L’aquaporine-2 (AQP2) est le canal responsable de la réabsorption finale d’eau au niveau du tubule collecteur du rein. À la base, contenue dans des vésicules internes, l’AQP2 est acheminée à la membrane apicale des cellules principales du tubule collecteur suite à une stimulation par l’hormone antidiurétique (ADH). L’incapacité à accomplir cette fonction entraîne le diabète insipide néphrogénique (DIN), une maladie caractérisée par l’inhabileté du rein à concentrer l’urine, entraînant une production de volumes urinaires élevés. Alors que les mutations récessives génèrent des protéines mal structurées et incapables de former des tétramères, les mutations dominantes sont capables de s’associer à leurs homologues sauvages, engendrant ainsi un DIN même chez les patients hétérozygotes. Ce mémoire présente l’analyse biochimique et fonctionnelle d’une nouvelle mutation naturelle de l’AQP2, la mutation T179N, aussi responsable du DIN. Cette dernière est particulièrement intéressante de par son génotype qui implique un caractère dominant, et sa position extracellulaire habituellement réservée aux mutations récessives. Les études comparatives de T179N à deux modèles de mutation récessive et dominante démontrent, tant en ovocytes de Xenopus laevis qu’en lignée cellulaire mpkCCDc14, le caractère récessif de cette nouvelle mutation. Les tests d’immunobuvardage de lysats d’ovocytes en membranes totales et membranes plasmiques purifiées ont révélé que seule la forme sauvage atteint la membrane plasmique alors que le mutant T179N est séquestré dans la cellule. En accord avec ce résultat, les analyses de perméabilité fonctionnelle démontrent aussi une absence d’activité pour T179N. En cellule mpkCCDc14, le mutant T179N exprimé seul n’atteint pas la membrane plasmique suite à l’action de la forskoline, contrairement à la forme sauvage. Cependant, ce mutant peut s’associer à son homologue sauvage en coexpression tant dans les ovocytes qu’en lignée mpkCCDc14 sans toutefois engendrer l’effet typique de dominance négative. En fait, dans ce contexte de coexpression, on remarque une augmentation de la Pf de 83±7 % et une récupération d’adressage à la membrane plasmique en cellule (immunofluorescence). En conclusion, T179N serait un mutant récessif fonctionnellement récupérable lorsqu’en présence de l’AQP2 sauvage. / Aquaporin-2 (AQP2) is the channel responsible for the final reabsorption of water in the collecting duct of the kidney. Basically, contained in internal vesicles, the AQP2 is delivered to the apical membrane of the principal cells of the collecting tubule after stimulation by the antidiuretic hormone (ADH). The failure to perform this function causes nephrogenic diabetes insipidus (NDI); a disease characterized by the inability of the kidney to concentrate urine and induces the production of high urinary volumes. While recessive mutations generate poorly structured proteins unable to form tetramers, dominant mutations are capable of associating with their wild counterparts, thus generating a NDI even in heterozygous patients. This paper presents the biochemical and functional analysis of T179N, a new NDI-causing mutation of the human AQP2. The mutant is particularly interesting because of its dominant genotype, despite its extracellular position usually restricted to recessive mutations. Here, we compare T179N against archetypal recessive and dominant mutations using both Xenopus laevis oocytes and in mpkCCDc14 cell model, and show the recessive nature of the mutation. The immunoblot tests on oocytes lysates in purified total and plasma membranes revealed that only the wild type protein reaches the plasma membrane while the T179N mutant is sequestered within cellular stores. Accordingly, functional analyzes indicate that T179N is inactive. In mpkCCDc14 cells, T179N expressed alone does not reach the plasma membrane in response to forskolin stimulation, unlike the wild-type. However, T179N does show the capacity to associate with its wild-type counterpart in both oocytes and in mpkCCDc14 cells, although without displaying the typical dominant negative effect. In fact, when coexpressed along wild-type, T179N gains back the functionality, with a Pf increase of 83±7 % and adequate plasma membrane targeting in cells (immunofluorescence). In conclusion, the mutant T179N is a mild recessive mutation that is susceptible to functional recovery when in presence of wild type AQP2.

Cette étude porte sur le contrôle moléculaire de l'expansion cellulaire dans l'apex de la racine du peuplier. L'étude a été focalisée sur la régulation de l'expression des aquaporines TIP1s sous deux niveaux de déficit hydrique. Un cadre conceptuel, combinant analyse de la densité de transcrits à haute résolution spatiale et un formalisme de la mécanique des fluides, a été établi pour décrire la régulation de l'expression des gènes dans le temps et l'espace, le long de l'apex. Deux états de croissance contrastés ont été étudiés : un où la croissance de l'apex de la racine est rétablie (après 3 jours de stress modéré, 80 mosmol kg-1, 100 g L-1 de polyéthylène glycol PEG à 3500 g mol-1) et un où elle est réduite (après 3 jours de stress fort, 250 mosmol kg-1, 200 g L-1 de PEG). L'analyse cinématique a révélé une sensibilité différentielle de la vitesse d'allongement relatif (REGR) selon le niveau de stress et la position le long de l'apex. Au niveau moléculaire, nous montrons que lorsque la croissance est réduite, les pics de densité de transcrits des TIP1s sont plus forts et décalés dans les premiers millimètres de l'apex, zone où la croissance des cellules est maintenue. Cependant les niveaux d'induction/répression n'étaient jamais plus élevés que dans la condition témoin, soulignant qu'une forte densité de transcrits n'est pas forcément synonyme d'une forte induction de l'expression. Focalisé sur la combinaison de deux processus dynamiques, l'expansion cellulaire et l'expression des gènes, cette étude a mis en évidence que les conclusions tirées de l'analyse de ces processus sont influencées par la façon dont le temps, l'espace sont considérés / This study consideres the molecular control of cell expansion in poplar root apex. The study was focused on the regulation of the TIP1 aquaporins expression under two levels of water deficit. A conceptual framework combining transcript density analysis (quantitative PCR) at a high spatial resolution and a fluid mechanics formalism was established to describe the regulation of gene expression in time and space along the root apex. Two contrasting growth status were both studied: root growth rate is either restored (after three days of moderate stress, 80 mmol kg-1, 100 g L-1 polyethylene glycol PEG 3500 g mol-1) or root growth rate is reduced (after 3 days of high stress, 250 mosmol kg-1, 200 g L-1 PEG). Kinematic analysis revealed a differential sensitivity of the relative elemental growth rate (REGR) according to the stress level and to the coordinate along the apex. At the molecular level, we showed that growth reduction was associated with a shift of maximal densities of transcripts towards the first millimeters of the apex, where cell expansion was maintained. Meanwhile the induction/repression levels were never stronger than in the control condition underlying that a high transcript density does not mean a high transcriptional induction. Focused on the combination of two dynamic processes, cell expansion and gene expression, my thesis showed that the conclusions issued from the analyses of these processes are influenced by the way time, space and age are considered

Le traumatisme crânien (TC) est la première cause de visite aux urgences pour la population pédiatrique. Indépendamment du niveau de sévérité du TC, les patients pédiatriques souffrent sur le long-terme de troubles cognitifs et émotionnels, cependant les mécanismes moléculaires et cellulaires sous-jacents sont encore peu connus, et il n’existe pas de traitement efficace disponible à ce jour. L’unité neurovasculaire est composée de vaisseaux sanguins, neurones et astrocytes. Les astrocytes sont essentiels à une variété de fonctions physiologiques assurées par cette unité tels que l’homéostasie cérébrale et le couplage neurovasculaire. Suite à une lésion, les astrocytes deviennent « réactifs », et cette « astrocytopatie » peut impacter leur rôle physiologique et empirer les conséquences de la lésion.Nous avons étudié le rôle de l’astrocytopatie dans le TC juvénile et fait l’hypothèse que : (1) les astrocytes réactifs contribuent à la propagation de l’œdème via les jonctions serrées connexines après un TC juvénile modéré ; (2) l’astrocytopatie se développe également après un TC juvénile léger avec des changements calciques qui pourraient contribuer à (3) une altération de la réactivité vasculaire, tout cela impactant sur les conséquences comportementales qui font suite à la lésion.Nous avons montré que :(1) Réduire l’astrocythopatie en sous-régulant la connexine 43 permettait d’améliorer les conséquences comportementales après un TC modéré juvénile, mais n’impactait pas la propagation de l’œdème.(2) Les astrocytes devenaient réactifs et subissaient des changements morphologiques après un TC juvénile léger avec des perturbations dans les signaux purinergiques-calciques liés à des changements dans l’expression du canal aqueux aquaporine 4 (AQP4).(3) Une dysfonction vasculaire majeure s’était développée après le TC juvénile léger avec des changements fonctionnels et morphologiques des vaisseaux intraparenchymaux parallèles aux altérations comportementales et précédant les dommages axonaux après la lésion.Ce travail apporte un nouvel aperçu de la pathophysiologie du TC juvénile et ouvre des possibilités pour développer des thérapies ciblant l’astrocytopatie après une lésion. / Traumatic brain injury (TBI) is the first cause for emergency department visits in the pediatric population. Regardless of the severity of TBI, pediatric patients suffer long-term cognitive and emotional impairments but the underlying cellular and molecular mechanisms are still poorly understood and there are no effective treatments available. The neurovascular unit is composed by blood vessels, neurons and astrocytes. Astrocytes are crucial for various physiological functions of this unit such as brain homeostasis and neurovascular coupling. In injuries astrocytes become “reactive”, and this “astrocytopathy” can impact their physiological roles and worsen the outcome after injury.We investigated astrocytopathy in juvenile TBI and hypothesized that: (1) reactive astrocytes contribute to spread of edema through connexin gap junctions after juvenile moderate TBI; and that (2) astrocytopathy also develops after juvenile mild TBI with calcium changes that could contribute to (3) impaired vascular reactivity, all of which impacts the behavioral outcome after injury.We have shown that:(1) Reducing astrocytopathy by downregulating the gap junction protein connexin 43 improved the behavioral outcome after juvenile moderate TBI, but did not impact the spread of edema.(2) Astrocytes became reactive and underwent morphological changes after juvenile mild TBI with disturbances in purinergic-calcium signaling related to expression changes of the water channel aquaporin 4 (AQP4).(3) Major vascular dysfunction developed after juvenile mild TBI with functional and morphological changes of the intraparenchymal vessels that paralleled behavioral impairments and preceded axonal damage after injury.This work brings new insights in the pathophysiology of juvenile TBI and opens prospects for developing therapeutics targeting astrocytopathy after injury.

Os pacientes que usam lítio (Li) para tratamento do transtorno bipolar freqüentemente apresentam poliúria e deficiência de concentração urinária, sintomas do Diabetes Insipidus nefrogênico (DIN). Animais tratados com Li apresentam baixos níveis de produção de adenosina monofosfato cíclico (AMPc) em resposta ao hormônio antidiurético (HAD). O Sildenafil (Sil), um inibidor da fosfodiesterase 5 (PDE5), eleva os níveis intracelulares de guanosina monofosfato cíclico (GMPc), levando a inserção de aquaporina 2 (AQP2) na membrana plasmática das células do ducto coletor. Portanto, inibidores de PDE podem promover a inserção de AQP2 na membrana plasmática mesmo sem a ativação do receptor de HAD, indicando a participação de uma via alternativa mediada pelo GMPc. Nós investigamos o efeito do Sil na expressão renal das proteínas de membrana AQP2, UT-A1, NKCC2, NHE3, P-ENaC em ratos com DIN induzido pelo Li. Ratos Wistar foram divididos nos seguintes grupos: grupo controle, recebendo dieta alimentar normal durante quatro semanas; grupo Li, recebendo dieta alimentar normal com 40 mmol Li por quilo de dieta durante quatro semanas; grupo Li + Sil, recebendo dieta alimentar normal com 40 mmol Li por quilo de dieta durante quatro semanas e 200 mg por quilo de dieta de Sil a partir da segunda semana; grupo Sil, recebendo dieta alimentar normal durante a primeira semana e a partir da segunda semana recebendo dieta normal com 200 mg de Sil por quilo de dieta. Os animais do grupo Li desenvolveram poliúria, diminuição da osmolalidade urinária e diminuição da expressão da AQP2. No grupo Li+Sil, o Sil foi capaz de reverter parcialmente a poliúria, diminuir o clearance de água livre, aumentar a osmolalidade urinária e aumentar a expressão da AQP2. A expressão de UTA1 foi completamente normalizada com o tratamento com Sil. A expressão das proteínas NKCC2 e NHE3 apresentaram-se aumentadas no grupo tratado com Li, e o Sil não foi capaz de reverter tal alteração. Além disso, o tratamento com Sil reverteu completamente o aumento da resistência vascular renal. Assim, concluímos que o tratamento com Sil em ratos com DIN melhora a poliúria, aumenta a smolalidade urinária e diminui o clearance de água livre pelo aumento da expressão de AQP2 e UT-A1. O tratamento com Sil pode ser benéfico para pacientes que sofrem com DIN induzida pelo Li. / Patients taking lithium to treat bipolar disorder often present polyuria and urinary concentrating defect. In addition, lithium-treated animals present lower cyclic adenosine monophosphate production in response to vasopressin. Sildenafil (Sil), a phosphodiesterase 5 (PDE5) inhibitor, elevates intracellular cyclic guanosine monophosphate (cGMP) levels, leading to plasma membrane accumulation of aquaporin 2 (AQP2). Therefore, PDE inhibitors might induce AQP2 membrane insertion even without vasopressin receptor activation by activating a parallel cGMP-mediated signal transduction pathway. We investigated the effect of sildenafil on renal expression of AQP2, UT-A1, sodium/hydrogen exchanger (NHE3), type 1 bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2), and the epithelial sodium channel alpha subunit (P-ENaC). Wistar rats received lithium (40 mmol/kg food) or not for 4 weeks (Li or control), some rats also receiving sildenafil (200 mg/kg food) in weeks 2-4, with or without lithium (Li+Sil or Sil). In Li+Sil rats, urine output was markedly lower, as was water free clearance, whereas urine osmolality was higher. Semiquantitative immunoblotting revealed the following: AQP2 expression was partially normalized; UT-A1 expression was completely normalized; expression of NKCC2 and NHE3 was significantly higher in Li rats (although not significantly different between Li+Sil rats and Li rats); and P-ENaC protein expression was unaltered in all groups. Sildenafil treatment completely reversed the lithium-induced increase in renal vascular resistance. In conclusion, sildenafil treatment of lithium-induced nephrogenic diabetes insipidus (NDI) improves polyuria, increases urinary osmolality, and decreases free water clearance via upregulation of renal AQP2 and UT-A1. Sildenafil treatment could be beneficial in patients with lithium-induced NDI.

As estratégias terapêuticas empregadas na ressuscitação do choque hemorrágico (CH) são capazes de evitar a morte, mas seus efeitos colaterais podem causar muitas alterações orgânicas, inclusive nos rins. Sabemos que a reposição volêmica feita com solução cristaloide restaura a hemodinâmica, diminui a mortalidade, mas pode levar a edema de órgãos e tecidos, entre outras alterações. Sendo assim, o objetivo do atual estudo foi comparar os efeitos da ressuscitação com Ringer lactato (RL) ou terlipressina (TLP) na função renal em modelo experimental de CH. Com este intuito, foi induzido o CH em suínos de 20-30 kg, por meio de sangramento pressão-controlada, até a obtenção e manutenção da pressão arterial média (PAM) em 40mmHg por 30 minutos. Os animais foram divididos em quatro grupos, sendo eles, o grupo Sham (feito apenas o procedimento anestésico), grupo Choque (indução de choque hemorrágico por 30 minutos) e os de instituição de estratégias de ressuscitação: grupo RL (indução de choque hemorrágico, e administração de RL de três vezes o volume de sangue retirado) e o grupo TLP (indução do choque e administração em bolus de 2 mg de TLP). Os parâmetros hemodinâmicos, de função renal e tubular foram avaliados nos momentos: basal; imediatamente após o CH; 30, 60, 90 e 120 minutos após tratamento. Ao final do estudo, os animais foram eutanasiados aos 60 ou aos 120 minutos. Foram coletadas amostras de tecido renal para avaliação de histologia e de western blott. Observamos uma melhora na microcirculação dos animais tratados com RL; entretanto, houve uma normalização da expressão da proteína NKCC2 e aquaporina 2 no grupo TLP. Observamos que o escore de lesão renal foi igual nos dois grupos de intervenção, mas a expressão de Bax estava mais diminuída no grupo TLP. Concluímos que a TLP mostrou ser uma estratégia tão eficaz quanto o RL no resgate do choque hemorrágico, com um provável potencial de maior proteção renal / Therapeutic strategies employed hemorrhagic shock (HS) resuscitation are able to prevent death, but its side effects can cause many organic dysfunction, including injury to the kidneys. It has been know that volume replacement with crystalloid solution can restore the hemodynamic status and decreases mortality; however, it can lead to organs and tissues edema, among other changes. Thus, the goal of this study was to compare the effects of resuscitation with lactated Ringer\'s (RL) or terlipressin (TLP) on renal function in an experimental model of HS. For this purpose, HS was induced in 20-30 kg swine, with pressure-controlled bleeding, to obtain and maintain the mean arterial pressure (MAP) of 40 mmHg for 30 minutes. The animals were divided into four groups, namely, the Sham group (made only the anesthetic procedure), Shock group (induction of hemorrhagic shock for 30 minutes) and the institution of resuscitation strategies: RL group (induction of hemorrhagic shock, and RL administration of three times the volume of removed blood) and TLP group (induction of shock and administration in bolus of 2 mg TLP). Hemodynamic parameters, renal and tubular function were evaluated at baseline; immediately after the HS; 30, 60, 90 and 120 minutes after treatment. At the end of the study, animals were euthanized after 60 or 120 minutes. Kidney tissue samples were collected for evaluation of histology and western blotting. We observed an improvement in microcirculation of animals treated with RL; however, there was a normalization of NKCC2 and aquaporin 2 protein expression in the TLP group. We observe that the kidney injury score was similar in both intervention groups, but Bax protein expression was more reduced than in the TLP group. We conclude that the TLP proved to be an effective strategy as RL in the rescue of hemorrhagic shock, with a likely potential for protect renal function

In marine teleosts that spawn pelagic eggs (pelagophils), the process of oocyte hydration that occurs during meiosis resumption is a key physiological process for the survival of the eggs in the ocean. Previous studies have discovered the role of a teleost-specific aquaporin water channel (Aqp1b) during fish oocyte hydration, but direct experimental evidence for the function of Aqp1b in oocytes is still lacking. In addition, the molecular regulation of the Aqp1b-mediated mechanism remains poorly understood. In this context, the main objectives of the present thesis were to investigate the evolutionary origin of aqp1ab in teleosts, to provide functional evidence of the role of Aqp1b during oocyte hydration, and to begin to dissect the molecular mechanisms involved in the transcriptional regulation of aqp1b in the oocyte of marine teleosts. By integrating the molecular phylogeny with synteny and structural analyses we show that the teleost aqp1aa and -1ab paralogs (previously annotated as aqp1a and -1b, respectively) arose by tandem duplication, and that the Aqp1ab C-terminus is the most rapidly evolving subdomain within the vertebrate aquaporin superfamily. The functional role of Aqp1ab was investigated in Atlantic halibut (Hippoglossus hippoglossus), a marine acanthomorph teleost that spawns one of the largest pelagic eggs known. Using immunological inhibition of Aqp1ab in halibut oocytes and artificial expression of the halibut paralog Aqp1aa, we demonstrate that Aqp1ab is required for full hydration of oocytes undergoing meiotic maturation. To investigate the aqp1ab transcriptional regulation in oocytes, we isolated the 5’-flanking region of the gilthead seabream (Sparus aurata) aqp1ab gene which contains regulatory cis-elements for the nuclear progestin receptor (Pgr) and SOX transcription factors. The Pgr, as well as sox3 and -8b transcripts, are co-expressed in seabream oo-gonia, whereas in primary growth oocytes, when aqp1ab mRNA and protein are synthesized, the Pgr is translocated into the nucleus. In contrast, sox9b is highly expressed in more advanced oocytes showing the depletion of aqp1ab transcripts. In the seabream, four different pgr transcript variants are expressed in primary growth ovaries which are generated by alternative pre-mRNA splicing. Seabream wild-type Pgr shows the highest transactivation response to progestins such as 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P) and 17α,20β,21-trihydroxy-4-pregnen-3-one (17,20β,21-P), whereas two of the N-terminally truncated Pgr isoforms regulate novel nuclear and cytosolic mechanisms of dominant-negative repression of Pgr-mediated transcription. Transactivation assays on the aqp1ab promoter demonstrated that aqp1ab transcription is dependent on wild-type Pgr, with Sox3 and -8b acting synergistically, while Sox9b acts as a repressor. Incubation of primary ovarian explants in vitro with 17,20β-P, followed by chromatin immunoprecipitation, confirmed that 17,20β-P-activated Pgr enhanced aqp1ab promoter activation. The production of 17,20β-P in seabream primary growth ovaries in vivo was consistent with the expression of P450c17-II (Cyp17a2) and 20β-hydroxysteroid dehydrogenase (Cbr1), enzymes needed for progestins synthesis, in granulosa cells associated with primary growth oocytes, and with a high concentration of 17,20β-P. Incubation of primary ovarian explants with recombinant piscine follicle-stimulating hormone (rFsh) in vitro stimulated 17,20β-P synthesis, which was reduced in the presence of Cbr1 inhibitors. The rFsh-mediated production of 17,20β-P correlated with the up-regulation of cyp17a2 and cbr1 transcription, as well as of wild-type pgr mRNA and protein levels. Altogether, these data suggest that aqp1ab transcription in seabream primary growth oocytes is under Fsh regulation through the synthesis of progestins. The results of this thesis show that the Aqp1ab mediated mechanism for oocyte hy-dration is likely conserved in marine teleosts. In addition, the tight transcriptional reg-ulation of Aqp1ab during oogenesis highlights the essential physiological role of this water channel and opens new research avenues for understanding the molecular basis of egg formation in marine fish. / La hidratación de los oocitos de teleósteos marinos que producen huevos pelágicos es clave para la supervivencia de los embriones en el océano. Estudios previos han descu-bierto el papel de una acuaporina específica de teleósteos (Aqp1b) durante este proceso, pero se carece todavía de evidencias experimentales directas, así como información sobre la regulación molecular de la Aqp1ab. En la presente tesis, estudios iniciales con el fletan Atlántico (Hippoglossus hippoglossus) confirman que los parálogos aqp1aa y -1ab de teleósteos han surgido probablemente por duplicación génica en tándem, y han demostrado el papel esencial de la Aqp1ab durante la hidratación del oocito. Para investigar el control transcripcional del gen aqp1ab en los oocitos de la dorada (Sparus aurata) se ha aislado su región promotora, la cual contiene elementos cis-reguladores de unión al receptor nuclear de progestinas (Pgr), como la 17α,20β-dihidroxi-4-pregnen-3-ona (17,20β-P), y factores de transcripción Sox. Estudios de localización subcelular indican que el Pgr aparece en el citoplasma de las oogonias, así como en el núcleo de oocitos en crecimiento primario (pre-vitelogénicos) coincidiendo con la activación de la expresión de aqp1ab. En este estadio también se expresan cuatro isoformas diferentes del Pgr, dos de las cuales pueden inhibir la transcripción mediada por el Pgr de forma dominante negativa. Las oogonias también expresan sox3 y -8b, mientras que el sox9b aparece en el estadio de alveolo cortical, cuando se reduce la expresión de aqp1ab. Ex-perimentos de transactivación indican que el Pgr activa la transcripción de aqp1ab, con Sox3 y -8b actuando de forma sinérgica, mientras que el Sox9b reprime este mecanismo. El papel del Pgr se ha investigado sobre explantes ováricos pre-vitelogénicos incubados in vitro, lo cual ha demostrado que la 17,20β-P, producida en las células de la granulosa en respuesta a la hormona folículo estimulante, activa el promotor de aqp1ab en el oocito induciendo la síntesis de Aqp1ab. Los resultados de esta tesis revelan por primera vez una estricta regulación transcripcional del gen aqp1ab durante la oogénesis de teleósteos marinos, lo cual remarca la función fisiológica esencial de este canal de agua durante la formación de los huevos pelágicos.

Cette thèse décrit diverses situations liées au transport fluidique aux nano-échelles. Le premier chapitre est une introduction à la nanofluidique qui contient une revue des longueurs caractéristiques, des forces et des phénomènes présents aux nano-échelles. Le deuxième chapitre est une étude de l'impact de la géométrie sur la perméabilité hydrodynamique d'un nanopore. Inspirée par la forme des aquaporines, cette étude suggère une optimisation possible pour des canaux biconiques. Le troisième chapitre est une étude du remplissage capillaire dans des canaux sub-nanométriques en carbone. Cette étude montre l'importance de la pression de disjonction induite par la structure du fluide sur le remplissage. Le quatrième chapitre est une étude d'une diode nanofluidique, un composant connu pour imiter le comportement d'une diode à semi-conducteur. On montre qu'un fort couplage entre l'eau et la dynamique des ions entraîne une rectification du flux d'eau à l'intérieur de la diode. Le cinquième et dernier chapitre est une étude de l'origine du bruit rose (1=f) communément observé lors des mesures de courant ionique dans les nanopores / This thesis discusses various situations linked to transport at the nanoscale. The first chapter is an introduction to nanofluidics, containing a review of characteristic lengths, forces, or phenomena existing at the nanoscale. The second chapter is a study of the impact of geometry on the hydrodynamic permeability of a nanopore. This study, inspired by the shape of aquaporins, suggests a possible optimisation of permeability for bi-conical channels. The third chapter is a study of capillary filing inside subnanometric carbon channels which highlights the importance of the disjoining pressure induced by the fluid structuring inside the nanochannel. The fourth chapter is a study of nanofluidic diode, a component known to mimic the behaviour of semiconductor diode. The study highlights a strong coupling between water and ion dynamics which leads to a water flow rectification inside the diode. The fifth and last chapter is a study of the origin of commonly observed pink noise (1=f) in ionic current measurements through nanopores

As estratégias terapêuticas empregadas na ressuscitação do choque hemorrágico (CH) são capazes de evitar a morte, mas seus efeitos colaterais podem causar muitas alterações orgânicas, inclusive nos rins. Sabemos que a reposição volêmica feita com solução cristaloide restaura a hemodinâmica, diminui a mortalidade, mas pode levar a edema de órgãos e tecidos, entre outras alterações. Sendo assim, o objetivo do atual estudo foi comparar os efeitos da ressuscitação com Ringer lactato (RL) ou terlipressina (TLP) na função renal em modelo experimental de CH. Com este intuito, foi induzido o CH em suínos de 20-30 kg, por meio de sangramento pressão-controlada, até a obtenção e manutenção da pressão arterial média (PAM) em 40mmHg por 30 minutos. Os animais foram divididos em quatro grupos, sendo eles, o grupo Sham (feito apenas o procedimento anestésico), grupo Choque (indução de choque hemorrágico por 30 minutos) e os de instituição de estratégias de ressuscitação: grupo RL (indução de choque hemorrágico, e administração de RL de três vezes o volume de sangue retirado) e o grupo TLP (indução do choque e administração em bolus de 2 mg de TLP). Os parâmetros hemodinâmicos, de função renal e tubular foram avaliados nos momentos: basal; imediatamente após o CH; 30, 60, 90 e 120 minutos após tratamento. Ao final do estudo, os animais foram eutanasiados aos 60 ou aos 120 minutos. Foram coletadas amostras de tecido renal para avaliação de histologia e de western blott. Observamos uma melhora na microcirculação dos animais tratados com RL; entretanto, houve uma normalização da expressão da proteína NKCC2 e aquaporina 2 no grupo TLP. Observamos que o escore de lesão renal foi igual nos dois grupos de intervenção, mas a expressão de Bax estava mais diminuída no grupo TLP. Concluímos que a TLP mostrou ser uma estratégia tão eficaz quanto o RL no resgate do choque hemorrágico, com um provável potencial de maior proteção renal / Therapeutic strategies employed hemorrhagic shock (HS) resuscitation are able to prevent death, but its side effects can cause many organic dysfunction, including injury to the kidneys. It has been know that volume replacement with crystalloid solution can restore the hemodynamic status and decreases mortality; however, it can lead to organs and tissues edema, among other changes. Thus, the goal of this study was to compare the effects of resuscitation with lactated Ringer\'s (RL) or terlipressin (TLP) on renal function in an experimental model of HS. For this purpose, HS was induced in 20-30 kg swine, with pressure-controlled bleeding, to obtain and maintain the mean arterial pressure (MAP) of 40 mmHg for 30 minutes. The animals were divided into four groups, namely, the Sham group (made only the anesthetic procedure), Shock group (induction of hemorrhagic shock for 30 minutes) and the institution of resuscitation strategies: RL group (induction of hemorrhagic shock, and RL administration of three times the volume of removed blood) and TLP group (induction of shock and administration in bolus of 2 mg TLP). Hemodynamic parameters, renal and tubular function were evaluated at baseline; immediately after the HS; 30, 60, 90 and 120 minutes after treatment. At the end of the study, animals were euthanized after 60 or 120 minutes. Kidney tissue samples were collected for evaluation of histology and western blotting. We observed an improvement in microcirculation of animals treated with RL; however, there was a normalization of NKCC2 and aquaporin 2 protein expression in the TLP group. We observe that the kidney injury score was similar in both intervention groups, but Bax protein expression was more reduced than in the TLP group. We conclude that the TLP proved to be an effective strategy as RL in the rescue of hemorrhagic shock, with a likely potential for protect renal function

Os pacientes que usam lítio (Li) para tratamento do transtorno bipolar freqüentemente apresentam poliúria e deficiência de concentração urinária, sintomas do Diabetes Insipidus nefrogênico (DIN). Animais tratados com Li apresentam baixos níveis de produção de adenosina monofosfato cíclico (AMPc) em resposta ao hormônio antidiurético (HAD). O Sildenafil (Sil), um inibidor da fosfodiesterase 5 (PDE5), eleva os níveis intracelulares de guanosina monofosfato cíclico (GMPc), levando a inserção de aquaporina 2 (AQP2) na membrana plasmática das células do ducto coletor. Portanto, inibidores de PDE podem promover a inserção de AQP2 na membrana plasmática mesmo sem a ativação do receptor de HAD, indicando a participação de uma via alternativa mediada pelo GMPc. Nós investigamos o efeito do Sil na expressão renal das proteínas de membrana AQP2, UT-A1, NKCC2, NHE3, P-ENaC em ratos com DIN induzido pelo Li. Ratos Wistar foram divididos nos seguintes grupos: grupo controle, recebendo dieta alimentar normal durante quatro semanas; grupo Li, recebendo dieta alimentar normal com 40 mmol Li por quilo de dieta durante quatro semanas; grupo Li + Sil, recebendo dieta alimentar normal com 40 mmol Li por quilo de dieta durante quatro semanas e 200 mg por quilo de dieta de Sil a partir da segunda semana; grupo Sil, recebendo dieta alimentar normal durante a primeira semana e a partir da segunda semana recebendo dieta normal com 200 mg de Sil por quilo de dieta. Os animais do grupo Li desenvolveram poliúria, diminuição da osmolalidade urinária e diminuição da expressão da AQP2. No grupo Li+Sil, o Sil foi capaz de reverter parcialmente a poliúria, diminuir o clearance de água livre, aumentar a osmolalidade urinária e aumentar a expressão da AQP2. A expressão de UTA1 foi completamente normalizada com o tratamento com Sil. A expressão das proteínas NKCC2 e NHE3 apresentaram-se aumentadas no grupo tratado com Li, e o Sil não foi capaz de reverter tal alteração. Além disso, o tratamento com Sil reverteu completamente o aumento da resistência vascular renal. Assim, concluímos que o tratamento com Sil em ratos com DIN melhora a poliúria, aumenta a smolalidade urinária e diminui o clearance de água livre pelo aumento da expressão de AQP2 e UT-A1. O tratamento com Sil pode ser benéfico para pacientes que sofrem com DIN induzida pelo Li. / Patients taking lithium to treat bipolar disorder often present polyuria and urinary concentrating defect. In addition, lithium-treated animals present lower cyclic adenosine monophosphate production in response to vasopressin. Sildenafil (Sil), a phosphodiesterase 5 (PDE5) inhibitor, elevates intracellular cyclic guanosine monophosphate (cGMP) levels, leading to plasma membrane accumulation of aquaporin 2 (AQP2). Therefore, PDE inhibitors might induce AQP2 membrane insertion even without vasopressin receptor activation by activating a parallel cGMP-mediated signal transduction pathway. We investigated the effect of sildenafil on renal expression of AQP2, UT-A1, sodium/hydrogen exchanger (NHE3), type 1 bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2), and the epithelial sodium channel alpha subunit (P-ENaC). Wistar rats received lithium (40 mmol/kg food) or not for 4 weeks (Li or control), some rats also receiving sildenafil (200 mg/kg food) in weeks 2-4, with or without lithium (Li+Sil or Sil). In Li+Sil rats, urine output was markedly lower, as was water free clearance, whereas urine osmolality was higher. Semiquantitative immunoblotting revealed the following: AQP2 expression was partially normalized; UT-A1 expression was completely normalized; expression of NKCC2 and NHE3 was significantly higher in Li rats (although not significantly different between Li+Sil rats and Li rats); and P-ENaC protein expression was unaltered in all groups. Sildenafil treatment completely reversed the lithium-induced increase in renal vascular resistance. In conclusion, sildenafil treatment of lithium-induced nephrogenic diabetes insipidus (NDI) improves polyuria, increases urinary osmolality, and decreases free water clearance via upregulation of renal AQP2 and UT-A1. Sildenafil treatment could be beneficial in patients with lithium-induced NDI.

La conductivité hydraulique foliaire (KF) est une composante majeure du transport d’eau dans toute la plante. Dans les feuilles de noyer, la KF est stimulée à la lumière et est étroitement liée à l’accroissement du taux des transcrits d’aquaporines JrPIP2s. Par ailleurs, la corrélation entre la stimulation de la KF et des transcrits d’aquaporines à la lumière, n’est pas générale et dépend de l’espèce. Ici, nous étudions cette corrélation chez cinq espèces forestières (Juglans regia, Fagus sylvitica, Quercus robur, Salix alba et Populus tremula) différant par leur réponse à la lumière. Nous démontrons seulement chez le noyer (Juglans regia), la contribution des deux familles d’aquaporines PIP1s et PIP2s. Afin de mieux comprendre le rôle des JrPIP1s et JrPIP2 dans la réponse à la lumière, nous avons isolé 8 nouvelles isoformes dans les feuilles de noyer et nous avons étudié leurs profils d’expression sur une cinétique lumière. Toutes les isoformes étudiées sont accumulées à la lumière et réprimées à l’obscurité. De plus, la KF est dépendante de la qualité de lumière. Elle est réduite de 65% en absence de lumière bleue. Cette diminution serait liée à l’inhibition des transcrits d’aquaporines. Afin de caractériser les mécanismes moléculaires précoces impliqués dans la modulation de KF par la lumière, l’approche globale cDNA-AFLP a été menée sur des feuilles de noyer sous différentes conditions d’éclairement. Nous obtenons 12000 transcrits différentiels dérivés (TDFs) générés par les 128 couples d’amorces. Parmi les 187 séquences obtenues, 93 d’entre elles ont une fonction putative. Leur classification fonctionnelle montre que les gènes relatifs à la régulation cellulaire représentent environ 58% des TDFs identifiés. Les feuilles exposées à la lumière, montrent des changements dans les voies de : signalisation calcique, protéolyse, trafic vésiculaire et l’expression de divers facteurs de transcription et protéines de régulation. Pour mieux comprendre le rôle potentiel de la signalisation calcique dans la modulation de la KF par la lumière, nous avons étudié l’effet d’un inhibiteur des canaux calciques [LaCl3] et d’un antagoniste de calmoduline [W7] sur la KF et les transcrits des 10 JrPIPs. Comparées aux feuilles témoins, les inhibiteurs calciques provoquent une réduction de la KF et de la majorité des JrPIPs étudiées à la lumière. Nos résultats confirment l’implication du complexe Ca2+ /calmoduline dans la transduction du signal lumineux responsable de la stimulation de la KF et des transcrits d’aquaporines chez le noyer. / Leaf hydraulic conductance (Kleaf) takes a significant part in plant water relations. In walnut leaves, Kleaf was stimulated by light and tightly related to accumulation of JrPIP2s aquaporin transcripts. However, the light effect on Kleaf value is not systematically related to aquaporin regulation. Here we investigated the relationship between light, Kleaf and transcript levels of aquaporin in five species (Juglans regia, Fagus sylvitica, Quercus robur, Salix alba and Populus tremula) differing by the response of their Kleaf to light. Only for walnut leaves, we showed that light-increased Kleaf value is closely related to higher stimulation of both PIP1s and PIP2s aquaporins. To further investigate the involvement of aquporins (JrPIP1s and JrPIP2) in the light Kleaf modulation, 8 new full length aquaporins have been identified in walnut leaves and their expression pattern was monitored. All the aquaporin tested was turned up to be upregulated under light condition and downregulated under darkness. Moreover, we showed that the Kleaf response to light is quality-dependant, since it was reduced of 65% in the absence of blue light. Interstingly, this Kleaf reduction was correlated with a high downregulation of almost all aquaporins tested. To give an insight into the early molecular events involved in the light-induced Kleaf regulation, a large-scale transcriptomic analysis consisting of the cDNA-AFLP procedure was carried out on walnut leaves, kept at different light conditions. We obtained a total of 12,000 transcript-derived fragments (TDFs) by cDNA-AFLP with 128 primer pairs. Reliable sequences were obtained for 187 of these TDFs, and functions were attributed to 93 TDFs through BLAST searches in GenBank databases. Most of the 93 TDFs corresponded to genes encoding proteins involved in cellular regulation (58%). Leaves exposed to light showed changes in the Ca2+-signaling pathway, the ubiquitin-proteasome pathway, vesicle trafficking process and the expression of multiple transcription factors and protein regulators. To progress in understanding of a potential role for calcium signalling in light-modulated Kleaf, Kleaf values and transcript accumulation of 10 JrPIPs were monitored in leaves perfused with either a blocker of calcium channels [LaCl3] or a CaM antagonist [W7]. Compared to control, these Ca2+ -effectors led to a significant reduction in Kleaf and transcripts accumulation of almost all JrPIPs under light conditions. Our results indicate that Ca2+ /calmodulin complex may transduce the light signal required for stimulation of Kleaf and its correlated aquaporin expression.