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DISC1, a strong genetic candidate for psychiatric illness, is a molecular scaffold residing in multiple subcellular compartments, where it regulates the function of interacting proteins with key roles in neurodevelopment and plasticity. Both common and rare DISC1 missense variants are associated with risk of mental illness and/or brain abnormalities in healthy carriers, but the underlying mechanisms are unclear. In this thesis, I initially examine the effect of a panel of common and rare single amino acid substitutions on DISC1 subcellular targeting, establishing that the rare mutation R37W and the common variant L607F disrupt DISC1 nuclear targeting in a dominant-negative fashion. This finding predicts that DISC1 nuclear expression is severely impaired in 37W and 607F carriers. In addition, I show that the L607F substitution results in aberrant cytoplasmic and cytoskeletal distribution of DISC1. In the nucleus, DISC1 interacts with the transcription factor ATF4, which is involved in the regulation of cellular stress responses and memory consolidation. Here I show that at basal cAMP levels, wild-type DISC1 strongly inhibits the transcriptional activity of ATF4, and this effect is ablated by 37W and 607F, most likely as a consequence of their defective nuclear targeting. 607F additionally reduces DISC1/ATF4 interaction, which likely contributes to its weakened inhibitory effect. I also demonstrate that DISC1 modulates transcriptional responses to endoplasmic reticulum stress, and that this modulatory effect is also ablated by 37W and 607F. By providing evidence that single amino acid substitutions of DISC1 associated with psychiatric illness impair its regulatory function on ATF4-dependent transcription, I highlight a potential mechanism by which these protein variants may impact on molecular pathways underlying cognition and stress responses, two processes of direct relevance to psychiatric disease.

The onset of fire within a compartment can pose a hazard to the occupants and the structure containing the compartment. Fire suppression systems aim to either extinguish or suppress an incipient fire before loss of life or damage to the structure can occur. The geometry and use of the compartment as well as the fuel packages within must be taken into account when choosing an appropriate fire suppression system. This thesis explores novel suppression methods inside of compartments. Los Alamos National Laboratories came to the University of Texas Fire Research Group (UTFRG) to characterize and investigate the fire danger inside of nuclear gloveboxes. The first suppression method discussed explores activation tests of a commercial automatic fire suppression system (Fire Foe [superscript TM]) containing heptaflouropropane (FE-36) fire suppressant conducted within a glovebox at the UTFRG's burn structure. Temperature and time to activation data of ten tests at four different fire sizes, three 13 kW, one 20 kW, three 25 kW, and three 50 kW, was taken. Gas temperatures from experiments were compared against NIST's Fire Dynamics Simulator (FDS) gas temperatures with good agreement. The time and spatially averaged net heat flux on a virtual Fire Foe [superscript TM] tube from the FDS simulations were passed to a thermo-physical, semi-empirical, sub-model to predict activation with poor agreement from experimental activation times. A Bayesian parameter inference was later run on the sub-model. While the Bayesian inference approach is able to match sub-model temperatures to experimental temperatures, some non-physical values for heat transfer coefficients and view factors were observed at the lower heat release rate fires. Micro combustion calorimetry (MCC) was used to determine heat of combustion of glovebox glove material and cone calorimetry tests were run to find ignition time versus incident heat flux. Using standard ignition time models, effective model parameters were calibrated. Thermal characterization of the glove material showed that the heat of combustion found from MCC was within the range of heats of combustion for other non-halogenated materials found in the literature. Analysis of the time to ignition tests showed that the glove material should be modeled as thermally thick when one would expect thin behavior. This behavior was attributed to possible heat losses from the back of the glove material. Dry water is expected to have similar suppression characteristics as water mist systems because the dry water particle sizes are on the order of water mist droplet sizes. The major benefit with dry water is the low pressures needed to drive the aerosol. An issue encountered with the dry water was flowing it in the way one would flow normal water. It was found that at low normal and shear stresses, the dry water clathrates would release the water held inside. A possible low shear delivery mechanism was discussed that avoids the ratholing effect. A continuous dry water production system was also designed. Filter loading tests were conducted to determine the quality of the dry water collected from the batch and continuous cases. It was observed that the ratio of water to silica for the continuous case reaches the batch value and is similar to results found in the literature. For the batch dry water it was observed that the particle size of the dried clathrates does vary with rotational speed of the blender and is independent of the type of water used (tap or deionized). / text

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Ectotherms are living creatures whose body temperature varies with the environment in which they live. Their physiology and metabolism have to rapidly respond to environmental changes in order to stay viable at across their tolerable thermal range (Lithgow et al. 1994). In nematodes such as Caenorhabditis elegans, temperature is an important factor that defines the fertility of the worm. A feature that delimits an ectotherm’s thermal range is the maximum temperature at which its germ line can produce gametes. How germ cells withstand high environmental stressors such as limiting temperatures is not well understood, especially when considering the thermodynamical principles that dominate the biochemical processes of the cytoplasm (Hyman and Brangwynne 2011). Previous studies in C. elegans have shown that the thermodynamic effects of temperature on the cell cycle rate in nematodes follows an Arrhenius relationship and defines the thermal range where worms can be fertile. At the limits of this relationship a breakdown of the Arrhenius trend is observed (Begasse et al. 2015a). It was hypothesized that some type of discontinuous phase transition occurred in the embryonic cells of C. elegans (Begasse et al 2015). However, it remains unknown if there is the physiological link between a drop off in fertility and the embryonic breakdown of the Arrhenius trend. This work finds the link between a temperature driven phase separation of P granules and fertility. P granules are important for germ line development and the fertility of C. elegans (Kawasaki et al. 1998b). Here it is shown that P granules mix with the cytoplasm upon a temperature quench of 27ºC to T=18ºC and de-mix from the cytoplasm forming droplets upon a temperature downshift of temperature from 18ºC to 27ºC. P granules also show a reversible behavior mixing and de-mixing with changes in temperature in vivo, having a strong dependence of these liquid-like compartments with entropy. These results were further confirmed using a minimally reconstituted, in vitro P granule system and showed that PGL-3, a constitutive component of P granules, can phase separate and form liquid compartments in a similar way as happens in vivo. Additionally, here it is shown that P granule phase separation does not require the chemical activity of other cytoplasmic factors to drive the phase separation of compartments in vivo and in vitro, instead their formation is strongly driven to mix and de-mix with changes in temperature. Furthermore, a binary phase diagram was constructed in order to compare the response of P granules in vivo and in vitro, showing that P granules form and function as a temperature driven liquid phaseseparation. Altogether, this indicates that P granules in vivo and PGL-3 liquid-like compartments in vitro, share the same temperature of mixing and de-mixing which coincides with the fertile temperature range over which Caenorhabditis elegans can reproduce. This suggests that P granule phase separation could define the thermal range of the worm.

Chez les eucaryotes, la photosynthèse a lieu dans le chloroplaste, un organite spécifique de la cellule végétale et caractérisé par différents compartiments : (i) l'enveloppe, la double membrane qui délimite le chloroplaste ; (ii) le stroma, phase aqueuse principalement composée de protéines solubles et (iii) un système membranaire interne, les thylacoïdes, qui contiennent les complexes photosynthétiques. Les thylakoïdes forment un réseau tridimensionnel continu et sont différenciés en deux domaines physiques distincts : des empilements de vésicules de membrane (appelés granas ou BBY) et des extensions de membrane simple (lamelles stromales). Les complexes majeurs de la photosynthèse ne sont pas distribués de manière égale dans cette membrane à cause de contraintes électrostatiques et stériques. Ainsi, le photosystème I et l'ATP-synthétase sont enrichis dans les granas, le photosystème II dans les lamelles stromales alors que d'autres complexes, comme le cytochrome b6f, auraient une répartition équivalente entre granas et lamelles stromales. Pour faire face aux variations environnementales de lumière (en qualité et quantité), les plantes ont développé des processus pour moduler leur capacité d'absorption et d'utilisation de la lumière par les photosystèmes, processus regroupés sous le terme de « quenching non photosynthétique ou NPQ ». Dans le cadre de ma thèse, je me suis intéressé à deux composants du NPQ, les états de transition et la dissipation sous forme de chaleur (partie qE).Le premier objectif de ma thèse a été d'identifier de nouveaux acteurs impliqués dans les transitions d'état et ceci en étudiant la relocalisation de protéines au sein des sous-compartiments des thylacoïdes par une approche protéomique. En effet, il a été montré que certaines antennes collectrices de lumière sont réorganisées dans les membranes photosynthétiques lors des transitions d'état. Jusqu'à présent, aucune description exhaustive de la composition et distribution des protéines dans les sous-compartiments de thylacoïdes n'avait été réalisée. J'ai donc dans un premier temps développé des protocoles de purification des sous-compartiments des thylakoïdes (granas et lamelles stromales) à partir de chloroplastes de plantes sauvage d'Arabidopsis thaliana. Ensuite, grâce à une approche d'analyse protéomique semi-quantitative, nous avons pu déterminer la localisation d'environ 300 protéines des thylacoïdes. Les résultats suggèrent que la localisation de complexes photosynthétiques est beaucoup plus dynamique que celle jusqu'à lors proposée. En effet, même s'ils sont préférentiellement identifiés dans un sous-compartiment, certains complexes photosynthétiques présentent une double localisation qui était inattendue. De plus, la composition en sous-unités de ces complexes diffère selon leur localisation, dans les granas et dans les lamelles stromales, suggérant l'existence de processus de régulation de la photosynthèse jusqu'à lors insoupçonnés. Cette approche a ensuite été appliquée sur des plantes mutantes d'Arabidopsis affectées dans les transitions d'état afin d'identifier des protéines pouvant être impliquées dans ce processus d'adaptation. En parallèle, je me suis intéressé au qE . L'activation de ce mécanisme n'est pas constitutive et nécessite la formation d'un gradient de pH entre le stroma et le lumen des thylacoïdes (ΔpH). L'objectif de l'étude a été d'identifier des acteurs pouvant contrôler la formation de ce gradient de pH. Pour cela, nous nous somme focalisés sur le rôle d'un transporteur de potassium récemment caractérisé, TPK3. Grâce à des approches biophysiques et biochimiques, nous avons démontré que TPK3 est impliqué, in vivo, dans la modulation des deux composantes de la force proton motrice (pmf), le gradient de pH (ΔpH) et la différence de potentiel (Δψ). En contrôlant la répartition de la force proton motrice,TPK3, permet une utilisation correcte de la lumière en dissipant l'excès d'énergie. / Within higher plants and algae, photosynthesis is carried out in the chloroplast. Structurally, chloroplasts are organized in (i) the envelope, a double membrane system surrounding the chloroplast (ii) the stroma, the aqueous space which mainly contains soluble proteins and the (iii) thylakoids, a three-dimensional membrane network where photosynthetic electron transport reactions occur. Thylakoids are non-homogeneously folded, and comprise two major domains: (i) the grana-BBY, which are stacks of thylakoids particularly enriched in photosystem II, LHCII (the antenna-protein complex responsible for light harvesting) and (ii) the stroma lamellae, which are unstacked thylakoids connecting grana stacks enriched in photosystem I and ATP synthase. Plants can respond to changes in the environmental light conditions by several means as those which are collectively called non-photochemical quenching or NPQ. During my thesis, I mainly focused on two components of the NPQ: state transition (qT) and high-energy state quenching (qE).State transitions is the process by which PSII-antenna proteins are re-organized between stroma-lamellae and grana-BBY following changes in ambient light both of intensity and spectral composition. State transitions play a key role in the plant adaptation but many aspects of this process remain unclear. The main objective of my thesis was to study the thylakoid protein re-localization between stroma-lamellae and grana-BBY during state transitions using a proteomic-based approach. At this aim I firstly focused on the sub-thylakoid protein localization in Arabidopsis WT and I developed different protocols for the purification of the two sub-compartments (stroma-lamellae and grana-BBY) starting from intact chloroplasts. Later, thanks to a semi-quantitative proteomic approach, I determined the precise localization of around 300 thylakoid proteins in Arabidopsis WT. Results suggested that the localization of the different photosynthetic complexes is much more dynamics than previously hypothesized. In fact, even if characterized by a preferential localization, some photosynthetic complexes displayed an unexpected double localization. Moreover the subunit composition of these complexes was found to vary according to their localization (BBY or stroma-lamellae) suggesting the existence of mechanisms of regulation which have never been evidenced before. Later, we used the same mass-spectrometry-based approach on two different Arabidopsis mutants unable to perform state transitions. The objective was to highlight the involvement of other proteins (other than LHCII) which could possibly be re-localized within the photosynthetic membrane during state transitions. In the second part of my thesis, I focused on the high-energy state quenching component of the NPQ. qE allows the plant to dissipate excessive light energy as heat. This process it's not constitutive but need to be activated by the formation of a difference in the pH between the stroma and the thylakoid lumen (ΔpH). The objective of the study was to identify new possible actors in the regulation of the ΔpH formation. At this purpose I focused on a recently characterized potassium channel, TPK3. Thanks to a biophysical and biochemical approach, we demonstrated that TPK3 is involved, in vivo, in the modulation of the two components of the proton motive force (pmf), the ΔpH and the difference in the electric field Δψ. By controlling the repartition of the pmf, TPK3, controls also the formation of the NPQ and directly affects light utilization and dissipation in vivo. This avoids serious damages to the photosynthetic chain when plants are exposed to high-light conditions