Introduction. The glial cell water channel aquaporin-4 (AQP4) plays an important ro le in brain oedema, astrocyte migration and neuronal excitability. Current theories of AQP4 function are based largely on experiments using AQP4 -1- mice. These mice have only been partially characterized. I therefore undertook a detailed investigation of baseline brain properties in AQP4 -1- mice. In the second part of my experiments I investigated the role of AQP4 in brain oedema in a mouse model of subarachnoid haemorrhage. Method. Gross anatomical measurements included estimates of brain and ventricle size. Neurons, astrocytes and oligodendrocytes were assessed using the neuronal nuclear marker NeuN, the astrocyte marker GFAP, and the myelin stain Luxol Fast Blue. The blood brain barrier was studied by electron microscopy and the horseradish peroxidase extravasation technique. A mouse model in which 30~1 of autologous blood was injected into the basal cisterns was used to reproduce subarachnoid haemorrhage. Brain water content, intracranial pressure and neurological score were compared in wildtype and AQP4 -/- mice. I also measured blood brain barrier permeability and the osmotic permeability of the glia lim itans, one of the routes of oedema elimination.

O nematoide de vida livre Panagrolaimus superbus é uma espécie anidrobiótica, ou seja, possui a capacidade de sobreviver ao estresse hídrico extremo adentrando no estado de anidrobiose. Durante tal estado adquiri tolerância a extremos de temperatura (~0 K a +151 °C), pressões hidrostáticas (1,2 GPa) e radiação ionizante. Entretanto, não se sabe qual a tolerância a água deuterada, molécula que possui dois átomos de deutério (isótopo estável e natural do hidrogênio) ao invés do hidrogênio, que em altas concentrações afeta negativamente sistemas biológicos. Além disso, uma vez que o processo de anidrobiose depende do movimento de moléculas de água pela membrana plasmática da célula, não se sabe se os canais responsáveis por este transporte, as aquaporinas, de espécies anidrobióticas possuem alguma particularidade na sua estrutura. Em vista disso, o objetivo deste trabalho visa investigar se em concentrações de 10%, 40% e 99,9% D2O, tanto de modo crônico quanto agudo, P. superbus tem sua viabilidade, crescimento populacional e desenvolvimento alterados. Além disso, a comparação in silico da sequência de aminoácidos (estrutura primária) entre aquaporinas de espécies anidrobióticas e não anidrobióticas, com ênfase nas sequências genéticas de P. superbus foi feita. Os resultados encontrados demonstram a alta tolerância de P. superbus a concentrações relativamente elevadas de D2O, não tendo sua viabilidade e desenvolvimento alterados em nenhum cenário, mesmo com uma troca total, homogênea e instantânea de sua matriz aquosa. Efeitos negativos foram encontrados apenas no crescimento populacional após exposição 10%, 40% e 99,9% D2O, contudo não o inviabilizaram. Ademais, não foram encontradas grandes diferenças entre as sequências primárias de aquaporinas de anidrobiotos e não anidrobiotos, sugerindo que estes canais de água não divergem em estrutura terciária entre tais grupos. Dos dois ESTs encontrados em P. superbus (números de acesso no NCBI: GW411914.1 e GW408200.1) o primeiro deles é o provável representante do gene da aquaporina na espécie, enquanto que o segundo aparenta ser um transcrito não codificante de proteínas. / The free-living nematode Panagrolaimus superbus is an anhydrobiotic species, it means that this species has the capacity to survive extreme water stress entering into the state of anhydrobiosis. During such a state, it acquires tolerance to extremes of temperature (~ 0 K to +151 °C), hydrostatic pressures (1.2 GPa) and ionizing radiation. However, the tolerance to deuterium oxide is poorly investigated. This molecule has two atoms of deuterium (natural and stable isotope of hydrogen) rather than hydrogen and in high concentrations negatively affects biological systems. Furthermore, since the process of anhydrobiosis depends on the movement of water molecules across the cell membrane, it is unclear whether the channels responsible for this transport, aquaporins, of anhydrobiotic species have some particularity in their structures. In view of this, the work aims to investigate whether P. superbus has its viability, population growth and development altered at concentrations of 10%, 40% and 99.9% D2O in chronic and acute expositions. In addition, the in silico analyses of amino acid sequence (primary structure) of aquaporins between anhydrobiotic and non-anhydrobiotic species, with emphasis at the P. superbus genetic sequences, were performed. The results demonstrated the high tolerance of P. superbus at high concentrations of D2O, their viability and development did not change in any scenario, even with a total, homogeneous and instantaneous exchange of their aqueous milieu. Negative effects were found only on population growth after exposure to 10%, 40% and 99.9% D2O, although not hindering the procedure. Furthermore, no significant differences were found between the primary aquaporin sequences of anhydrobiotic and non-anhydrobiotic species, suggesting that these water channels do not differ in tertiary structure between such groups. Two ESTs found in P. superbus, (NCBI access numbers: GW411914.1 and GW408200.1): the first likely corresponds to the aquaporin gene in the species, while the second appears to be a noncoding transcript.

Les protéines membranaires occupent en moyenne 50% de la masse des membranes cellulaires. Cependant, certaines membranes spécialisées peuvent avoir de 20 à 90% de leur masse en protéines. Dans ce cadre, l'importance de l'assemblage des protéines membranaires dans des complexes cohérents, dynamiques et fonctionnels n'est plus à démontrer.Mon projet s'inscrit dans la compréhension des forces qui mènent à l'assemblage des protéines membranaires. J'utilise pour cela le modèle de l'Aquaporine Z (AqpZ) d'Escherichia coli. En premier lieu, j'ai mis en oeuvre une approche de dynamique moléculaire gros grains avec des forces de biais adaptatifs pour étudier les relations entre orientations de deux monomères d'AqpZ. Il existe, de façon surprenante, des forces se propageant à longue distance vraisemblablement par les lipides qui biaisent les orientations relatives entre les protéines.Un deuxième axe de mon travail est l'étude des enrichissements lipidiques autour de l'AqpZ native ou mutée, à différentes distances, avec l'utilisation d'une membrane complexe rendant compte de la diversité lipidique de la membrane interne d'E.coli. Dans cette analyse, la cardiolipine est enrichie à proximité de la protéine. Enfin, j'ai construit un système contenant 125 monomères d'AqpZ dans une membranes simple ou complexe, qui représentent 50% en masse en protéines. Ce système m'a permis de questionner l'évolution spontanée d'un tel système encombré, mais aussi le devenir des forces à longue distance et des lipides enrichis à la surface de la protéine dans ce contexte. / Membrane proteins represent on average 50% of the mass of cellular membranes. However, specialized membranes can have from 20 to 90% of their mass in proteins. In this context, the importance of the assembly of membrane proteins in coherent, dynamic and functional complexes isn't to be proven anymore. The goal of my project is to understand the different forces that lead to the assembly of membrane proteins. For this aim, I am using the Aquaporin Z (AqpZ) model protein from Escherichia coli, which is studied in our laboratory. First, I use a coarsed grain molecular dynamics approach with adaptive biasing forces to study the relations between orientations of two AqpZ monomers. Surprisingly, there are forces propagating at long distance, presumably by the lipids which in turn bias the relative orientations between the proteins. The second axis of my work is the study of lipid enrichments around native or mutated AqpZ, at different distances, with the use of a complex membrane accounting for the lipid diversity of the inner membrane of E.coli. In this analysis, cardiolipin is enriched near the protein. Finally, I built a system containing 125 AqpZ monomers in a simple or complex membrane, which represents 50% protein by weight. This system allowed me to examine the spontaneous evolution of such a crowded system, but also to investigate the fate of the long distance forces and the lipid enrichments at the protein surface in this context.

Touchant près de 3 à 10 % des nouveau-nés, les hémangiomes infantiles (HI) sont des tumeurs vasculaires bénignes, les plus fréquentes chez les nourrissons. Les HI sévères sont actuellement traités par un bêtabloqueur, le propranolol, dont l’efficacité a été découverte de manière fortuite. Ainsi, son mécanisme d’action est méconnu. Le propranolol se fixe sur les récepteurs beta-adrénergiques et empêche leur activation par les catécholamines comme la noradrénaline. Nous nous sommes donc interrogés sur la relation entre le propranolol et la noradrénaline dans cette tumeur. Nous avons montré une forte expression de la noradrénaline et des enzymes de synthèse des catécholamines dans les HI, comparés aux hémangiomes congénitaux, qui diminuent lorsque la tumeur involue ou est traitée par le propranolol. Nous avons ensuite réalisé un modèle in vitro ressemblant à l’HI à partir de cellules isolées d’HI capables de synthétiser les catécholamines : les cellules endothéliales et les péricytes. Ce modèle nous permettra d’étudier l’impact de la noradrénaline et du propranolol sur ces cellules. Parallèlement, notre équipe a réalisé un modèle in vivo qui a permis de mettre en évidence le rôle clé de la protéine quaporine-1 (AQP1) dans la réponse antitumorale du propranolol. Nous avons également étudié l’expression de l’AQP1 dans les HI et les hémangiomes congénitaux et découvert un type cellulaire adventitiel exprimant l’AQP1 dans les HI, le télocyte. Au total, notre travail sur l’HI a mis en évidence d’une part une possibilité de production endogène accrue de noradrénaline, probablement antagonisée avec succès par le propranolol, et la découverte de télocytes AQP1+ qui pourraient avoir un rôle dans la spécificité de la réponse des HI au propranolol. / Affecting nearly 3 to 10 % of newborns, infantile hemangiomas (HI) are the most common benign vascular tumors in infants. Severe HIs are currently treated with a beta-blocker, propranolol, whose efficacy was discovered by serendipidity. Propranolol binds to beta-adrenergic receptors and prevents their activation by catecholamines such as noradrenaline. We therefore wondered about the relationship between propranolol and noradrenaline in this tumor. We showed a strong expression of noradrenaline and catecholamine synthesis enzymes in HI, compared to congenital hemangiomas, which decrease when the tumor involutes or is treated with propranolol. We then realize an in vitro model resembling HI from cells isolated from HI capable of synthesizing catecholamines: endothelial cells and pericytes. This model will permit to study the impact of noradrenaline and propranolol on these cells. At the same time, our team created an in vivo model that highlighted the key role of aquaporin-1 protein (AQP1) in the antitumor response to propranolol. We have also studied the expression of AQP1 in HI and congenital hemangiomas, and discovered an adventitious cell type expressing AQP1 in HI, the telocyte. Altogether, our work on HI has revealed firstly the possibility of increased endogenous production of norepinephrine, probably successfully antagonized by propranolol, and secondly the presence of AQP1 + cells which could have a central role central in the specificity of HI response to propranolol.

<p>While constituting a well functioning interface with the aqueous environment in utero, the skin offers a poor barrier after very preterm birth. As a result, transepidermal water loss (TEWL) is high, a fact which has important clinical consequences in these infants. To investigate the transport of water through perinatal skin and the potential role of aquaporin (AQP), a water channel protein, in this process, we determined TEWL in a group of extremely preterm infants, and in an experimental rat model we analyzed the expression and distribution of AQP in perinatal skin in relation to TEWL, skin surface hydration and water content. The effects of antenatal corticosteroids (ANS) and of restricted intake of fluids and nutrients on barrier characteristics of the perinatal skin and its AQP expression were also studied.</p><p>In infants born at 24 and 25 weeks of gestation TEWL was very high in the first days after birth and decreased with increasing postnatal age. At a postnatal age of 4 weeks, TEWL was still twice as high as previously reported in infants born at a gestational age of 25-27 weeks and four times higher than in infants born at term. In the rat model, immunohistochemical analysis revealed that AQP1 and AQP3 are abundantly expressed in the skin. AQP1 was expressed exclusively in dermal capillaries and AQP3 in basal layers of the epidermis. AQP1 and AQP3 mRNA as assessed by semiquantitative RT-PCR was higher in fetal than in adult skin. As in infants, TEWL and skin surface hydration were inversely related to gestational age in the rat. In preterm rat pups exposed to ANS, TEWL and skin surface hydration were lower than in unexposed controls, and AQP3 expression was selectively induced by ANS. In term newborn rat pups, restriction of fluid and nutrient intake resulted in a higher skin water content and higher TEWL early after birth, while at an age of 7 days TEWL was lower in fasting rat pups than in controls, although skin water content was still higher.</p><p>To conclude, TEWL is very high in extremely preterm infants early after birth and then decreases at a slower rate than previously reported for a group of slightly more mature infants. </p><p>This is the first time that the distribution and gene expression of AQP1 and AQP3 have been demonstrated in perinatal skin. The localization and expression of AQP in the skin might indicate that these water channels are involved in the regulation of skin hydration and transepidermal water transport in the fetus and newborn infant.</p>

While constituting a well functioning interface with the aqueous environment in utero, the skin offers a poor barrier after very preterm birth. As a result, transepidermal water loss (TEWL) is high, a fact which has important clinical consequences in these infants. To investigate the transport of water through perinatal skin and the potential role of aquaporin (AQP), a water channel protein, in this process, we determined TEWL in a group of extremely preterm infants, and in an experimental rat model we analyzed the expression and distribution of AQP in perinatal skin in relation to TEWL, skin surface hydration and water content. The effects of antenatal corticosteroids (ANS) and of restricted intake of fluids and nutrients on barrier characteristics of the perinatal skin and its AQP expression were also studied. In infants born at 24 and 25 weeks of gestation TEWL was very high in the first days after birth and decreased with increasing postnatal age. At a postnatal age of 4 weeks, TEWL was still twice as high as previously reported in infants born at a gestational age of 25-27 weeks and four times higher than in infants born at term. In the rat model, immunohistochemical analysis revealed that AQP1 and AQP3 are abundantly expressed in the skin. AQP1 was expressed exclusively in dermal capillaries and AQP3 in basal layers of the epidermis. AQP1 and AQP3 mRNA as assessed by semiquantitative RT-PCR was higher in fetal than in adult skin. As in infants, TEWL and skin surface hydration were inversely related to gestational age in the rat. In preterm rat pups exposed to ANS, TEWL and skin surface hydration were lower than in unexposed controls, and AQP3 expression was selectively induced by ANS. In term newborn rat pups, restriction of fluid and nutrient intake resulted in a higher skin water content and higher TEWL early after birth, while at an age of 7 days TEWL was lower in fasting rat pups than in controls, although skin water content was still higher. To conclude, TEWL is very high in extremely preterm infants early after birth and then decreases at a slower rate than previously reported for a group of slightly more mature infants. This is the first time that the distribution and gene expression of AQP1 and AQP3 have been demonstrated in perinatal skin. The localization and expression of AQP in the skin might indicate that these water channels are involved in the regulation of skin hydration and transepidermal water transport in the fetus and newborn infant.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009. / Härtill 4 uppsatser.

Aquaporin proteins significantly increase water permeability across tissues and cell membranes. Ocular tissues, including the trabecular meshwork (TM) and retinal pigment epithelium (RPE), are especially reliant on aquaporin mediated water movement for ocular homeostasis. Even though bulk fluid movement is paracellular through the TM and transcellular through the RPE, both express aquaporin-1 (AQP1). The role and regulation of AQP1 as it relates to homeostasis in different ocular tissues is not well understood. I hypothesized that ocular tissues respond to external mechanical and molecular cues by altering AQP1 expression and function in order to regulate ocular fluid movement and maintain homeostasis.To test how AQP1 function is altered in response to external cues in order to maintain tissue-specific homeostasis, I addressed the following two aims. The first aim was directed at determining how mechanical strain, an external stimulus that routinely affects TM function, influences AQP1 expression and TM homeostasis. Primary cultures of human TM were subjected to static and cyclic stretch and then analyzed for changes in AQP1 expression by western blot and cell damage by activity of lactate dehydrogense (LDH) in conditioned media. The results show AQP1 expression and LDH release significantly increased with static stretch. Analysis of LDH release with respect to AQP1 expression revealed an inverse linear relationship (r² = 0.7780).The second aim was directed at characterizing signaling mechanisms responsible for regulating fluid transport in RPE, previously shown to be dependent upon AQP1. I treated primary cultures of human RPE with either atrial natriuretic peptide (ANP) or 8-bromo-cyclic guanosine monophosphate (8-Br-cGMP) in the presence or absence of Anantin (ANP-receptor inhibitor) or H-8 (Protein Kinase G inhibitor). The results show that ANP and 8-Br-cGMP significantly increased apical to basal net fluid movement (p < 0.05, n = 3). Inhibition of these effects was successful with Anantin treatment but not with application of H-8.The data presented demonstrate a novel role of protection for AQP1 in TM, and also expand upon cGMP dependent regulation of RPE fluid transport. The combined studies indicate tissue specific AQP1 regulation may offer new avenues to target water movement in treatment of ocular pathologies.

Pacific hagfish (Eptatretus stoutii) slime mucin vesicles are released by holocrine secretion with membranes that remain intact until the vesicle contacts seawater and ruptures. This thesis is an investigation of the mechanisms that drive mucin vesicle rupture for mucin release. Using isolated mucin vesicles collected from the slime glands of the hagfish, I tested the effects of a variety of solutions and drugs on vesicle rupture. I found that there are two categories of mucin vesicle that differ in their sensitivity to calcium ions, and that calcium-dependent vesicle rupture was inhibited with anion channel inhibitors. I also found that vesicle swelling rate was reduced by the aquaporin inhibitor mercuric chloride. Together, these data suggest that mucin vesicle rupture is partially dependent on the movement of chloride ions from seawater through calcium-activated anion channels and the rapid influx of water through aquaporin-like proteins in the vesicle membrane. / NSERC Discovery Grant, NSERC CGSM scholarship, Canada Foundation for Innovation, Ontario Ministry of Research and Innovation

Vaginal atrophy symptoms such as dryness, irritation, and itching, are common after menopause. Vaginal estrogen therapy is the most effective treatment but not appropriate for all women. Women with estrogen-responsive breast cancer treated with aromatase inhibitor (AI) treatment, suppressing estrogen levels, often suffer from more pronounced vaginal atrophy symptoms. However, vaginal estrogen treatment is not recommended, leaving them without effective treatment options. The aim of this thesis was to study the effect of long-term anti-estrogen therapy on circulating estrogen levels and biochemical factors in vaginal mucosa in relation to morphological changes and clinical signs of vaginal atrophy. Circulating estrogen levels were analyzed by use of mass spectrometry and radioimmunoassay. Immunohistochemistry was used to study vaginal proliferation and steroid hormone receptors in vaginal mucosa. Vaginal gene expression was studied by use of microarray technology and bioinformatic tools, and validated by use of quantitative real-time PCR and immunohistochemistry. An estrogenic regulation of aquaporins and a possible role in vaginal dryness was investigated in vaginal mucosa and in Vk2E6E7 cells. Aromatase inhibitor-treated women had higher than expected estradiol and estrone levels but still significantly lower than other postmenopausal women. Aromatase was detected in vaginal tissue, the slightly stronger staining in vaginal mucosa from AI-treated women, suggest a local inhibition of vaginal aromatase in addition to the systemic suppression. Vaginal mucosa from AI-treated women had weak progesterone receptor, and strong androgen receptor staining intensity. Low estrogen levels lead to low expression of genes involved in cell adhesion, proliferation, and differentiation as well as weak aquaporin 3 protein immunostaining. The higher than expected estrogen levels in AI-treated women suggest that estrogen levels might previously have been underestimated. Systemic estrogen suppression by treatment with AIs, and possibly also by local inhibition of vaginal aromatase, results in reduced cell adhesion, proliferation, differentiation, and weak aquaporin 3 protein staining. Low proliferation and poor differentiation leads to fewer and less differentiated superficial cells affecting epithelial function and possibly also causing vaginal symptoms. Aquaporin 3 with a possible role in vaginal dryness, cell proliferation, and differentiation should be further explored for the development of non-hormonal treatment options for vaginal symptoms.