasis maintenance. In a general fashion the organism has five means to regulate the homeostasis. Self-renewal and quiescence, often used in order to maintain a functional progenitors pool. Differentiation enhances effector cells production. Apoptosis and senescence can limit cell production and reduce cell number in case of excess. These regulation mechanisms can be performed in an extrinsic fashion using different signaling pathways combined with the action of transcription factors like Ikaros and GATA1. The transcription factor Ikaros is critical for progenitor cells fate and hematopoietic lineages differentiation. However, Ikaros is mostly known for its influence on Notch signaling in lymphoid cells, notably T lymphocytes. Erythroid cells are highly sensitive to the environment thus, particularly adapted to study homeostasis maintenance regulation. Results obtained in different studies showed Ikaros and Notch signaling influencing erythropoiesis. However, the detail of their effect remains mainly unknown to day. Our aim was to determine Ikaros effect on erythroid cells homeostasis maintenance and if its role involved a cross-talk with Notch signaling. We will decipher transcription regulation mechanisms used by Ikaros and Notch during erythropoiesis and their effects. We show Ikaros uses GATA1 to repress Hes1, a major Notch target by recruiting a Polycomb family complex, the PRC2 (Polycomb Repressive Complex 2). This repression promotes erythroid cells differentiation. At the apoptosis mediated control of homeostasis level, Ikaros is known to target Bcl2l1 in lymphocytes. As GATA1, Ikaros preferential partner, targets Bcl2l1 in erythroid cells, we assessed their effect on Bcl2l1 expression. We discovered Ikaros directly activates Bcl2l1 iv and recruits two elongation associated complexes: one from SET1/MLL complex family, and the P-TEFb-NuRD complex. In the absence of Ikaros, the intracellular fragment of Notch (NICD) and its cofactor RBP-J replace Ikaros and favors Bcl2l1 overactivation. This is associated with a switch of recruited elongation associated complex and the establishment of distinct epigenetic modifications from those observed with Ikaros, which modifies the gene transcriptional elongation. Ikaros and Notch are frequently mutated or present altered functions in leukemia. Our works present an Ikaros/Notch cross-talk influencing as well differentiation as apoptosis and reveal the existence of a genetic circuit for which a malfunction could favor hematologic disorders. Keywords : transcription, homeostasis, erythropoiesis