Carbamazepina (Carba) é um anticonvulsivante, uma droga psicotrópica muito utilizada no tratamento de pacientes com distúrbios intelectuais. Esta droga foi utilizada para diminuir o volume urinário no Diabetes Insípido (DI), pois possui um efeito antidiurético, mas a incidência de hiponatremia é uma ocorrência comum. O lítio é uma das drogas mais importantes para o tratamento do distúrbio bipolar. No entanto, ele tem uma grande capacidade de induzir DI dificultando o seu uso em pacientes debilitados psicologicamente. Atualmente, a associação destas drogas é frequentemente utilizada para o tratamento de pacientes com distúrbios psiquiátricos e neurológicos. O objetivo deste trabalho foi investigar o efeito da Carba no ducto coletor medular interno (DCMI) de ratos normais e elucidar a sua ação no DI induzido pelo lítio: 1) Estudos in vitro- A) Estudos com microperfusão de segmentos isolados do néfron onde a permeabilidade à água (Pf, m/sec) foi determinada em DCMI perfundidos de ratos normais (n=20) na presença de Carba à 10-5 M, e na ausência de HAD. B) estudos com a técnica de Imunoblotting para avaliar a expressão da proteína Aquaporina 2 (AQP2) em suspensão de túbulos de DCMI de ratos normais incubados com 10-5 M de Carba por 30 minutos. 2) Estudos in vivo A) quatro grupos foram formados: a) Controle (n=5); b) Li (40 mmol/Kg/dieta por 3 semanas; n=4); c) Li+Carba (40 mmol Li/Kg/dieta + 400 mg carba/Kg/dia por 3 semanas; n=5); d) Carba (400 mg /Kg/dia por 3 semanas; n=5); - B) estudo da expressão da AQP2 no DCMI destes quatro grupos pela técnica de Western Blot. Resultados: 1) Estudos in vitro A) nos estudos de microperfusão a carba adicionada ao banho aumentou a Pf dos DCMI, na ausência de HAD (n=6) Controle12,3+ 3,6, Carba-62,6+14,8 (p<0,01), Recuperação (Rec)-17,4+5,5 (p<0,01). Com o intuito de estudar o mecanismo pelo qual a Carba ativa a cascata do HAD, foi utilizado o inibidor do receptor V2 do HAD (AV2; n=10): Controle-23,5+5,2, Carba-37,4±4,4 (p<0,01), Carba+AV2-19,6±5,0 (p<0,05), Rec-21,4+5,5; e Controle-18,6+7,0, AV2-27,3+7,2, AV2+Carba-25,3+5,7, Rec-32,6+6,4. O inibidor da PKA (H8; n=4) também foi utilizado: Controle-15,0+9,0, Carba-106,1+12,3 (p<0,01), Carba+H8-60,3+16,4 (p<0,01), Rec- 44,5+13,2. B) a análise densitométrica mostrou um aumento de 38,8% na expressão da AQP2 (Controle-100,0+8,3 vs. Carba-138,8+12,12, p<0,05); 1) Estudos in vivo Volume urinário (UV, mL/24h) Controle-10,7+3,0, Li-62,6+6,0 (p<0,001), Li+Carba-28,5+4,9 (p<0,001), Carba-23,3+3,0 (<0,001). Osmolalidade urinária (mOsm/Kg/H2O) Controle-819,6+175,7, Li-149,4+18,0 (p<0,01), Li+Carba-251,5+39,7 (p<0,05), Carba-396,2+75,5 (p<0,01). FENa+ (%) - Controle-0,15+0,01, Li-0,10+0,02, Li+Carba-0,12+0,02, Carba-0,11+0,02. Expressão da AQP2 (%) Controle-100,0+6,7, Li-55,8+5,4 (p<0,01), Li+Carba-75,8+9,6 (p<0,01), Carba- 99,7+4,7 (p<0,05). Não houve diferenças significantes no Na+, K+ e Osmolalidade plasmáticas. Em resumo, nossos dados revelaram que a Carba diminui o UV, aumenta a osmolalidade urinária e a expressão da AQP2 no DI induzido pelo lítio, e aumenta a permeabilidade à água, provavelmente agindo diretamente no receptor da vasopressina (V2). Estes resultados enfatizam que a hiponatremia encontrada nos pacientes que fazem uso da Carba pode ser explicada, pelo menos em parte, pelo aumento da permeabilidade osmótica no DCMI e que a poliúria do DI ocasionado pelo uso do lítio pode ser diminuída com a associação da Carba. / Carbamazepine (Carba) is an anticonvulsant and a psychotropic medication commonly used in the treatment of patients with intellectual disability (ID). This drug has been used to try to decrease the urinary volume in Diabetes Insipidus (DI) because Carba presents an antidiuretic effect, but the incidence of the hyponatremia in neurological patients is a common ocurrence. Lithium (Li) is one of the most important drugs used to treat bipolar mood disorders. However, Li has the undesirable capacity to induce DI complicating its usage in patients psychologically weakened. Nowadays, the association of these drugs is used in the treatment of patients with psychiatric and neurological problems. Our objective was to investigate the effect of Carba in the Inner Medullary Collecting Duct (IMCD) and elucidate its effect in the lithium-induced DI: 1) In vitro study: A) Microperfusion studies the water permeability (Pf, m/sec) was determined in normal rats IMCD isolated and perfused by the standard methods. Carba 10-5M was added to the bath fluid. B) Immunoblotting studies for AQP2 protein expression in IMCD tubule suspension from normal rats incubated with Carba 10-5M for 30 minutes. 2) In vivo study A) four groups of normal rats were done - a) Control (C, n=5); b) Li (40 mmol/kg/food/3 weeks n=4) c) Li+Carba (40 mmol Li/kg/food/3 weeks and 400 mg Carba/kg/bw/2 last weeks, n=5); and Carba (400 mg Carba/kg/bw/3weeks, n=5); - B) AQP2 expression in IMCD from the four groups, by Western Blot. Results: 1) In vitro study A) in microperfusion, Carba added to the bath in Vasopressin (Vp) absence (n=6) increased Pf Control-12.3+3.6, Carba-62.6+14.8 (p<0.01), Recuperation (Rec)-17.4+5.5 (p<0.01). In order to study the mechanism by which Carba activates the Vp cascade, the antagonist of the Vp receptor 2 (AV2; n=10) was used: Control-23.5+5.2, Carba-37.4±4.4 (p<0.01), Carba+AV2-19.6±5.0 (p<0.05), Rec-21.4+5.5; and Control-18.6+7.0, AV2- 27.3+7.2, AV2+Carba-25.3+5.7, Rec-32.6+6.4. The PKA inhibitor (H8; n=4) was also used: Control-15.0+9.0, Carba-106.1+12.3 (p<0,01), Carba+H8-60.3+16.4 (p<0.01), Rec-44.5+13.2. B) the densitometric analysis showed an increased of 38.8% in AQP2 expression (Control- 100.0+8.3 vs. Carba-138.8+12.12, p<0.05); B) In vivo study Urinary volume (UV, mL/24h) Control-10.7+3.0, Li-62.6+6.0 (p<0.001), Li+Carba-28.5+4.9 (p<0.001), Carba-23.3+3.0 (p<0.001). Urinary Osmolality (mOsm/Kg/H2O) Control-819.6+175.7, Li-149.4+18.0 (p<0.01), Li+Carba-251.5+39.7 (p<0.05), Carba-396.2+75.5 (p<0.01). FENa+ (%) - Control- 0.15+0.01, Li-0.10+0.03, Li+Carba-0.12+0.02, Carba-0.11+0.02. AQP2 expression (%) Control-100.0+6.7, Li-55.8+5,4 (p<0.01), Li+Carba-75.8+9.6 (p<0.01), Carba-99.7+4.7 (p<0.05). There were no significant differences in Na+, K+ and plasma osmolality. In summary, our data showed that Carba decreased the UV, increased the UOsm and the AQP2 expression in Li-induced DI, increased the water permeability, probably acting directly in the Vp V2 receptor-Protein G complex, since its action was blocked by the specific Vp V2 receptor antagonist. These results emphazise that the hyponatremia found in patients using Carba could be explained, at least in part, by increased osmotic permeability of IMCD. In addition, poliuria observed in lithium-induced DI can be decreased with Carba-treatment.

Made available in DSpace on 2017-07-10T14:17:04Z (GMT). No. of bitstreams: 1 Leonardo.pdf: 2065969 bytes, checksum: ecaa0d3dc812ef2017f1f16fc1f49d2a (MD5) Previous issue date: 2013-07-01 / The induction of obesity in experimental animals with cafeteria diet simulates the high-calorie food that humans eat, nowadays, thus enabling to study obesity and comorbidities resulting from this condition. The male reproductive system is directly affected by obesity with increased lipid peroxidation, increase of apoptotic bodies, changes in parameters espermatobioscópicos and reduced fertility. The aim of the study was to evaluate whether there are changes caused by obesity in epithelia of efferent ducts and epididymis , both in relation to morphology and expression of aquaporins 1 and 9 ( AQPs 1 and 9 ) in adult Wistar rats . After weaning , the animals were randomly divided into two groups : control group ( CON ) received standard diet and water ad libitum ( n = 10 ) and group cafeteria ( CAF ) , which received cafeteria diet soda and degassed ad libitum ( n = 10). The treatment group CAF began after the 12th week of life, totaling 40 weeks of consumption of cafeteria diet, being all dead animals with 52 weeks of age. The efferent ducts , along with initial segments ( Si ) , head ( Cç ) , body ( Co ) and tail ( Cd ) epididymis were submitted to routine histological and immunohistochemical staining for AQP- 1 and AQP- 9 . In the present study we evaluated the data: body mass, length nasoanal, waist circumference, Lee index and retroperitoneal fat mass and perigonadais; also evaluated dimensions, using morphometry and morphology of the epithelia of the efferent ducts and segments Si, Cç, Co and Cd epididymis and analyzed the expression of AQP- 9 and AQP- 1 in the efferent ducts and epididymal segments above. Changes in expression of AQPs in the efferent ducts and the epididymal epithelium were observed with increased expression of AQP- 1 and AQP- 9 reduction in efferent ducts and epididymal cauda, respectively, in animals treated with cafeteria diet. Also, there were morphological changes in the epididymal epithelium, such as increasing the number and size of cells and clear halo; increased endocytic organelles, and heterogeneous distribution of stereocilia, in animals with diet-induced obesity cafeteria. Given the results, it is believed that these changes should cause a profound impact on the luminal environment and interfere in the transport process, maturation, maintenance, protection and storage of sperm, being responsible for obesity alter the expression of AQP- 1 in efferent ducts, AQP- 9 in the epididymal cauda, the relative distribution of cells and clear halo and morphological characteristics of these parts via the sperm. However, more studies are needed to elucidate the specific mechanisms by which the cafeteria diet affects sperm morphology via specifically efferent ducts and epididymal ducts / A indução à obesidade, no modelo experimental animal com dieta de cafeteria, simula a alimentação com alto teor calórico que os seres humanos ingerem, na atualidade, possibilitando, assim, estudar a obesidade bem como as comorbidades advindas desta condição. O sistema genital masculino é afetado diretamente pela obesidade com aumento de peroxidação lipídica, aumento de corpos apoptóticos, mudanças nos parâmetros espermatobioscópicos e redução de fertilidade. O objetivo do estudo foi avaliar se há alterações causadas pela obesidade nos epitélios dos ductos eferentes e epididimário, tanto em relação à morfologia quanto à expressão de aquaporinas 1 e 9 (AQPs 1 e 9) em ratos Wistar adultos. Após o desmame, os animais foram divididos aleatoriamente em dois grupos: grupo controle (CON), que recebeu dieta padrão e água ad libitum (n=10) e grupo cafeteria (CAF), que recebeu dieta de cafeteria e refrigerante desgaseificado ad libitum (n=10). O tratamento do grupo CAF teve início depois da 12º semana de vida, totalizando 40 semanas de consumo da dieta de cafeteria, sendo todos os animais mortos com 52º semanas de idade. Os ductos eferentes, juntamente com segmentos inicial (Si), cabeça (Cç), corpo (Co) e cauda (Cd) do epidídimo, foram submetidos à rotina histológica e imunohistoquímica para AQP-1 e AQP-9. No presente estudo foram avaliados os dados: massa corporal, comprimento nasoanal, circunferência abdominal, índice de Lee e massa das gorduras retroperitoniais e perigonadais; também, avaliou-se dimensões, através de morfometria, e características morfológicas dos epitélios dos ductos eferentes e segmentos Si, Cç, Co e Cd do epidídimo, bem como foram analisadas as expressões de AQP-9 e AQP-1 nos ductos eferentes e segmentos epididimários supracitados. Alterações na expressão das AQPs, nos ductos eferentes e parte do epitélio epididimário, foram observadas com aumento na expressão de AQP-1 e redução de AQP-9 nos ductos eferentes e cauda epididimária, respectivamente, dos animais tratados com dieta de cafeteria. Também, ocorreram alterações morfológicas no epitélio epididimário, como aumento do número e tamanho de células halo e claras; aumento de organelas endocíticas; e distribuição heterogênea de estereocílios, nos animais com obesidade induzida por dieta de cafeteria. Em vista dos resultados, acredita-se que essas alterações devem causar um profundo impacto sobre o ambiente luminal e interferir no processo de transporte, maturação, manutenção, proteção e armazenamento dos espermatozoides, sendo a obesidade responsável por alterar a expressão de AQP-1 nos ductos eferentes, da AQP-9 na cauda epididimária, a distribuição relativa de células claras e halo e características morfológicas destas partes da via espermática. No entanto, mais estudos são necessários para esclarecer os mecanismos específicos pelos quais a dieta de cafeteria afeta a morfologia da via espermática, especificamente dos ductos eferentes e ductos epididimários

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Carbamazepina (Carba) é um anticonvulsivante, uma droga psicotrópica muito utilizada no tratamento de pacientes com distúrbios intelectuais. Esta droga foi utilizada para diminuir o volume urinário no Diabetes Insípido (DI), pois possui um efeito antidiurético, mas a incidência de hiponatremia é uma ocorrência comum. O lítio é uma das drogas mais importantes para o tratamento do distúrbio bipolar. No entanto, ele tem uma grande capacidade de induzir DI dificultando o seu uso em pacientes debilitados psicologicamente. Atualmente, a associação destas drogas é frequentemente utilizada para o tratamento de pacientes com distúrbios psiquiátricos e neurológicos. O objetivo deste trabalho foi investigar o efeito da Carba no ducto coletor medular interno (DCMI) de ratos normais e elucidar a sua ação no DI induzido pelo lítio: 1) Estudos in vitro- A) Estudos com microperfusão de segmentos isolados do néfron onde a permeabilidade à água (Pf, m/sec) foi determinada em DCMI perfundidos de ratos normais (n=20) na presença de Carba à 10-5 M, e na ausência de HAD. B) estudos com a técnica de Imunoblotting para avaliar a expressão da proteína Aquaporina 2 (AQP2) em suspensão de túbulos de DCMI de ratos normais incubados com 10-5 M de Carba por 30 minutos. 2) Estudos in vivo A) quatro grupos foram formados: a) Controle (n=5); b) Li (40 mmol/Kg/dieta por 3 semanas; n=4); c) Li+Carba (40 mmol Li/Kg/dieta + 400 mg carba/Kg/dia por 3 semanas; n=5); d) Carba (400 mg /Kg/dia por 3 semanas; n=5); - B) estudo da expressão da AQP2 no DCMI destes quatro grupos pela técnica de Western Blot. Resultados: 1) Estudos in vitro A) nos estudos de microperfusão a carba adicionada ao banho aumentou a Pf dos DCMI, na ausência de HAD (n=6) Controle12,3+ 3,6, Carba-62,6+14,8 (p<0,01), Recuperação (Rec)-17,4+5,5 (p<0,01). Com o intuito de estudar o mecanismo pelo qual a Carba ativa a cascata do HAD, foi utilizado o inibidor do receptor V2 do HAD (AV2; n=10): Controle-23,5+5,2, Carba-37,4±4,4 (p<0,01), Carba+AV2-19,6±5,0 (p<0,05), Rec-21,4+5,5; e Controle-18,6+7,0, AV2-27,3+7,2, AV2+Carba-25,3+5,7, Rec-32,6+6,4. O inibidor da PKA (H8; n=4) também foi utilizado: Controle-15,0+9,0, Carba-106,1+12,3 (p<0,01), Carba+H8-60,3+16,4 (p<0,01), Rec- 44,5+13,2. B) a análise densitométrica mostrou um aumento de 38,8% na expressão da AQP2 (Controle-100,0+8,3 vs. Carba-138,8+12,12, p<0,05); 1) Estudos in vivo Volume urinário (UV, mL/24h) Controle-10,7+3,0, Li-62,6+6,0 (p<0,001), Li+Carba-28,5+4,9 (p<0,001), Carba-23,3+3,0 (<0,001). Osmolalidade urinária (mOsm/Kg/H2O) Controle-819,6+175,7, Li-149,4+18,0 (p<0,01), Li+Carba-251,5+39,7 (p<0,05), Carba-396,2+75,5 (p<0,01). FENa+ (%) - Controle-0,15+0,01, Li-0,10+0,02, Li+Carba-0,12+0,02, Carba-0,11+0,02. Expressão da AQP2 (%) Controle-100,0+6,7, Li-55,8+5,4 (p<0,01), Li+Carba-75,8+9,6 (p<0,01), Carba- 99,7+4,7 (p<0,05). Não houve diferenças significantes no Na+, K+ e Osmolalidade plasmáticas. Em resumo, nossos dados revelaram que a Carba diminui o UV, aumenta a osmolalidade urinária e a expressão da AQP2 no DI induzido pelo lítio, e aumenta a permeabilidade à água, provavelmente agindo diretamente no receptor da vasopressina (V2). Estes resultados enfatizam que a hiponatremia encontrada nos pacientes que fazem uso da Carba pode ser explicada, pelo menos em parte, pelo aumento da permeabilidade osmótica no DCMI e que a poliúria do DI ocasionado pelo uso do lítio pode ser diminuída com a associação da Carba. / Carbamazepine (Carba) is an anticonvulsant and a psychotropic medication commonly used in the treatment of patients with intellectual disability (ID). This drug has been used to try to decrease the urinary volume in Diabetes Insipidus (DI) because Carba presents an antidiuretic effect, but the incidence of the hyponatremia in neurological patients is a common ocurrence. Lithium (Li) is one of the most important drugs used to treat bipolar mood disorders. However, Li has the undesirable capacity to induce DI complicating its usage in patients psychologically weakened. Nowadays, the association of these drugs is used in the treatment of patients with psychiatric and neurological problems. Our objective was to investigate the effect of Carba in the Inner Medullary Collecting Duct (IMCD) and elucidate its effect in the lithium-induced DI: 1) In vitro study: A) Microperfusion studies the water permeability (Pf, m/sec) was determined in normal rats IMCD isolated and perfused by the standard methods. Carba 10-5M was added to the bath fluid. B) Immunoblotting studies for AQP2 protein expression in IMCD tubule suspension from normal rats incubated with Carba 10-5M for 30 minutes. 2) In vivo study A) four groups of normal rats were done - a) Control (C, n=5); b) Li (40 mmol/kg/food/3 weeks n=4) c) Li+Carba (40 mmol Li/kg/food/3 weeks and 400 mg Carba/kg/bw/2 last weeks, n=5); and Carba (400 mg Carba/kg/bw/3weeks, n=5); - B) AQP2 expression in IMCD from the four groups, by Western Blot. Results: 1) In vitro study A) in microperfusion, Carba added to the bath in Vasopressin (Vp) absence (n=6) increased Pf Control-12.3+3.6, Carba-62.6+14.8 (p<0.01), Recuperation (Rec)-17.4+5.5 (p<0.01). In order to study the mechanism by which Carba activates the Vp cascade, the antagonist of the Vp receptor 2 (AV2; n=10) was used: Control-23.5+5.2, Carba-37.4±4.4 (p<0.01), Carba+AV2-19.6±5.0 (p<0.05), Rec-21.4+5.5; and Control-18.6+7.0, AV2- 27.3+7.2, AV2+Carba-25.3+5.7, Rec-32.6+6.4. The PKA inhibitor (H8; n=4) was also used: Control-15.0+9.0, Carba-106.1+12.3 (p<0,01), Carba+H8-60.3+16.4 (p<0.01), Rec-44.5+13.2. B) the densitometric analysis showed an increased of 38.8% in AQP2 expression (Control- 100.0+8.3 vs. Carba-138.8+12.12, p<0.05); B) In vivo study Urinary volume (UV, mL/24h) Control-10.7+3.0, Li-62.6+6.0 (p<0.001), Li+Carba-28.5+4.9 (p<0.001), Carba-23.3+3.0 (p<0.001). Urinary Osmolality (mOsm/Kg/H2O) Control-819.6+175.7, Li-149.4+18.0 (p<0.01), Li+Carba-251.5+39.7 (p<0.05), Carba-396.2+75.5 (p<0.01). FENa+ (%) - Control- 0.15+0.01, Li-0.10+0.03, Li+Carba-0.12+0.02, Carba-0.11+0.02. AQP2 expression (%) Control-100.0+6.7, Li-55.8+5,4 (p<0.01), Li+Carba-75.8+9.6 (p<0.01), Carba-99.7+4.7 (p<0.05). There were no significant differences in Na+, K+ and plasma osmolality. In summary, our data showed that Carba decreased the UV, increased the UOsm and the AQP2 expression in Li-induced DI, increased the water permeability, probably acting directly in the Vp V2 receptor-Protein G complex, since its action was blocked by the specific Vp V2 receptor antagonist. These results emphazise that the hyponatremia found in patients using Carba could be explained, at least in part, by increased osmotic permeability of IMCD. In addition, poliuria observed in lithium-induced DI can be decreased with Carba-treatment.

Le sujet de l’adaptation aux changements climatiques est devenu l’un des sujets contemporains les plus importants dans la vigne. Une grande focalisation a été mise sur la compréhension des effets du porte-greffe sur la croissance du scion, l’absorption des nutriments, et la tolérance au stress, dans l’objectif final de développer de nouveaux porte-greffes qui facilitent l’adaptation au changement climatique. L’objectif de cette thèse est d’examiner comment les différences dans la résistance à la sécheresse entre les génotypes peut résulter en de grandes différences dans leur capacité à maintenir leur croissance racinaire en situation de stress. Une meilleure compréhension sur la manière dont la structure, la croissance racinaire et l’absorption d’eau répondent au stress nous permettra de mieux comprendre quels sont les aspects de la physiologie racinaire qui contribuent à la tolérance face à la sécheresse. Des recherches précédentes qui s’étaient focalisées sur l’absorption d’eau racinaire chez la vigne ont suggéré que l’absorption d’eau racinaire pouvait être fortement liée à la vitesse de croissance racinaire instantanée (voir Gambetta et al. 2013). Cette observation implique que des différences entre les génotypes dans la résistance face à la sécheresse pourrait largement résulter de leur capacité à maintenir la croissance racinaire en conditions de stress. Deux porte-greffes de vigne avec des capacités contrastées en matière de résistance à la sécheresse, le Riparia Gloire de Montpellier (RGM) et le 110 Richter (110R) ont été sélectionnés pour étudier dans cette thèse. RGM est considéré comme sensible à la sécheresse, tandis que 110R est fortement résistant à la sécheresse (Carbonneau 1985). La thèse a examiné la relation entre la croissance racinaire et la capacité de résistance à la sécheresse en évaluant la vitesse de croissance racinaire, la conductivité hydraulique à travers deux variétés de porte-greffe en conditions de déficit en eau. Le niveau de l’expression des gènes d’aquaporines (via la qPCR et l’ARNseq) et leur contribution à la conductivité hydraulique racinaire ont été analysés dans les radicelles afin d’obtenir une meilleure compréhension sur les mécanismes impliqués dans la régulation de l’absorption de l’eau racinaire et la conductivité hydraulique au cours du développement et en réponse à un manque d’eau.Le traitement de stress d’eau prolongé a diminué le potentiel hydraulique de la plante. La croissance racinaire individuelle est très hétérogène : bien que le traitement de sécheresse réduise l’élongation racinaire en moyenne, la vitesse de croissance racinaire varie tout de même énormément. Un haut niveau de stress hydrique a réduit significativement la vitesse de croissance racinaire moyenne à la fois pour RGM et 110R. Globalement, la vitesse de croissance racinaire moyenne a montré une tendance réduite au cours du développement de la plante. La température du sol est aussi un facteur qui affecte la croissance racinaire. Pour RGM et 110R, en conditionsIIde bon arrosage et de stress hydrique, la vitesse de croissance quotidienne moyenne a été positivement corrélée avec la température du sol quotidienne moyenne. En conditions de bon arrosage, des vitesses de croissance racinaires plus importantes ont été constamment observées chez 110R par rapport à RGM, ce qui pourrait être une explication possible de sa meilleure résistance à la sécheresse par rapport à 110R. [...] / The subject of adaptation to climate change has become one of the most important contemporary topics in grapevine. Much focus has been placed on the understanding of rootstocks effects on scion growth, nutrient uptake, and tolerance to stress, with the ultimate goal of developing novel rootstocks that facilitate adaptation to a changing climate. The purpose of this thesis is to examine how differences in drought resistance between genotypes could result largely from differences in their ability to maintain root growth under stress. A better understanding of how root structure, growth, and water uptake respond to stress will allow us to better understand what aspects of root physiology contribute to drought tolerance. Previous research focused on root water uptake in grapevine suggested that root water uptake could be tightly coupled to a root’s instantaneous rate of growth (see Gambetta et al. 2013). This observation implies that differences in drought resistance between genotypes could result largely from their ability to maintain root growth under stress. Two grapevine rootstocks with contrasting drought resistance capacity, Riparia Gloire de Montpellier (RGM) and 110 Richter (110R), were selected to study in this thesis. RGM is considered as sensitive to drought, while 110R is highly resistant to drought (Carbonneau 1985). The thesis examined the relationship between root growth and drought resistant capacity by assessing root growth rate, hydraulic conductivity across two rootstock varieties subjected to water deficit. The role of aquaporin gene expression (via qPCR and RNAseq) and their contribution to root hydraulic conductivity were analyzed in fine roots in order to obtain a better understanding on the mechanisms involved in the regulation of root water uptake and hydraulic conductivity across development and in response to water deficit.Prolonged water stress treatment decreased plant water potential. Individual root growth is very heterogeneous, although drought treatment reduces root elongation on average, individual root growth rate still varies enormously. High level of water stress significantly reduced average root growth rate for both RGM and 110R. Globally, average root growth rate showed a decreased trend over plant development. Soil temperature is also a factor that affects root growth. For both RGM and 110R, under both well-watered and water-stressed conditions, average daily root growth rate was positively correlated with average daily soil temperature. Under well-watered conditions, higher root growth rates were constantly observed in 110R compared to RGM, which could be one possible explanation for the higher capacity in drought resistance of 110R.Root hydraulic conductivity (Lpr) was influenced by both water stress treatment and plant developmental stage. Generally, for both RGM and 110R, Lpr was significantly reduced under water stress in early stage. In mid and late stages, no significant differences in Lpr were observedIVbetween well-watered and water-stressed plants. Changes in individual root Lpr in response to pre-dawn leaf water potential (ᴪpredawn) were investigated as well. Lpr showed a fast drop in the beginning of water stress treatment when ᴪpredawn was higher than -0.5 MPa. However, with ᴪpredawn getting more negative, e.g. from -0.4 MPa to -2.0 MPa, the range of Lpr values measured in our study maintained constant. Lpr of well-watered plants decreased as well even though their ᴪpredawn was maintained at a high level (< 0.1 MPa) during the period of the experiment. [...]

Le syndrome de Sjögren (SS) est une maladie auto-immunitaire caractérisée par une infiltration lymphocytaire des glandes exocrines menant le plus souvent, à une xérophtalmie et à une xérostomie. La physiopathologie de la maladie est complexe et malgré les progrès realisés, il existe beaucoup de questions à repondre. Classiquement, le syndrome sec qui caractérise la maladie résulterait d’un double processus où dans un premier temps, la glande serait envahie par des cellules lymphoplasmocytaires puis secondairement détruite. Des avancées récentes dans la physiopathologie de la maladie ont démontré le rôle de nouvelles molécules, Aquaporine 5 (AQP5) et anticorps muscariniques, qui peuvent contribuer au syndrome sec. Dans ce travail, nous avons étudié des marqueurs diagnostiques de la maladie. Nous avons montré que 2 alarmines, HMGB1 et S100A8/A9 sont augmentés mais ne présentent pas de corrélation avec le score d’activité de la maladie. Utilisant différents modèles animaux de SS, nous avons montré une modification de la distribution de l’AQP5. De plus, nous avons montré que la modification de la distribution de l’AQP5 dans les glandes salivaires était liée à la présence des infiltrats inflammatoires. Utilisant un modèle non-immun de souris qui présente un syndrome sec, l’expression de l’AQP5 n’était pas modifiée en l’absence d’infiltrats inflammatoires. Ces résultats montrent que la modification de l’AQP5 dans le SS est liée à la présence des infiltrats inflammatoires. / Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished

Abstract The function of skeletal muscle is to generate force and produce movement. These tasks are carried out by long multinucleated cells, the skeletal myofibres. The membrane system and the cytoskeleton of these cells are uniquely organized to respond rapidly to neuronal stimuli and to achieve efficient contraction. In the present study the organization and distribution of selected protein/lipid based microdomains that reside in the plasma membrane and sarcoplasmic reticulum of isolated rat skeletal myofibres, were investigated. Aquaporin 4 (AQP4) water channels are arranged as higher order oligomers of several sizes in the sarcolemma and in the T tubules. These oligomers, however, were absent from many specialized micro- and- macrodomains. The distribution of AQP4 coincided with that of a highly organized protein assembly, the dystrophin glycoprotein complex (DGC), in the sarcolemma. A chimaeric venus-AQP4 was equally mobile in the T tubules and sarcolemma, but the anchoring mechanisms of the protein appeared to be different. In contrast to AQP4, the proteins resident in cholesterol and sphingolipid-based microdomains, known as rafts, also occupied DGC deficient areas, which surround the T tubule openings. Indeed, flotillin-1 rafts were located in the neck portions of the T tubules. The rafts defined by the influenza haemagglutinin (HA) also resided in DGC deficient areas, but at the borders of the DGC area. Importantly, of the raft proteins, only the localization of caveolin 3 (CAV3) was dependent on the cholesterol enriched lipid environment, as evidenced by cholesterol depletion experiments and localization studies on a non-raft associated variant of HA. The organization and distribution of membrane associated rough ER (RER) proteins were also analysed. Biochemical detergent extraction analyses and immunofluorescence staining indicated that the ER proteins were assembled as microdomains within the sarcoplasmic reticulum (SR). The microdomains were distributed throughout the SR network and they were capable of protein translocation. Taken together, skeletal myofibres comprise visually distinct microdomains both in the plasma membrane and in the SR. In the plasma membrane, different types of microdomains are not homogenously distributed and function in diverse locations. This may have important physiological implications concerning, among other things, local regulation of ion concentrations and cell signalling cascades. Different constraints ranging from protein-protein interactions to the surrounding lipid environment are important for dictating the observed distribution patterns. / Tiivistelmä Luustolihaksen toimintojen perustana ovat supistumiskykyiset lihassolut, joiden kalvorakenne on järjestynyt erityisellä tavalla ohjaamaan supistusta. Tässä tutkimuksessa analysoitiin proteiini- ja lipidiperustaisten mikroalueiden järjestäytymistä ja tähän vaikuttavia tekijöitä luustolihassolun solukalvolla sekä lihassolun sisäisessä kalvojärjestelmässä, sarkoplasmisessa verkossa (SR). Ensin analysoitiin vesikanavatyyppiä 4 (AQP4), joka oligomerisoituessaan muodostaa erikokoisia mikroalueita. Havaittiin, että AQP4-mikroalueita esiintyy kaikkialla solukalvolla lukuun ottamatta eräitä erilaistuneita mikro- ja makroalueita. AQP4-oligomeerien jakauma solukalvon lateraalisessa osassa, sarkolemmalla, noudatti dystrofiini-glykoproteiinikompleksin jakaumaa. Fluoresoivan venus-AQP4-proteiinin avulla osoitettiin, että proteiinin liikkuvuus oli samanlainen solun sisään ulottuvissa poikkiputkistoissa ja sarkolemmalla, mutta liikkuvuutta rajoittavat tekijät olivat erilaisia näissä solukalvon osissa. Toiseksi analysoitiin kolesteroli- ja sfingolipidipitoisia mikroalueita, kalvolauttoja. Flotilliini-1- ja influenssaviruksen hemagglutiniini (HA) -proteiinia sisältäviä lauttoja esiintyi vain poikkiputkien suuaukkojen alueella, mutta lauttojen jakauma oli erilainen. Lauttojen lipidiympäristöllä ei ollut vaikutusta proteiinien sijaintiin. Tämä osoitettiin kolesterolin poistokokeilla sekä kokeilla, joissa käytettiin mutatoitua HA-proteiinia, joka ei hakeudu kolesteroliympäristöön. Kaveoliini-3-proteiinin sijainti poikkeaa edellä mainituista, ja kolesterolin poisto vaikutti merkittävästi sijainnin määräytymiseen. Kolmanneksi analysoitiin, miten karkean endoplasmakalvoston proteiinit ovat järjestäytyneet SR:ssä. Havaittiin, että endoplasmiset kalvoproteiinit eivät ole homogeenisesti levittäytyneet SR-kalvostoon vaan muodostavat pieniä mikroalueita. Detergenttiuuttoanalyysit osoittivat lisäksi, että näissä mikroalueissa on erilainen lipidikoostumus kuin SR:ssä yleensä. Huomattavaa oli myös, että mikroalueet olivat toiminnallisia kaikkialla SR-kalvostossa. Tulosten perusteella luustolihassolujen kalvojärjestelmä sisältää mikroalueita, joiden jakautuminen vaikuttaa hyvin organisoituneelta. Erityisesti solukalvon mikroalueet esiintyvät tietyillä spesifeillä alueilla, joissa niiden voidaan olettaa toimivan mm. erilaisissa solusignalointitapahtumissa ja paikallisessa ionipitoisuuksien säätelyssä. Eräissä tapauksissa lipidiympäristöllä on merkitystä mikroalueiden sijainnin määräytymisessä, mutta proteiinien sitoutuminen solukalvo- tai solukalvon alaisiin rakenteisiin saattaa myös olla määräävä tekijä.

No description available.

Les aquaporines (AQPs) sont des petites protéines formant des canaux hydriques àtravers les membranes cellulaires. Les AQPs 0, 1, 2 ,4 ,5 ,6 et 8 assurent le transport sélectif de l’eautandis que les AQPs 3, 7, 9 et 10 permettent également le passage du glycérol. Nous avons étudié leurexpression dans l’épiderme, la couche supérieure de notre peau supposée imperméable. Dans lesmélanocytes, des cellules dendritiques responsables de la pigmentation, seule l’AQP1 est exprimée.Nous avons montré que les kératinocytes, les cellules majoritaires de l’épiderme, expriment enprolifération les AQPs 3 et 10, alors que les kératinocytes différenciés expriment les AQPs 3 et 9. Lalocalisation de l'AQP3 a été précédemment rapportée à la membrane plasmique des kératinocytes, de lacouche basales à la couche épineuse. Nous avons localisé l'AQP9 dans les kératinocytes différenciés dela couche granuleuse au contenu riche en glycérol et réduit en eau. De fait nous pensons que l'AQP9 ysert de transporteur de glycérol. Enfin contrairement à d'autres auteurs, nous n’avons pu mettre enévidence de lien entre prolifération tumorale et expression des aquaporines. / Aquaporins (AQPS) are a family of small proteins forming water channels across cell membranes.AQPs 0, 1, 2, 4, 5, 6 and 8 are strictly water channel whereas AQPs 3, 7, 9 and 10 allow transport ofwater and glycerol. We have studied their expression in the epidermis, the outer-most water-impermeablelayer of the skin. In melanocytes, dendritic cells responsible for pigmentation, only AQP1 is expressed, invitro and ex vivo. We have shown that keratinocytes, principal cells of the epidermis, express AQPs 3 and10 in proliferation, whereas differentiated keratinocytes express AQPs 3 and 9. The localisation of AQP3to plasma membrane of keratinocytes was previously reported from the basal layer to the spinous layer ofthe skin. We localised AQP9 in the fully differentiated keratinocytes of the granular layer, where there is ahigh glycerol and low water content. So we think that AQP9 likely functions as a glycerol transporter.Unlike other authors, we were unable to identify a link between tumorous proliferation and the expressionof aquaporins.

Dans le contexte de changement climatique, la compréhension des mécanismes régissant les pertes en eau de la vigne peut permettre d'adapter le matériel végétal pour maintenir la productivité de la vigne et la qualité du vin. L'adaptation à la sécheresse est un caractère complexe faisant intervenir des mécanismes physiologiques liés aux génotypes du greffon et du porte-greffe. Mais les effets du porte-greffe sur la régulation stomatique du greffon sont mal connus. La production par les racines de signaux chimiques tels que l'ABA et/ou hydraulique pourraient y contribuer. La réponse physiologique et moléculaire à la contrainte hydrique a été analysée sur de jeunes boutures pour 7 porte-greffes plus ou moins adaptés à la sécheresse et 2 cépages connus pour leur caractère iso ou anisohydrique. Puis 23 combinaisons greffon/porte-greffe issues de ces génotypes ont été étudiées. Une analyse métabolique sur l'accumulation de l'ABA et ses dérivés a été menée sur feuilles, racines et dans la sève xylémienne. Ces informations ont été couplées à des analyses transcriptomiques sur des gènes du métabolisme et de la signalisation de ABA, et codant des aquaporines de type PIP. L‘analyse conjointe des données physiologiques, métabolomiques et transcriptomiques ont permis d'identifier des composants moléculaires discriminant les porte-greffes selon leur fond génétique et leur adaptation à la sécheresse. Les réponses globales à la contrainte hydrique sont mieux coordonnées au sein d‘un même tissu qu‘entre racines et feuilles. A l‘échelle de la plante greffée, une prépondérance du signal hydraulique est probable. Certains gènes répondent spécifiquement aux interactions greffon/porte-greffe. / In the context of climate change, understanding the mechanisms governing the water loss of the vine is necessary to adapt the plant material to maintain the productivity of the vine and wine quality. The adaptation to drought is a complex trait involving physiological mechanisms related to scion and rootstock genotypes. But the effects of the rootstock on stomatal regulation graft are still unknown. Production by roots of chemical signals such as ABA and / or hydraulic ones be involved. Molecular and physiological responses to water stress were analyzed on young cuttings for 7 rootstocks more or less adapted to drought and 2 varieties known for their iso or anisohydric behaviour. Then 23 combinations scion / rootstock from these genotypes were investigated. Metabolic analyses for ABA and its derivatives was conducted in leaves, roots and in the xylem sap. The information was integrated with transcriptomic analyzes for genes involved in ABA metabolism and signaling, and encoding PIP aquaporins. Joint analyses of physiological data, metabolomic and transcriptomic allow the identification of the molecular components discriminating rootstocks according to their genetic background and their adaptation to drought. Global responses to water stress are better coordinated within the same tissue between roots and leaves. At the scale of the grafted plant, a preponderance of the hydraulic signal is likely. Some genes specifically respond to the scion / rootstock interactions.