To date, the structural significance of the skin of fish larvae in osmoregulation has received little attention and the evidence for salt secretion by cutaneous chloride cells is based largely on morphological observations. Thus, in the present study, a combination of microscopical and electrophysiological techniques were utilised to determine the role of the skin of early post-hatch turbot larvae in osmoregulation. A number of specialised structural features were revealed in the skin of the turbot larva with electron microscopy which would appear to provide some protection against the high osmotic and ionic gradients tending to dehydrate and salt load the body tissue and fluids. In the heterogenous epidermis, consisting of both transporting and non-transporting cells, only the shallow junctions between chloride cells and accessory cells were believed to permit ion influx and/or water loss via the paracellular pathway; the extensive junctions between adjacent pavement cells and pavement cells and neighbouring chloride cells effectively occluding the passage of ions and water through the extracellular space. Chloride cells were revealed in the skin and prebranchial epithelium of the turbot larva from hatching, but accessory cells, and thus "leaky" junctions, were only observed in association with the closely juxtaposed chloride cells in the prebranchial epithelium which, although densely packed, represented just a small area of the otherwise "tight" skin. Water and ion permeation through the external plasma membrane of the superficial pavement cells might further be impeded by the extracellular glycocalyx coat observed in TEM. In addition, the large numbers of mucous cells, which were a characteristic feature of the skin of the turbot larva, may produce a protective mucus coating of low permeability. The apparent "tightness" of the skin was reflected by the measurements of diffusional water permeability (Pdiff) from early stage larvae which suggested that the larvae of turbot were relative impermeable to water compared with the gills of adults. Nevertheless, the rates of water turnover were still sufficiently high that a net osmotic loss of water must be replaced by water uptake through drinking. The observation that the Pdiff of early stage turbot larvae increased with development substantiates earlier supposition that the drinking rates of larvae are a direct function of the permeability of the larva to water. A study of the chronology of chloride cell development utilising specific fluorochromes and electron microscopy revealed that the prebranchial chloride cells, which closely resembled the chloride cells described in the branchial epithelium of juveniles, were both numerous and well equipped to participate in active salt extrusion in turbot larvae even at hatching. In view of the early hypertrophy and proliferation of the prebranchial cells, their rapid increase in Na+,K+-ATPase binding sites, and the subsequent degeneration of the cutaneous chloride cells observed with larval development, it was concluded that the prebranchial chloride cells are the primary site for active ion excretion shortly after yolksac absorption. The potential importance of the cutaneous chloride cells in salt extrusion was also considered, but in view of the apparent lack of accessory cell associations and the small number of apical pits observed in SEM and TEM sections, questions were raised as to the significance of these cells in ion excretion. Measurements of the trans epithelial electrical potential (TEP) from early stage turbot using intracellular micro electrode techniques confirmed that the larvae of turbot maintain ionic gradients by the active extrusion of ions that enter into the body cavity down electrical or chemical gradients. The TEP was found to be largely the result of a Na+ diffusion potential with an additive electrogenic potential due to CI- transport, which was somehow functionally connected to Na+,K+ -ATPase. Furthermore, the concentration of Na+ in the external bathing medium was found to have a direct regulatory influence on the rate of CI- secretion, suggesting that the active secretion of Cl across the skin must be coupled to Na+. These conclusions are consistent with the current theories proposed for salt extrusion by the chloride cells in the adult teleost.

Development is a dynamic process characterized by critical periods in which organ systems are sensitive to changes in the surrounding environment. In the current study, critical windows of embryonic growth and kidney development were assessed in the embryonic chicken. All‐trans retinoic acid (tRA) influences not only organogenesis and cell proliferation, but also targets metanephric kidney nephrogenesis. Embryonic chickens were given a single injection of tRA on embryonic day 8. tRA decreased embryo, kidney, and heart mass from day 16 to day 18. However, mass specific kidney and heart masses showed no differences. Whole blood, plasma, and allantoic fluid osmolality were altered in tRA treated embryos from day 16 to day 18. In addition, hematocrit, red blood cell count, and hemoglobin concentration were altered in tRA treated embryos. The results suggest that although nephrogenesis was not affected by tRA, the developing osmoregulatory system was altered in tRA treated embryos.

Les épinoches à 3 épines (Gasterosteus aculeatus), pêchées dans différentes régions de Camargue, ont été étudiées et soumises à différentes salinités. La pression osmotique du sang de poissons gardés en eau douce (FW : 5 ppm), en eau saumâtre (BW : 15 ppm) et en eau de mer (SW : 30ppm) était différente (147, 457 et 911 mOsmkg-1, respectivement). Cependant, les résultats de consommation d’oxygène montrent des valeurs similaires pour les poissons capturés dans la l’étang du Marteau (SW), le canal du Versadou (FW) et l’étang mésohalin (eau saumâtre) du Vaccarès (BW). Ainsi, ces épinoches peuvent rapidement s'acclimater à différentes salinités et se déplacer librement entre ces différents habitats avec des dépenses énergétiques limitées. Pour les épinoches du Vaccarès, le taux de consommation d'O2 diminue immédiatement après le transfert en FW jusqu'à 1 heure après transfert. L'activité branchiale Na+/ K+-ATPase (NKA) des épinoches collectées dans l’étang du Marteau et directement transférées en FW a révélé une augmentation significative 24h et 48h après le transfert (28% et 40%, respectivement). Cependant, l'activité NKA des branchies est réduite de manière significative de 36%, 24h suivant le transfert en SW.La morphologie corporelle des épinoches a également été étudiée mais des variations morphologiques limitées ont pu être observées. Le nombre moyen de plaques latérales est de 4,45 ± 0,31. Cependant, une différence morphologique significative est observée pour les branchiospines.Le marquage NKA a révélé des ionocytes avec différentes morphologies. Ces cellules sont également deux fois plus nombreuses chez les poissons FW que chez les poissons SW. Deux types d'ionocytes ont été identifiés chez les poissons FW: les cellules avec un dôme apical et les ionocytes avec une structure en nid d'abeille. Ces cellules sont bien des ionocytes du fait de leurs structures internes (présence de nombreuses mitochondries et d’un système tubulo-vésiculaire dense avec une membrane basale profondément invaginée). Chez les poissons acclimatés en SW, seuls des ionocytes avec une grande crypte apicale ont été observés le long des filaments branchiaux à la base des lamelles branchiales. Aucun ionocyte n'a pu être observé le long des lamelles branchiales. L'expression relative de l'ARNm de NKA α1 est apparue plus élevée dans les branchies des poissons acclimatés en SW que chez les poissons en BW. L’expression de la NKA α1b est significativement surexprimée dans les branchies des poissons SW par rapport aux poissons BW et FW. Cependant, pour la NKAα1a, les expressions relatives dans les branchies ne sont pas différentes selon la salinité. Pour l'expression du CFTR et de la V-ATPase, nous n'avons trouvé aucune différence significative. Par contre, NKCC1 est significativement plus élevé chez les épinoches acclimatées en SW.Les analyses histologiques du rein et le marquage de la NKA au niveau des tubules proximaux du rein n'ont révélé aucune différence pour les poissons en FW et en SW. Pour l'intestin, un fort marquage a été observé le long des membranes basolatérales des entérocytes. Cependant, là encore, nous n'avons observé aucune différence due aux conditions de salinité. Ces résultats suggèrent un haut niveau de plasticité pour ces organes.Globalement, ces résultats indiquent que les épinoches de Camargue sont homogènes au niveau morphologique et physiologique et possèdent de fortes capacités euryhalines. Cependant, des différences ont été identifiées au niveau des branchiospines entre les poissons capturés dans les habitats FW et les habitats BW/SW, probablement en raison de régimes alimentaires différents. Par conséquent, ces populations peuvent ne pas se mélanger même si les différents environnements aquatiques de Camargue sont interconnectés. / Threespined sticklebacks (Gasterosteus aculeatus) fished from different areas of the Camargue were studied and challenged to various salinity conditions. Blood osmotic pressures for fish kept in FW (5‰), BW (15‰) and SW (30‰) were different (147, 457 and 911 mOsm kg-1, respectively). However, oxygen consumption results revealed similar values for fish collected from the seawater Marteau lagoon (SW), the freshwater Versadou canal (FW) and the mesohaline/brackish water Vaccarès lagoon (BW). Therefore, sticklebacks of the Camargue can rapidly acclimate to different salinity conditions and move freely among these different habitats with limited energy expenditure. For sticklebacks from the Vaccarès lagoon, a significant reduction in O2 consumption rate occurred immediately after transfer to FW and up to 1h after transfer. The branchial Na+/K+-ATPase (NKA) activity of sticklebacks collected from the SW Marteau lagoon directly transferred from SW to FW, revealed a significant increase 24 h and 48 h after transfer (28% and 40%, respectively). However, gill NKA activity was significantly decreased by 36% within 24 h following immediate transfer to SW.Body morphology of sticklebacks was also studied but limited morphological variations could be observed for fish collected from different habitats. The average lateral plate number is 4.45±0.31. However, a significant morphological difference is observed for gill rakers.NKA labeling on histological sections revealed different ionocyte morphologies. In the gills of FW sticklebacks, ionocytes were observed along the gill filaments as well as the gill lamellae. Ionocytes were only identified along the gill filaments for SW fish. For SW fish, the ionocytes were round in shape and slightly smaller than the ionocytes of the FW fish. The ionocytes of the FW fish along the gill filaments were elongated but ionocytes along the gill lamellae were flattened and wider. Ionocytes were also twice more numerous for FW fish compared to SW fish.Also, two types of ionocyte were identified in FW fish: cells with an apical dome shape and ionocytes with a honeycomb-like structure. All these cell types were confirmed as ionocytes due to their internal structures filled with numerous mitochondria and a dense tubulo-vesicular system, with deeply invaginated basal membrane. In SW-acclimated fish, only ionocytes with a large apical crypt were observed along the gill filaments at the base of the gill lamellae. No ionocytes could be observed along the gill lamellae.Relative NKA α1 mRNA expression was higher in the gills of SW acclimated fish compared to BW fish and NKA α1b was significantly overexpressed in the gills of SW acclimated fish compared to the FW and BW fish. However, for NKAα1a, mRNA relative gene expressions in the gills are not different due to salinity. Whereas, for CFTR and V-ATPase expression, we did not find any significant difference. On the other hand, NKCC1 was significantly higher in SW acclimated sticklebacks.For sticklebacks kept in FW and for those acclimated to SW, analyses of histological sections from the kidney and NKA immunolabeling of the kidney proximal tubules revealed no difference. For the intestine, a strong immunofluorescence was observed along the basolateral membranes of the enterocytes for both the anterior and posterior intestine of FW and SW-acclimated sticklebacks but, again, we did not observe any difference due to salinity conditions. These results suggest a high level of plasticity for these organs.Altogether, these results indicate that sticklebacks of the Camargue area are morphologically and physiologically homogenous and have strong euryhaline capacities. Some differences were identified however for the gill rakers between fish collected from the FW and mesohaline / euryhaline habitats most probably due to different feeding regimes. Therefore, these fish populations may not mix even if the different Camargue aquatic environments are interconnected.

Die Aufrechterhaltung des Zellvolumens unter variablen osmotischen Bedingungen stellt für nahezu alle tierischen Zellen eine essenzielle Aufgabe dar. Um regulatorische Volumenanpassungen vorzunehmen besitzen sie daher effektive Mechanismen, mit deren Hilfe der zelluläre Gehalt an organischen und anorganischen Osmolyten erhöht (= regulatorische Volumenzunahme; RVI) oder gesenkt (= regulatorische Volumenabnahme; RVD) werden kann. Trotz langjähriger Forschung auf diesem Gebiet konnten die hieran beteiligten Transportwege für Osmolyte bisher nur unvollständig aufgeklärt werden. Insbesondere bei T-Lymphozyten sind wichtige Zellfunktionen wie die Proliferation, Migration und die T-Zell-Aktivierung eng mit volumenregulatorischen Mechanismen verbunden. Bei all diesen Prozessen sind u. a. unterschiedliche Kaliumkanäle beteiligt, die insbesondere für die pharmakologische Manipulation von Immunsystemprozessen von wissenschaftlichem Interesse sind. Bisherige Modelle der hypotonen Volumenregulation von T-Lymphozyten berücksichtigen lediglich den spannungsabhängigen KV1.3 sowie den Ca2+-aktivierten IKCa1-Kanal, die zur Klasse der 6TM/P-K+-Kanäle gehören. Im ersten Teil der vorliegenden Arbeit wurde eine potentielle Rolle von kürzlich entdeckten Zwei-Poren Domänen Kaliumkanälen (K2P) am RVD von murinen und humanen primären CD4+-T-Lymphozyten untersucht. In einem kombinierten genetischen und pharmakologischen Ansatz mittels knockout-Tiermodellen und dem Einsatz kanalspezifischer Inhibitoren konnte mithilfe zellvolumetrischer Analysen gezeigt werden, dass die K2P-Vertreter TASK1, TASK2, TASK3 und TRESK maßgeblich am schwellungsaktivierten Efflux von K+ beteiligt sind. Beurteilt an den Ergebnissen dieser Untersuchung sind der spannungsabhängige TASK2- und der Ca2+-aktivierte TRESK-Kanal für die hypotone Volumenregulation in T-Zellen deutlich bedeutender als TASK1 und TASK3. Der Beitrag der Kanäle TASK2 und TRESK am RVD-Prozess war über dies vergleichbar mit dessen des bisher bekannten KV1.3-Kanals. In dieser Arbeit wurde damit erstmals eine Beteiligung der K2P-Kanäle am RVD muriner und humaner CD4+-Lymphozyten identifiziert. Aufgrund der engen Verbindung zwischen T-Zell-Funktion und der Volumenregulation können Zwei-Poren Domänen K+-Kanäle damit in den engeren Kreis potentieller immunmodulierende Angriffspunkte aufgefasst werden. Im zweiten und umfangreicheren Teil dieser Arbeit wurden darüber hinaus die schwellungsaktivierten Transportwege für organische Osmolyte (small organic osmolytes; SOOs) untersucht. SOOs stellen chemisch inerte Verbindungen dar, zu denen vor allem Polyole (Sorbitol, myo-Inositol), Methylamine (Betain, α-Glycerophosphocholin) sowie Aminosäuren (α- bzw. β-Alanin und Prolin) und deren Derivate (Taurin) zählen. Da SOOs weder die zelluläre Struktur noch die Funktion von Makromolekülen beeinträchtigen, sind sie wichtige Instrumente der Volumenregulation, die sich in hohen Konzentrationen im Zytosol nahezu aller Zellen wiederfinden. Werden tierische Zellen mit hypotonen Bedingungen konfrontiert, dann ist bei nahezu allen Zellen die Freisetzung organischer Osmolyte zu beobachten, wodurch die zelluläre Osmolarität unabhängig von Elektrolyten angepasst werden kann. Trotz der wichtigen Funktion der SOOs in der Osmoregulation tierischer Zellen konnte die molekulare Identität beteiligter Effluxwege (Kanäle bzw. Transporter) bisher nicht aufgeklärt werden. Ungeachtet der molekularen Identität der SOO-Effluxwege war es aus zahlreichen biotechnologischen Anwendungen zu Beginn dieser Arbeit bekannt, dass die schwellungsaktivierten Transportwege für organische Osmolyte eine größenselektive Permeabilität für eine Reihe monomerer Zucker und verwandter Verbindungen aufweisen. Um diese Größenselektivität näher zu charakterisieren, wurde im ersten Schritt die schwellungsaktivierte Membranpermeabilität für eine Reihe strukturell homogener Polyethylenglykole unterschiedlicher Polymerlänge (PEG200–1500; hydrodynamische Radien zwischen ~0,5-1,5 nm) unter iso- und hypotonen Bedingungen in Jurkat-Lymphozyten untersucht. Unter milden hypotonen Bedingungen (200 mOsm) war die Plasmamembran der untersuchten Lymphozyten für PEG300-1500 undurchlässig, was aus der Fähigkeit der Zellen zur hypotonen Volumenregulation geschlossen werden konnte. Darüber hinaus wurde RVD in stark hypotonen Lösungen (100 mOsm) mit PEG600-1500 beobachtet, während PEG300-400 unter vergleichbaren osmotischen Bedingungen die Volumenregulation der Zellen inhibierten. Dieses Ergebnis deutet darauf hin, dass starkes hypotones Zellschwellen der Lymphozyten zur Permeabilisierung der Plasmamembran für PEG300-400, nicht jedoch für PEG600-1500, führt. Anhand der hydrodynamischen Radien Rh der verwendeten PEGs konnte ein cutoff-Radius von ~0,74 nm für schwellungsaktivierte Transportwege organischer Osmolyte bestimmt werden. Da diese schwellungsaktivierten Transportwege vielfältig für Zellbeladungstechniken verwendet werden, könnte dieses Ergebnis für zahlreiche biotechnologische und biomedizinische Anwendungen von Interesse sein. Im zweiten Schritt wurde der Versuch unternommen, potentielle Transportwege für organische Osmolyte im RVD-Prozess molekular zu identifizieren. Da es grundlegend ungeklärt war, wie viele unterschiedliche Transporter bzw. Kanäle am Efflux der zahlreichen organischen Osmolyte beteiligt sind, erfolgte zunächst die vergleichende Analyse des schwellungsaktivierten Membrantransports strukturell verschiedener SOOs einschließlich der Aminosulfonsäure Taurin und des Polyols myo-Inositol. Hierbei wurde erstmals gezeigt, dass die schwellungsaktivierten Transportwege für Taurin und myo-Inositol deutlich unterschiedliche Aktivitätsprofile aufweisen. Während der Taurintransport bereits unter milden hypotonen Bedingungen, d.h. nach einer geringen Absenkung der Osmolalität von 300 auf ~230 mOsm, aktiviert wurde, erfolgte die Aktivierung der Membranpermeabilität für myo-Inositol bei einer viel niedrigeren Osmolalität von ~150 mOsm. Darüber hinaus wiesen die beiden Transportwege unter vergleichbarem hypotonen Stress von 100 mOsm deutlich unterschiedliche Aktivitätsdauern auf (Transport von Taurin ~95 min und myo-Inositol ~40 min). Somit deuteten diese Ergebnisse erstmals auf substrat-spezifische Transportwege für SOOs hin, die voneinander stark abweichende osmotische Aktivierungsprofile besitzen. Als aussichtsreiche Kandidaten für diese Transportwege wurden zwei Mitglieder der Gruppe der Solute Carrier (SLC) untersucht, die klare Übereinstimmungen mit den gesuchten Transportern für SOOs aufweisen. Daher wurde im Weiteren eine RVD-Beteiligung dieser Transportergruppe mit einer Kombination aus molekularbiologischer und konventioneller bzw. hochaufgelöster mikroskopischen Techniken überprüft. Die semiqantitativen RT-PCR-Ergebnisse dieser Arbeit zeigen dabei, dass die Gentranskription der potentiellen SOO-Transporter SLC5A3 und SLC6A6 in den untersuchten Zelllinien Jurkat, HEK wie auch HepG2-Zellen durch hypotone Bedingungen deutlich verstärkt wird. Hierbei nimmt der zelluläre mRNA-Gehalt der Gene SLC5A3 zwischen 20-60% und SLC6A6 um 30-100% innerhalb von 10-20 min zu, was auf eine potentielle RVD-Beteiligung von SLC-Transportern hindeutet. Ausgehend von diesem Ergebnis wurde daraufhin die zelluläre Lokalisation des SLC5A3-Transporters unter isotonen und hypotonen Bedingungen mikroskopisch untersucht. Wie anhand der konfokalen lasermikroskopischen Untersuchung zu erkennen ist, findet unter hypotoner Stimulation eine zelluläre Umverteilung des mit EGFP fluoreszenzmarkierten Proteins SLC5A3 statt. Innerhalb von 10 min wird der Transporter dabei von intrazellulären Regionen in Richtung Plasmamembran verlagert. Darüber hinaus konnte mit Hilfe der hochauflösenden Mikroskopie-Technik dSTORM gezeigt werden, dass der Transporter SLC5A3 unter hypotoner Stimulation verstärkt mit der Plasmamembran assoziiert vorliegt. Diese verstärkte Membranassoziation des SLC5A3-Proteins deutet damit auf einen schwellungsinduzierten exozytotischen Einbau des Transporters hin. Die Ergebnisse dieser Arbeit zeigen damit erstmals, dass SLC-Transporter wie SLC5A3, SLC6A6 und vermutlich andere Vertreter der SLC-Superfamilie potentiell am Mechanismus der hypotonen Volumenregulation beteiligt sind. Da SLC-Transporter als wichtige Transportsysteme für Therapeutika angesehen werden und die Mechanismen der Volumenregulation bereits in zahlreichen biotechnologischen Anwendungen implementiert sind, könnte der hier aufgedeckte Zusammenhang einen Erkenntnisgewinn für zahlreiche biomedizinische Forschungsgebiete darstellen. / Cell volume homeostasis is critically important for the functional and structural integrity of mammalian cells. To counteract osmotically induced volume perturbations, cells possess efficient mechanisms that control the intracellular osmolyte composition. The volume regulatory mechanisms operating under hyper- and hypotonic conditions are known, respectively, as regulatory volume increase (RVI) and decrease (RVD). During both, RVI and RVD, cells adjust the cellular content of inorganic ions (most notably Na+, K+ and Cl-) and organic solutes in order to gain or lose osmotically obligated water. These mechanisms counteract osmotic cell damage and enable the adaptation of cells to a wide range of extracellular osmolarities. Despite decades of research in this field, many aspects of the mechanisms underlying RVD and RVI remain poorly understood. In case of T lymphocytes, various cellular functions, including proliferation, migration and T cell activation are closely associated with the cell volume regulatory machinery. Among other mechanisms, all these processes are tightly linked by a network of potassium channels. The identification of this network is of great biomedical interest as it provides a key to pharmacological manipulation of the immune system. Current models of hypotonic volume regulation (RVD) in T-lymphocytes consider primarily the voltage-gated KV1.3 and the calcium-activated IKCa1 channel. The first part of this thesis explores the potential role of two-pore domain (K2P) potassium channels in RVD in murine and human primary CD4+-T lymphocytes. Using a combined genetic and pharmacological approach, time-resolved cell volume analysis revealed an important role of the K2P channels TASK1, TASK2, TASK3 and TRESK in swelling activated K+ efflux from hypotonically swollen T cells. Based on the analysis carried out here, the voltage-gated TASK2 as well as the calcium-activated TRESK channel were found as the most important K2P channels involved in the RVD of both naïve and stimulated T cells. The importance of TASK2 and TRESK in the RVD process was comparable to that of KV1.3. In summary, the data provide first evidence that hypotonic volume regulation of murine and human CD4+-T lymphocytes relies on K2P channels. With respect to the close relationship of T-cell function and volume regulatory mechanisms K2P channels may thus be considered as potential targets for immunomodulation. In the second and major part of this thesis, the swelling-activated transport pathways for small organic osmolytes (SOOs) were investigated. Nearly all eukaryotic cells possess a considerable reservoir of SOOs, such as polyols (e.g. sorbitol, myo-inositol), methylamines (e.g. betaine, α-glycerophosphoryl choline) and small amino acids (e.g. α-/β- alanine, proline and the derivate taurine), which are synthesized within the cells or accumulated from the extracellular medium. Since SOOs do not interfere with the integrity of macromolecules and the membrane potential, cells tolerate great cytosolic fluctuations of these solutes without negative effects on cellular structure or function. Due to these properties, small organic osmolytes are important tools for cell volume regulatory mechanisms, by which the intracellular osmolarity can be adjusted independently of electrolytes. Although the importance of SOOs for hypotonic volume regulation has been known for long time, the molecular identity of participating membrane efflux pathways is far from being clear. Regardless of the involved transporters, swelling-activated pathways have been reported to exhibit a size selective permeability for a wide range of sugars and related compounds. To gain a deeper insight into this issue, in a first step the impact of the molecular size on the permeation of low-molecular-weight polyethylene glycols (PEG200–1500) through the plasma membrane of Jurkat cells under iso- and hypotonic conditions was analyzed. Upon moderate swelling in slightly hypotonic solutions (200 mOsm), the lymphocyte membrane was found to remain impermeable to PEG300–1500, which allowed the cells to accomplish regulatory volume decrease. RVD also occurred in strongly hypotonic solutions (100 mOsm) of PEG600–1500, whereas 100 mOsm solutions of PEG300–400 inhibited RVD. These findings suggest that extensive hypotonic swelling rendered the cell membrane highly permeable to PEG300–400, but not to PEG600–1500. Using the values of hydrodynamic radii Rh for PEGs, the observed size-selectivity of membrane permeation yielded an estimate of ∼0.74 nm for the cut-off radius of the swelling-activated pathway for organic osmolytes. This result may be of interest for many biotechnological and biomedical applications, where swelling-activated SOO-pathways are widely used for cell-loading techniques. As a second step, an attempt was made to elucidate the molecular identity of transporters for organic osmolytes potentially involved in RVD. Since it was not clear whether RVD-related efflux of SOOs is mediated by one common or several distinct transporter(s), at first, the plasma membrane permeability profiles for two structurally dissimilar SOOs, including the amino sulfonic acid taurine and the polyol myo-inositol were analyzed. The results of the time resolved volumetric measurements clearly showed that the membrane permeability to taurine was activated upon moderate cell swelling (by ~15%) in mildly hypotonic solutions (~230 mOsm). In sharp contrast, the membrane permeability to myo-inositol was activated after a much larger swelling (~50%) in strongly hypotonic media (<150 mOsm). Moreover, the swelling-activated permittivity to taurine during RVD in 100 mOsm medium persisted for about twice as long as that for myo-inositol (taurine ~95 min, myo-inositol ~40 min). These findings clearly showed that, taurine and myo-inositol utilized separate, apparently substrate-specific pathways, which were activated at different hypotonic thresholds. Since many members of SLC-family proteins (Solute Carrier) are known for their substrate selectivity and also for their contribution to osmoregulatory mechanisms a participation of SLCs was investigated in the context of RVD. To this end, a combination of molecular biological (semiquantitative RT-PCR) and fluorescence microscopy techniques (confocal and super-resolution microscopy) was used. The semiquantitative RT-PCR data showed a transcriptional upregulation for the SLC proteins SLC5A3 (myo-inositol transporter; SMIT) and SLC6A6 (taurine transporter TauT) in hypotonically stressed Jurkat lymphocytes, HEK293, and HepG2 cells. In all three human cell lines strongly hypotonic solutions (100 mOsm) increased the mRNA level of the genes SLC5A3 and SLC6A6 between 20-60% and 30-100%, respectively, suggesting a potential participation of SLC transporters in RVD. In addition, confocal microscopy images clearly showed the intracellular displacement of EGFP-tagged SLC5A3 expressed in HEK293 cells following strongly hypotonic stress (100 mOsm). Within 10 min the fluorescence of EGFP was shifted from intracellular regions towards the plasma membrane. Furthermore, super-resolution microscopy by means of dSTORM revealed a considerably increased membrane association of SLC5A3 in strongly hypotonic stressed (100 mOsm) HEK293 and Jurkat cells. This finding suggests that SLC5A3 is integrated into the plasma membrane by swelling-induced exocytosis. Taken together, the results of this investigation provided first evidence that transporters such as SLC5A3, SLC6A6 and probably other SLC-proteins participate in the mechanism of hypotonic volume regulation. Due to the relevance of SLC-proteins as potential drug delivery systems the possible role of these transporters might be of great interest for many biomedical research areas.

Live juvenile cobia (Rachycentron canadum) transport methods were examined to determine opportunities for increasing packing density in closed containers for temporal durations up to 24 hours. Juvenile cobia (27 to 46 days-post-hatch (dph)) were tested for salinity tolerance following abrupt transfer from 35 ppt salinity water to salinities ranging from 0 ppt to 55 ppt. Results indicate a wide range of tolerance, with 100% survival at 24 hours post-transfer in salinities between 11 ppt and 45 ppt. Salinity preference was also tested to determine a possible correlation between acclimation salinity and salinity preference using an experimental horizontal salinity gradient with juvenile cobia (87 dph) over a period of 24 hours. Results of the salinity preference trials showed that salinity preference was directly related to acclimation salinity. Using two different salinities within the range tested in the tolerance trials (12 ppt and 32 ppt), a 24 hour simulated shipping trial was conducted comparing final survival between the two salinities at each of four packing densities (5 kg/m3, 10 kg/m3, 15 kg/m3, and 20 kg/m3). Results indicated a significant relationship between salinity and stocking density on survival of juvenile cobia following a 24 hour simulated shipment. At packing densities above 10 kg/m3, survival was significantly higher in the low salinity (12 ppt) treatments as compared to survival rates in the higher salinity (32 ppt) treatments. To help aquaculture professionals make accurate and economical decisions regarding the shipment of live juvenile cobia in closed containers, a bioeconomic model was constructed using survival data at different packing densities (1 kg/m3 to 20 kg/m3) and salinities (12 ppt and 32 ppt) obtained in the experimental trials combined with shipping cost and fingerling price data. The resulting model enables cobia fingerling producers to optimize their shipping methods and protocols, allowing for reductions in labor and material costs.

No description available.

No description available.

No description available.

Thesis (M. Med. Sc.)--University of Hong Kong, 2007.

Thesis (Ph. D.)--Ohio State University, 2008. / Title from first page of PDF file. Includes bibliographical references (p. 118-123).