O processo de secreção salivar é dependente de energia, consome glicose e pode mobilizar glicogênio na glândula submandibular. Nos ratos diabéticos a produção de saliva estimulada é reduzida e ocorre acúmulo de glicogênio nas glândulas parótida e submandibular. O objetivo deste trabalho foi avaliar in vivo o metabolismo de glicogênio das glândulas salivares, submandibular e parótida, de ratos diabéticos após o estimulo com agonistas colinérgico e adrenérgico e também analisar se os animais alimentados ou com restrição alimentar overnight apresentam diferenças no metabolismo de glicogênio das glândulas salivares nas condições estudadas. Os ratos foram divididos em grupos controles (C) e diabéticos (D). Após 30 dias da indução do diabete com estreptozotocina i.p. 60mg/kg p.c., os animais foram subdivididos em alimentados ou em jejum, anestesiados com pentobarbital 50mg/kg p.c. e hidrato de cloral 400mg/kg p.c, administrado i.p. 7,5 mg/kg p.c de pilocarpina ou 5 mg/kg p.c. de isoproterenol, os ratos foram eutanasiados 0(T0), 30(T30), 60(T60) and 120(T120) minutos após a injeção do agonista. As glândulas SM e P foram removidas e analisadas quanto ao conteúdo de proteína total, glicogênio, atividade da glicogênio sintase (GS) e da glicogênio fosforilase (GP), ativa (a) e total (t). Os dados foram analisados estatisticamente pelo ANOVA e o teste de Tukey (p>0,05). A concentração de proteína total não foi afetada pela doença diabetes, nem pela administração dos agonistas, mas apresentou-se maior nos grupos em jejum quando comparados aos grupos alimentados. A concentração de glicogênio inicial foi maior nos ratos diabéticos quando comparados ao controles nas glândulas SM e P. O estímulo com a pilocarpina e com o isoproterenol na SM dos ratos alimentados e em jejum promoveu a degradação do glicogênio observada em T30 e posterior recuperação do conteúdo até o T120 nos grupos controles e diabéticos. Na P os agonistas não mobilizaram o glicogênio no grupo controle e sim no grupo diabético. As enzimas GP e GS tiveram a atividade alterada pelos agonistas e pela doença diabetes, porém não apresentaram um padrão nas condições estudadas. Os animais em jejum apresentaram menor conteúdo de glicogênio que os diabéticos nas glândulas SM e parótida e as enzimas GS apresentou um aumento na relação da forma ativa e total nos grupos em jejum e a GP apresentou menores valores que foi mais evidente na glândula SM. A injeção dos sialogogos apresentou efeitos diferentes no metabolismo de glicogênio das glândulas P e SM, assim como nos animais diabéticos / The salivary secretion process is energydependent, consumes glucose and might mobilize glycogen in the submandibular glands. In diabetics rats the stimulated saliva flow rate is reduced and accumulate glycogen in submandibular (SM) and parotid (P). The aim of this work was evaluated in vivo glycogen metabolism in the SM and P of diabetic rats stimulated with adrenergic or cholinergic agonists, and to analyze if there are any differences in the glycogen metabolism in fed or unfed (alimentary fasting overnight) animals.The rats were divided in control (C) and diabetic (D) groups. Thirty days after diabetes induction with streptozotocin (60mg/kg b.w. i.p.), the animals were subdivided in fed or unfed, anaesthetized with pentobarbital (50mg/kg b.w. i.p.). and chloral hydrate (400mg/kg b.w. i.p.), injected pilocarpine (7.5mg/kg b.w.) or isoproterenol (5mg/kg b.w.) intraperitoneally, and euthanized 0(T0), 30(T30), 60(T60) and 120(T120) minutes post-injection of the agonists. SM and P were excised and assessed for glycogen and protein content and glycogen synthase (GS) and phosphorylase (GP) active (a) and total (t) activities. Data was statistically analyzed by ANOVA and Tukeys test (p<0.05). Protein concentration didn´t alter by diabetes or agonist injections but was higher in unfed when compared to the fed rats. Increased initial glycogen content was found in both groups of glands in diabetic rats when compared to the control group. Pilocarpine and isoproterenol stimulus promoted glycogen degradation in SM of fed and unfed rats on T30 and the T120 SM control and diabetic groups recovered glycogen content as the initial T0 values. In P the agonist mobilized glycogen just in diabetic group. The GP and GS activities were different and didnt present a pattern in this study´s condition. The unfed animals present glycogen content diminished when compared to fed animal in both glands and the relation of active and total glycogen synthase was higher in fast animals and lesser specific activities of active and total glycogen phosphorilase that were more evident in submandibular glands. The secretagogues injection presents different effects on glycogen metabolism of P and SM even so in diabetic animals.

Orientador: Jaime Amaya-Farfán / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-21T17:15:26Z (GMT). No. of bitstreams: 1 Morato_PriscilaNeder_D.pdf: 813172 bytes, checksum: 96f9ce7da352faefcc39166ef5fe4fa9 (MD5) Previous issue date: 2012 / Resumo: As proteínas do soro do leite apresentam propriedades nutricionais e funcionais que influenciam a modulação de funções bioquímicas e fisiológicas.Estudos têm demonstrado que as proteínas do soro do leite (PSL), principalmente na forma hidrolisada (PSLH) possuem a capacidade de aumentar os níveis de glicogênio muscular. Considerando que a captação de glicose pela célula do músculo esquelético relaciona-se diretamente à atividade de proteínas transportadoras de glicose, este estudo se propôs realizar dois experimentos para conhecer os efeitos da PSL e da PSLH e de alguns dos seus produtos de hidrólise nos transportadores de glicose em músculos de ratos. No experimento 1, o objetivo foi verificar se o consumo de PSL e PSLH modulam a concentração de transportadores de glicose GLUT-1 e GLUT-4 na membrana plasmática (MP) de células musculares de animais sedentários e exercitados. Foram utilizados 48 ratos Wistar machos divididos em dois grupos: sedentários e exercitados, e cada um desses subdivididos em outros três, de acordo com a dieta, totalizando 6 grupos (n=8 por grupo). Os animais foram mantidos por 9 dias recebendo as dietas experimentais baseadas na AIN-93G, com as seguintes fontes protéicas: caseína (CAS), utilizada como controle, proteína do soro do leite (PSL), proteína do soro do leite hidrolisada (PSLH), e o animais exercitados foram submetidos a uma única sessão de exercício a 15m/min durante 60min um dia anterior ao sacrifício. Após o período experimental os animais foram sacrificados, os transportadores de glicose no músculo, GLUT-1 e GLUT-4, foram analisados por western blot. Adicionalmente, glicogênio, aminoácidos livres plasmáticos, insulina e indicadores bioquímicos de saúde foram determinados por métodos padrões. O consumo de PSLH elevou significativamente as concentrações de GLUT-4 na MP e de glicogênio, enquanto GLUT-1, insulina e os indicadores de saúde não apresentaram alterações. Baseado nas evidências do experimento 1, de que o consumo de PSLH eleva os estoques de glicogênio muscular e que também aumenta a concentração do transportador de glicose GLUT-4 na membrana plasmática, o experimento 2 teve como objetivo identificar quais componentes da PSLH poderiam modular a translocação do transportador de glicose GLUT-4 para a MP em músculo esquelético. Foram utilizados 49 ratos Wistar machos divididos em 7 grupos (n=7), que receberam soluções orais de glicose 30% mais 0,55 g/kg de peso corpóreo os seguintes componentes da PSLH: a) glicose (controle); b) PSLH; c) L-isoleucina; d) L-leucina; e) L-leucina mais L-isoleucina; f) peptídeo Lisoleucil- L-leucina; g) peptídeo L-leucil-L-isoleucina. Após receberem as soluções, os animais foram sacrificados, o transportador de glicose GLUT-4 no músculo foi analisado por western blot. Também foram analisados glicogênio, glicemia, insulina, aminoácidos livres plasmáticos e musculares, e indicadores bioquímicos de saúde por métodos clássicos. Entre os componentes testados da PSLH, o peptídeo leucil-isoleucina e o aminoácido isoleucina se mostraram mais eficientes em translocar GLUT-4 para a MP, favorecendo a captação de glicose pelo músculo esquelético. Os resultados obtidos nos experimentos indicam que o consumo da PSLH e de seus componentes ao aumentarem a translocação de GLUT-4 para a membrana plasmática, poderiam auxiliar no tratamento ou prevenção do diabetes do tipo II / Abstract: The milk whey proteins (WP) exhibit nutritional and functional properties which result in the modulation of the biochemical and physiological functions. Studies have shown that the WP, especially those in the hydrolyzed form (WPH),has the capacity to increase muscle glycogen levels. Considering that glucose uptake by the skeletal muscle cell is directly related to the activity of the glucose transporter proteins, the present study proposed to carry out two experiments to determine the effects of WP and WPH and of some of their hydrolysis products on the glucose transporters in rat muscles. The objective of experiment 1 was to verify if the consumption of WP and WPH are able to modulate the concentration of the glucose transporters GLUT-1 and GLUT-4 in the plasma membrane (PM) of muscle cells in sedentary and exercised animals. Forty-eight male Wistar rats were used, divided into sedentary and exercised groups, each of which was sub-divided into three sub-groups according to the diet, giving a total of 6 groups (n=8 per group). The animals were maintained for 9 days on experimental diets based on AIN-93G with the following protein sources: casein (CAS) used as the control, whey protein (WP) and whey protein hydrolysate (WPH). The exercised animals were submitted to a single exercise session for 60 min at 15m/min one day prior to euthanasia. After the experimental period, the animals were euthanized, and the muscle glucose transporters GLUT-1 and GLUT-4 analyzed by western blot. In addition, glycogen, free plasma amino acids, insulin and the biochemical health indicators were analyzed by standard techniques. The consumption of WPH significantly increased the concentrations of GLUT-4 in the PM and of glycogen, whereas GLUT-1, insulin and the health indicators remained unaltered. Based on evidence from experiment 1 that the consumption of WPH raised the muscle glycogen reserves and also the concentration of the glucose transporter GLUT-4 in the plasma membrane, the second experiment was designed to identify which WPH components could modulate translocation of the glucose transporter GLUT-4 to the PM in the skeletal muscle of the animals. Forty-nine male Wistar rats were used, divided into 7 groups (n=7), who were orally fed 30% glucose solutions plus 0.55 g/kg of body weight of the following WPH components: a) glucose (control); b) WPH; c) L-isoleucine; d) L-leucine; e) L-leucine plus L-isoleucine (50:50 mixture of both amino acids); f) L-isoleucyl-L-leucine peptide or g) L-leucyl-L-isoleucine peptide. After receiving the solutions, the animals were euthanized and the GLUT-4 determined by western blot. Glycogen, glycemia, insulin, free plasma and muscle amino acids, and the biochemical health indicators were also analyzed by classical methods. Of the WPH components tested, the peptide L-leucyl-L-isoleucine and the amino acid L-isoleucine were shown to be more efficient in translocating GLUT-4 to the PM, favoring the capture of glucose by the skeletal muscle. The results obtained from these experiments indicated that the consumption of WPH and its components increased GLUT-4 translocation to the plasma membrane, and could aid in the treatment and prevention of type ll diabetes / Doutorado / Nutrição Experimental e Aplicada à Tecnologia de Alimentos / Doutora em Alimentos e Nutrição

REDD1 contribue au dialogue entre le métabolisme énergétique et la masse musculaire.REDD1 est une protéine ubiquitaire et conservée qui est exprimée en réponse à de nombreux stress et pathologies associés à une atrophie du muscle squelettique, un paramètre corrélé à la mortalité des patients. REDD1 est connue pour inhiber la voie Akt/mTORC1 qui contrôle la synthèse des protéines (composants majoritaires du muscle), mais également d'autres macromolécules tels les ribosomes, les nucléotides ou le glycogène. Nos travaux montrent, grâce à un modèle murin, que REDD1 est capable d'une part d'inhiber la synthèse protéique ce qui conduit à l'atrophie du muscle, et d'autre part de réduire le stockage du glycogène musculaire. Cependant, sa délétion est responsable d'une augmentation du métabolisme basal, d'une réduction de la capacité d'exercice et d'une aggravation de l'atrophie musculaire en situation d'hypoxie. Ces altérations du métabolisme ne sont pas liées à un dysfonctionnement mitochondrial, mais associées à une moindre inhibition de la signalisation d'Akt et/ou mTORC1, tous deux responsables de l'activation de processus anaboliques couteux en énergie. Pris ensembles, ces résultats suggèrent que REDD1 agit comme modérateur de la dépense en ATP dans des situations de stress énergétique. / REDD1 contributes to the crosstalk between energetic metabolism and skeletal muscle mass. REDD1 is a ubiquitous and conserved protein, which is expressed in response to numerous stresses and pathologies responsible of muscle atrophy, a parameter correlated with patient mortality. REDD1 is known to inhibit Akt/mTORC1 pathway which controls synthesis of proteins (the major component of muscle) and other macromolecules such as ribosome, nucleotide or glycogen. Our work shows on a mice model that REDD1 inhibits protein synthesis, leading to skeletal muscle atrophy, and reduces muscle glycogen storage. However, REDD1 deletion is responsible of an increase in basal metabolism, a reduction of exercise capacity and an exacerbation of hypoxia-induced skeletal muscle atrophy. These metabolic alterations are not associated with a mitochondrial dysfunction but rather with an hyper activation of the Akt/mTORC1 pathway which is responsible for the stimulation of energy demanding processes. Altogether, these results strongly suggest that REDD1 acts for moderating ATP demand in energetic stress conditions

Orientador: Denise Vaz de Macedo / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-26T20:57:41Z (GMT). No. of bitstreams: 1 Costa_KellGrandjean_M.pdf: 3663020 bytes, checksum: 9e2b6eee5b841b824157538fe4dee768 (MD5) Previous issue date: 2015 / Resumo: O objetivo do trabalho foi desenvolver uma escada com sistema de roldanas para aplicação da sobrecarga, para mimetizar condições semelhantes aos treinos em humanos, uma vez que o músculo esquelético é muito sensível as variáveis de treinamento (sobrecarga, repetições, pausas). Outro objetivo foi avaliar os efeitos de oito semanas de treinamento de força em escada com e sem suplementação antioxidante. Foram analisados marcadores morfológicos, metabólicos e de estresse oxidativo no sangue e no músculo flexor longo do hálux (FHL) em ratos divididos em grupo controle e treinado (Protocolo 1), e posteriormente em ratos submetidos a suplementação antioxidante (óleo de arroz) e treinamento (Protocolo 2). O sistema de roldanas gerou uma sobrecarga com menor interferência de atrito, propiciando sessões de treino com contribuição do metabolismo anaeróbico, e com dano tecidual nas fibras musculares, resultando em aumento de desempenho ao longo das 24 sessões de treino e adaptações características de um treino resistido tais como aumentos da AST, dos estoques de glicogênio e atividade da enzima lactato desidrogenase. Importante salientar que houve hipertrofia das fibras glicolíticas e oxidativas e aumento da atividade da enzima citrato sintase, demonstrando que além do aumento na produção de força o treino aumentou a capacidade oxidativa do músculo. Houve uma maior produção de EROs refletida em aumento significativo da atividade das enzimas antioxidantes glutationa redutase e catalase. Esse aumento foi suficiente para a proteção do músculo, uma vez que os valores do marcador de peroxidação lipídica (TBARs) estavam homogeneamente diminuídos após as 8 semanas de treino quando comparado ao grupo controle. A possível resposta ergogênica não ocorreu com a suplementação de óleo de arroz no FHL de ratos treinados divididos em 3 grupos: suplementados com água, 0.75 ml e 1.5 ml de óleo de arroz. Não encontramos diferenças significativas no desempenho e na hipertrofia entre os grupos. Houve diminuição significativa nas concentrações de TBARs nos grupos suplementados com 0,75mL e 1,5mL de óleo de arroz. No entanto, os efeitos adaptativos na capacidade oxidativa muscular e nas enzimas antioxidantes foi perdido com a suplementação. Esses dados sugerem cautela na utilização de antioxidantes com intuito de proteção do aumento de EROs induzido pelo treinamento, pois vias adaptativas são sinalizadas por EROs / Abstract: The main goal of this study was to develop a ladder for rats with pulley system for the application of overload, to mimic conditions similar to training in humans, since the skeletal muscle is very sensitive to the training variables (overload, repetitions, pauses). Other objective was to evaluate the effect of eight weeks of strength training in ladder in this system with and without antioxidant supplementation. It was performed morphological markers, metabolic and oxidative stress analyses in blood and in the flexor hallucis muscle (FHL) in rats divided into control group and trained group (Protocol 1), and in other group of rats subjected to antioxidant supplementation (rice oil) and training (Protocol 2). The pulley system generated an overload with less interference of friction, providing training sessions with anaerobic metabolism contribution, and tissue damage in the muscle fibers, resulting in increased performance over the 24 training sessions and adaptations of resistance training such as increases in (Cross-section-area) CSA, the glycogen stores and activity of lactate dehydrogenase. It is Important to note that there was hypertrophy of the glycolytic and oxidative fibers and increased activity of the citrate synthase enzyme, showing that besides the increase in force production, training also increased muscle oxidative capacity. There was a higher production of ROS reflected in a significant increase of antioxidant enzymes catalase and glutathione reductase. This increase was enough for muscle protection, since the values of lipid peroxidation marker (TBARS) were homogeneously decreased after 8 weeks of training compared to the control group. We did not find ergogenic response with the rice oil supplementation in rats trained FHL divided into 3 groups: supplemented with water, 1.5 ml and 0.75 ml rice bran oil. We found no significant differences in performance and hypertrophy among groups. There was a significant decrease in TBARS concentrations in the groups supplemented with 0,75mL and 1.5 mL of rice oil. However, the adaptive effects in muscle oxidative capacity and the antioxidant enzymes were lost with the supplementation. These data suggest caution with the use of antioxidants to protect against the increase of ROS induced by training, because adaptive pathways of training can be signalize by ROS / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

No description available.

Toll-like receptor 4 (TLR4), a key member of the TLR family, has been well characterized by its function in the induction of inflammatory products of innate immunity. However, the involvement of TLR4 in a variety of apoptotic events by an unknown mechanism has been the focus of great interest. Our investigation found that TLR4 promoted apoptotic signalling by affecting the glycogen synthase kinase-3β (GSK-3β) pathway in a serum-deprivation- induced apoptotic paradigm. Serum deprivation induces GSK-3β activation in a pathway that leads to subsequent cell apoptosis. Intriguingly, this apoptotic cascade is amplified in presence of TLR4 but greatly attenuated by β-arrestin 2, another critical molecule implicated in TLR4-mediated immune responses. Our data suggest that the association of β-arrestin 2 with GSK-3β contributes to the stabilization of phospho-GSK-3β, an inactive form of GSK-3β. It becomes a critical determinant for the attenuation of TLR4-initiated apoptosis by β-arrestin 2. Taken together, we demonstrate that the TLR4 possesses the capability of accelerating GSK-3β activation thereby deteriorating serum-deprivation-induced apoptosis; β-arrestin 2 represents an inhibitory effect on the TLR4-mediated apoptotic cascade, through controlling the homeostasis of activation and inactivation of GSK-3β.

It is generally accepted that a decrease in temperature causes the rates of chemical and biochemical reactions to slow down, usually resulting in poorer performance of biological wastewater treatment systems. Despite this, early researchers repeatedly showed that excess biological phosphorus removal (EBPR) was more efficient at colder temperatures. Recent studies, however, have demonstrated that the reaction rates of EBPR processes decrease with temperature in accordance with Arrhenius' Law, resulting in an apparent contradiction in the literature. The objective of this study was to investigate the EBPR temperature controversy. The experimental systems used were two, lab-scale UCT configuration plants fed with acetate as the sole volatile fatty acid (VFA) source. The results showed that EBPR systems do perform more efficiently at colder temperatures, i.e., at 5°C compared to 20°C. The reason for better system performance was determined to be related to reduced competition for substrate in the non-oxic zones that results in an increased population of phosphate accumulating organisms (PAOs) relative to non-PAOs and, therefore, greater EBPR efficiency even though the reaction rates are slower. The proliferation of PAOs relative to non-PAOs at cold temperature indicates that some of the PAOs are psychrophilic, i.e., they have alternate biochemical pathways that give them a competitive advantage over bacteria dependent upon glycogen metabolism. The activated sludge acclimated to 20°C had relatively high polyhydroxyvalerate (PHV) and glycogen contents relative to sludge acclimated to 5°C. It was initially hypothized that there is a significant competition between PAO and glycogen accumulating organisms (GAOs) at 20°C and cold temperature (5°C) nearly eliminates this competition in favor of the PAOs. A series of batch test experiments revealed that despite similar acetate utilization by the sludges grown at the two temperatures nearly 30% less PHA was produced by the sludge taken from the 20°C reactor, indicating that GAOs were a small fraction of the population at 20°C. Transmission electron microscopy pictures showed that the biomass acclimated to 20°C had a much more diverse bacterial population than the biomass acclimated to 5°C. However, no GAO population was detected in electron microscopy samples under any temperature conditions. The decreased P removal efficiency at 20°C was then attributed to the presence of fermentative or other non poly-P bacteria that are capable of utilizing substrate under anaerobic conditions. PHA production greatly increased at 5°C, whereas glycogen metabolism substantially reduced. Even though glycogen is an essential requirement for EBPR mechanism, the EBPR microorganisms have the ability to adapt their metabolic pathways to environmental conditions and greatly reduce their need for glycogen. It is apperant that cold temperature inhibits some of the key enzymes in glycogen metabolism resulting in lower glycogen accumulation that in turn increases the EBPR performance. Therefore temperature not only exerts selective pressure on the dominant population but also alters the metabolic pathways of the EBPR process. Increased glycogen accumulation, as observed in this study at 20°C, may not be related to GAO proliferation as suggested by Filipe et al. (2001) instead it may be related to EBPR bacteria to efficiently use glycogen metabolism. Current models (Brdjanovic et al. 1997; Filipe et al. 2002) consider that GAO metabolism is an integral part of EBPR metabolism and the performance of EBPR processes depends on PAO/GAO fraction in the EBPR system. No GAO proliferation was observed even the A/O process was operated without P addition for more than 3 weeks at 10°C. Therefore such important concept should be further investigated before it is included in EBPR models. EBPR stoichiometry was presumed to be insensitive to temperatures (Brdjanovic et al. 1997). However, observed stoichiometric values of PHA storage per unit glycogen utilization and PHA utilization per unit glycogen rephlenishment were quite different at different temperatures. Temperature, therefore, not only affects the kinetics of EBPR systems but also affects the EBPR stoichiometry. Most prokaryotic cells have the ability to alter their cellular membrane fatty acid composition as temperature decreases to counteract the adverse effects of temperature on membrane fluidity (Becker et al., 1996). This unique ability is known as "homeoviscous adaptation". In this study, homeoviscous adaptation by EBPR activated sludge was investigated for a series of temperatures ranging from 20°C to 5°C using one of the lab scale EBPR systems. The fatty acid analysis results showed that the unsaturated to saturated fatty acid ratio increased from 1.40 to 3.61 as temperature dropped from 20 to 5°C. The increased cis-9-hexadecanoic acid (C16:1) at 5°C strongly indicated the presence of homeoviscous adaptation in the EBPR bacterial community. Thus the cell membranes of the EBPR community were still in a fluid state, and solute transport and proton motive force mechanisms were operable even at 5°C. It was concluded that loss of EBPR performance at low temperatures, as reported by McClintock et al. (1992) was not related to the physical state of the cellular membranes, but was probably caused by unsuitable operational conditions. Even though the transport of volatile fatty acids (e.g. acetate) is an integral part of EBPR biochemistry and stoichiometry, this important concept has been ignored. Fleet (1997) concluded that acetate entry into bacterial cells in EBPR sludge was simple passive diffusion based upon the results of a single study (Baronofsky et al. 1984). However, this study showed that neither acetate nor propionate can cross the cell membrane via simple passive diffusion. The existence of apparent saturation curves when the substrate uptake rates (acetate and propionate) were plotted against the substrate concentrations suggested that transport of volatile fatty acids obey facilitated or active transport. Following from the above results, an investigation of the impacts of operational conditions such as low solids retention time (SRT), presence of electron acceptors in the non-oxic zones, low anaerobic detention time, and lack of acclimation was performed. The results showed that the "critical, i.e., wash-out" SRT increased as temperature decreased, but if the biomass was permitted to acclimate to the lower temperature, a major population shift would occur which would increase the capacity of the system for phosphorus (P) removal. When the 5 °C sludge was allowed to acclimate at a relatively high SRT (18 d), the system's P-removal capacity greatly surpassed that of the 20 °C system. The decrease in EBPR performance because of the presence of nitrates in the non-oxic zones was determined to be greater than what would be predicted based on accepted stoichiometry. / Ph. D.

The study of enzyme binding sites is an essential but rather demanding process of increased complexity since the amino acids lining these areas are not rigid. At the same time, the minimization of side effects and the specificity of new ligands is a great challenge in the structure-based drug design approach. Using glycogen phosphorylase - a validated target for the development of new antidiabetic agents - as a case study, this project focuses on the examination of side-chain conformations of amino acids that play a key role in the catalytic site of the enzyme. Specifically, different rotamers of each amino acid were collected to build a dataset of different conformations of the catalytic site. The rotamers were filtered by their probability of occurrence and subsequently, all rotamers that create steric clashes were rejected. Then, these conformations were clustered based on their similarity. Three different clustering algorithms and multiple numbers of clusters were tested using the silhouette scores evaluation for the clustering process. In order to measure the similarity, the Euclidean metric was used which due to the correspondence of the coordinates between the conformations was very similar to the cRMSD metric. Two-level clustering was applied to the dataset for more in-depth observations. According to the clustering results, specific aminoacids with major geometrical variations in their rotamers play the most important role in the separation of the clusters. Additionally, all rotamers of an amino acid can be grouped based on their structure, something that was confirmed using “Chimera” software as a visualization tool. To this end, the ultimate aim of this study is to examine whether the clustering of conformations produces clusters with points geometrically similar to each other, in order to identify near neighbors, i.e. conformations that are quite similar in structure but do not play a determinant role in the function and those that are quite diverse and could be further exploited.

Les modifications post-traductionnelles (MPTs) permettent un haut degré de régulation de l'expression des gènes en générant une diversité fonctionnelle au niveau du protéome. Dans le système nerveux, les MPTs régulent entre autres des facteurs de transcription permettant une adaptation rapide à un microenvironnement dynamique. Dans ce contexte, je me suis concentrée sur l’étude des Glycogène Synthase Kinases 3 (GSK3s). Elles sont au centre de la régulation de nombreuses voies de signalisation et contrôlent la stabilité de multiples cibles par phosphorylation. Au cours du développement du cerveau, les kinases GSK3 contrôlent la balance entre la prolifération et la différenciation. La dérégulation de l'activité des kinases GSK3 a un rôle clé dans les maladies neurodégénératives du cerveau. En revanche, le rôle important de ces kinases au cours du développement rétinien ainsi que dans les maladies neurodégénératives rétiniennes reste une question ouverte.L'objectif de ma thèse était d'étudier le rôle de ces kinases au cours du développement et de l'homéostasie rétinienne. J’ai montré que l'absence totale de Gsk3α et de Gsk3β très tôt au cours du développement rétinien entraîne une microphtalmie chez l'adulte. Les deux kinases jouent des rôles redondants puisque l'expression d'un seul allèle Gsk3 est suffisante pour prévenir le phénotype de microphtalmie. Cependant, une analyse phénotypique approfondie dans ce contexte génétique (un seul allèle Gsk3) a révélé une forte augmentation du nombre de cellules ganglionnaires déplacées (dRGCs) dans la couche nucléaire interne, associée à une modification des projections axonales des cellules ganglionnaires dans le cerveau par rapport aux contrôles. Dans l’ensemble, ces données suggèrent que les kinases GSK3s sont essentielles au maintien des progéniteurs rétiniens et sont impliquées dans la genèse des dRGCs. Compte tenu du très faible nombre de dRGCs en conditions normales, la fonction de ces cellules a été très peu étudiée à ce jour. Le modèle génétique que j’ai développé offre par conséquent un modèle de choix pour étudier l’ontogenèse et la fonction de ces cellules.Mes travaux de thèse se sont ensuite concentrés sur le rôle de GSK3 dans les photorécepteurs. En effet, des défauts de développement ou leur mort est l’une des principales causes de dégénérescence rétiniennes. Afin de mieux comprendre la fonction de ces kinases dans la maintenance des photorécepteurs, j'ai donc utilisé des souris invalidées de manière conditionnelle pour Gsk3α et Gsk3β spécifiquement dans les précurseurs des photorécepteurs. L’absence de GSK3 conduit à une altération de la maturation et de la fonction des photorécepteurs, suivie de leur dégénérescence. J’ai alors combiné des analyses transcriptomiques et des approches in vitro pour élucider les mécanismes sous-jacents. Mes données m’ont conduit à proposer un modèle selon lequel l’absence de GSK3 dans les photorécepteurs conduit à des défauts de phosphorylation de NRL (facteur de transcription nécessaire au développement des photorécepteurs de type bâtonnet), augmentant sa stabilité. Cette dérégulation post-traductionnelle conduit à la diminution d’expression d'un sous-ensemble de gènes cibles de NRL, co-régulés par CRX, et impliqués dans le développement et l'homéostasie des photorécepteurs. Cette dérégulation conduirait alors à la dégénérescence des photorécepteurs observée dans les mutants GSK3. Ce travail suggère donc que GSK3 joue un rôle essentiel dans la régulation de NRL pour contrôler la maturation et l'homéostasie des photorécepteurs. De telles données suggèrent également que ce mécanisme de régulation pourrait être déficient chez les patients atteints de rétinites pigmentaires dues à des mutations de NRL empêchant sa phosphorylation par GSK3. / Post-translational modifications (PTMs) allow a higher degree of regulation for the control of gene expression by generating functional diversity at the proteome level. In the central nervous system, PTMs regulate stability or activity of transcription factors allowing a rapid response to external signals and a quick adaptation to a dynamic cellular microenvironment. In this context, I focused on the ubiquitously expressed and highly conserved Glycogen Synthase Kinases 3 (GSK3s). They are at the crossroad of multifunctional signalling pathways. During mammalian brain development, GSK3 kinases control the balance between proliferation and differentiation. Deregulation of GSK3 kinases activity has also a key role in neurodegenerative diseases by causing the accumulation/aggregations of proteins causing neuronal cell death. Drugs targeting GSK3s hold a lot of promises to treat such diseases. Whether these kinases are also important during retinal development and involved in retinal diseases remains an open question. Several studies suggest the importance of regulating GSK3 function in photoreceptor under pathological conditions. Therefore, the main objective of my PhD was to investigate the role of these kinases during photoreceptor development and homeostasis. To better understand the role of these two kinases during retinal development and to highlight potential differences with the developing brain, we also investigated their function in the control of the balance between proliferation and differentiation of retinal progenitors. To achieve my work, I used conditional knockout mice for Gsk3α and Gsk3β specifically deleted either in photoreceptor precursors or in retinal progenitors during early development. The lack of GSK3 kinases in photoreceptor precursors led to impaired photoreceptor maturation and function followed by their degeneration. Transcriptomic analysis (RNAseq) 6, 10 and 14 days postnatally prior degeneration revealed several genes downregulated belonging to biological processes involved in eye development and visual functions. Among them, the expression of the transcription factor Nrl that is required for rod photoreceptor development was decreased. Astonishingly, NRL expression was highly increased at protein level. By in vitro approaches, I demonstrated that GSK3-dependent phosphorylation regulates NRL protein stability. Despite such increase, a large number of NRL target genes were downregulated leading to impaired photoreceptor maturation and function. Surprisingly, a vast majority of these downregulated genes were also target genes for CRX, another transcription factor working in synergy with NRL. This work demonstrates that PTMs of NRL play a critical role in fine tuning the expression of a subset of genes involved photoreceptor development and homeostasis. Such findings could allow the development of innovative therapeutic strategies for retinal dystrophies. The functional characterisation of GSK3 in the course of retinal development by invalidating both Gsk3α and Gsk3β in retinal progenitors early during development revealed their requirement for controlling cell cycle exit and neuronal differentiation. Indeed, the complete lack of Gsk3α and Gsk3β led to microphtalmia in adults. Interestingly, the expression of only one Gsk3 allele was enough to rescue the phenotype. However, further analysis revealed a large number of displaced ganglion cells in the inner nuclear layer. The function of these cells remains to be determined, but their timing of production corresponds to other ganglion cells. Strikingly, these displaced ganglion cells project in distinct brain regions than normal ganglion cells. Therefore, our work could provide the first step toward determining the function of the displaced ganglion cells, which appear at low number in wildtype but whose function remains to be clarified.

Angling practices subject Largemouth Bass Micropterus salmoides to multiple stressors, causing homeostatic physiological disturbances. The combined effects of ambient and live well temperature on stress responses from exercise have not been thoroughly examined. Large numbers of fish required for stress experiments can be produced by intensive culture, but hatchery fish may differ physiologically from wild fish due to dietary carbohydrates. Therefore, the effects of diet, population, and temperature on stress response and health were examined. Stress responses were similar among fish fed formulated and live diets and liver health improved within 4-6 weeks. Although cortisol responses of hatchery and wild fish differed, secondary stress responses were similar. Fish subjected to simulated angling at temperatures of 17, 25, 33 °C with live well temperature differentials of -4, 0, +4 °C, had the lowest resilience to stress at the warmest temperatures, exhausting energy supplies, coincident with metabolic acidosis and poor ion regulation.