[EN] The plasma membrane H+-ATPase (Pma1) is essential for yeast growth and is activated by glucose metabolism by an unknown mechanism involving double phosphorylation of a regulatory site at the C-terminus (Ser911 Thr912). In this thesis we have investigated in Saccharomyces cerevisiae the role of two protein phosphatases, type 1 Glc7 and type 2A Sit4, and of an essential atypical protein kinase, TORC1, in the activation of Pma1 by glucose. The regulatory site of activated Pma1 can be dephosphorylated "in vitro" by recombinant Glc7 and Sit4, but inhibition "in vivo" of these phosphatases does not activate Pma1. Inhibition of Glc7 by regulated expression of a dominant-negative truncated form (the null mutant is not viable) had no effect on Pma1 activity while deletion of SIT4 gene decreased both Pma1 activity and double phosphorylation of the regulatory site. Inhibition of TORC1 protein kinase by treatment of yeast cells with the drug rapamycin or by exposure to non-permissive temperature of a temperature-sensitive mutant (tor1¿ tor2ts) inhibited Pma1 and decreased double phosphorylation of the regulatory site. We conclude that Sit4 and TORC1 are required for full activation of Pma1 by glucose while Glc7 either does not participate or is redundant with other phosphatases. / [ES] La H+-ATPasa de la membrana plasmática (Pma1) es esencial para el crecimiento de la levadura y se activa por metabolismo de glucosa por un mecanismo desconocido que lleva consigo la doble fosforilación de un sitio regulador en el extremo C-terminal (Ser911 Thr912). En la presente tesis hemos investigado en Saccharomyces cerevisiae la participación de dos proteína fosfatasas, Glc7 de tipo 1 y Sit4 de tipo 2A, y de una proteína kinasa atípica esencial, TORC1, en la activación de Pma1 por glucosa. El sitio regulador de Pma1 en su estado activo puede defosforilarse "in vitro" por Glc7 y Sit4 recombinantes pero la inhibición "in vivo" de estas fosfatasas no activa Pma1. La inhibición de Glc7 mediante la expresión regulada de una forma truncada que actúa como dominante-negativa (el mutante nulo no es viable) no tiene efecto en la actividad de Pma1 mientras que la deleción del gen SIT4 disminuye tanto la actividad de Pma1 como la doble fosforilación del sitio regulador. Inhibición de la proteína kinasa TORC1 mediante tratamiento de las células de levadura con el fármaco rapamicina o exponiéndolas a temperatura no permisiva en el caso de un mutante termosensible (tor1¿ tor2ts) resulta en inhibición de Pma1 y disminución de la doble fosforilación del sitio regulador. Estos resultados indican que Sit4 y TORC1 son necesarias para la máxima activación de Pma1 por glucosa mientras que Glc7 podría no participar o hacerlo de forma redundante con otras fosfatasas. / [CAT] L'H+-ATPasa de la membrana plasmàtica (Pma1) és essencial per al creixement dels llevats i s'activa gràcies al metabolisme de glucosa per un mecanisme desconegut que porta associat la doble fosforilació d'una regió reguladora a l'extrem C-terminal (Ser911 Thr912). En aquesta tesi hem investigat en Saccharomyces cerevisiae la participació de dos proteïnes fosfatases, Glc7 de tipus 1 i Sit4 de tipus 2A, i d'una proteïna quinasa essencial atípica, TORC1, en l'activació de Pma1 per glucosa. La regió reguladora de Pma1, en seu estat activat, pot desfosforar-se "in vitro" per Glc7 i Sit4 recombinants, però la inhibició "in vivo" d'aquestes fosfatases no activa Pma1. La inhibició de Glc7 mitjançant l'expressió regulada d'una forma truncada que actua com a dominant-negativa (el mutant nul no és viable) no té cap efecte en l'activitat de Pma1 mentre que la deleció del gen SIT4 disminueix tant l'activitat de Pma1 com la doble fosforilació de la regió reguladora. La inhibició de la proteïna quinasa TORC mitjançant un tractament de cèl·lules de llevat amb el fàrmac rapamicina o la seua exposició a temperatures no permissives en el cas d'un mutant termosensible (tor1¿ tor2ts) resulta en la inhibició de Pma1 i la disminució de la doble fosforilació de la regió reguladora. Aquests resultats indiquen que Sit4 i TORC1 són necessàries per a l'activació màxima de Pma1 per glucosa, mentre que Glc7 podria no participar o fer-ho d'una forma redundant amb altres fosfatases. / Mahmoud Ali Ibrahim Hamouda, S. (2015). Protein kinases and phosphatases regulating the yeast proton pump [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/54131 / TESIS

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Conclusions The role of AUXIN BINDING PROTEIN 1 (ABP1) in the auxin management in plant cells was followed using simplified model material of suspension-cultured cells of tobacco BY-2 line. ABP1 is a putative auxin receptor considered to mediate fast non-genomic responses to auxin and it can be involved in every aspect of the regulation of auxin responses, metabolism and transport. There are four major conclusions that could be made based on the results presented in this thesis: 1) Auxin binding protein 1 mediates both cell division and expansion in tobacco BY-2 cells. In standard cultivation conditions or at lower concentrations of 2,4-D in culture medium, ABP1 overexpression had no detectable impact on cell division, cell elongation or cell growth.. 5- times increased 2,4-D concentration stimulated weakly cell elongation. . Antisense suppression of ABP1 expression resulted in disturbance in both cell expansion and cell division intensity, suggesting that ABP1 is essential for the control of balance between cell division and cell elongation during the growth cycle. ABP1 is localized in endoplasmic reticulum of cells cultivated in standard medium supplemented with 1 μM 2,4-D and it appeared also at the plasma membrane following the IAA application. 2) ABP1 mediates intercellular auxin transport. Cells...

L'interaction des glycosphingolipides de la membrane plasmique avec les protéines de liaison aux glucides (lectines) est d'une importance vitale pour l'infection de la cellule hôte par divers virus et bactéries. Dans ce travail, nous avons exploré l’interaction des lectines LecA de la bactérie P. aeruginosa et de la sous-unité B de la toxine Shiga (StxB) de S. dysenteriae avec son récepteur à membrane plasmatique, le globotriaosylcéramide (Gb3). De plus, nous avons étudié l'interaction de la bactérie complète P. aeruginosa avec Gb3. Afin de déchiffrer l'interaction lectine-Gb3 en l'absence d'autres composants cellulaires, nous avons utilisé les systèmes de membrane artificielle - vésicules unilamellaires géantes (GUV) et bicouches lipidiques supportées (SLB). Nous avons observé la liaison de la lectine en utilisant différents modes de microscopie à fluorescence (confocal, TIRF, etc.). Nous examinons la liaison des deux lectines aux domaines membranaires de différents ordres et compositions. Nous avons constaté que StxB préfère des domaines membranaires plus ordonnés alors que LecA est moins préférentiel. De plus, les deux lectines induisent la réorganisation des domaines membranaires: StxB stabilise les domaines ordonnés, les réduit et induit la formation des nouveaux domaines ordonnés. D'autre part, LecA ainsi que la bactérie P. aeruginosa induisent la dissolution des domaines ordonnés. Nous pensons que ces processus de réorganisation membranaire sont cruciaux pour l’infection bactérienne. / The interaction of plasma membrane glycosphingolipids with the carbohydrate binding proteins (lectins) is of vital importance for the infection of the host cell by various viruses and bacteria. In this work, we explored the interaction of the lectins LecA from the bacterium P. aeruginosa and B subunit of Shiga toxin (StxB) from S. dysenteriae with its plasma membrane receptor globotriaosylceramide (Gb3). Moreover, we studied the interaction of the complete bacterium P. aeruginosa with Gb3. In order to decipher the lectin-Gb3 interaction in absence of other cellular components we employed the artificial membrane systems – Giant unilamellar vesicles (GUVs) and Supported lipid bilayers (SLBs). We observed the lectin binding using different modes of fluorescence microscopy (confocal, TIRF, etc…). We examine the binding of both lectins to the membrane domains of different ordere and composition. We found that StxB prefers more ordered membrane domains whereas LecA is less preferential. Moreover, both lectins induce the reorganization of the membrane domains: StxB stabilizes ordered domains, shrinks them and induces the formation of the novel ordered domains. On the other hand LecA, as well as the bacterium P. aeruginosa induce the dissolution of the ordered domains. We believe, these membrane reorganization processes are crucial for the bacterial infection.

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This study provides information on the rates of DNA synthesis, sites of DNA synthesis, and DNA content of the avian salt gland during the osmoticstressing (plasma membrane synthesis) and destressing (plasma membrane turnover) cycle, in an effort to better understand the relationship of cell turnover to the initial events in plasma membrane amplification, differentiation, and turnover. The rate of DNA synthesis increases 12-24 h after the onset of osmotic stress, is maximal at about 24 h of osmotic stress, and decreases thereafter in fully stressed and destressed glands. The maximum DNA and protein content, and the maximum protein/DNA ratio are obtained after about 3 days of stress. Autoradiograms show that at 24 h of stress 70-80% of DNA synthesis occurs in connective tissue cells and 20-30% in parenchymal cells, but by 6 days of stress, synthesis occurs about equally in these cell groups. Because destressing is characterized by a large decrease in plasma membrane and in glandular protein, but by little DNA turnover or loss, the loss of plasma membrane is likely due to some type of cell dedifferentiation rather than cell turnover.

Pseudemoia pagenstecheri is a viviparous Australian scincid lizard in which the maternal-embryonic placental interface is differentiated into structurally distinct regions. The chorioallantoic placenta contains an elliptical-shaped region, the placentome, characterized by hypertrophied uterine and embryonic epithelial cells supported by dense vascular networks. The remainder of the chorioallantoic placenta, the paraplacentome, is also highly vascularized but uterine and chorionic epithelia are thin. An omphaloplacenta with hypertrophied epithelia is located in the abembryonic hemisphere of the egg. There is extensive placental transport of organic and inorganic nutrients, e.g., 85-90% of neonatal calcium is received via placental transfer. Calcium uptake by extraembryonic membranes of squamates correlates with expression of the intracellular calcium binding protein, calbindin-D 28K, and plasma membrane calcium ATPase (PMCA) is a marker for active calcium transport. We estimated expression of calbindin-D 28K and PMCA in the chorioallantoic membrane in a developmental series of embryos using immunoblotting and used immunohistochemistry to define the cellular localization of calbindin-D 28K to test the hypotheses that 1) expression of calcium transporting proteins is coincident with placental transport of calcium and 2) the placenta is functionally specialized for calcium transport in regions of structural differentiation. Calbindin-D 28K and PMCA were detected at low levels in early stages of development and increased significantly prior to birth, when embryonic calcium uptake peaks. These data support the hypothesis that placental calcium secretion occurs over an extended interval of gestation, with increasing activity as embryonic demand escalates in late development. In addition, calbindin-D 28K expression is localized in chorionic epithelial cells of the placentome and in the epithelium of the omphalopleure of the omphaloplacenta, which supports the hypothesis that regional structural differentiation in the placenta reflects functional specializations for calcium transport.

Intravenous administration of ovokinin(2-7), a cleavage peptide derived from ovalbumin, dose-dependently (0.1-5 mg/kg) lowered the mean arterial pressure (MAP) that was not accompanied by a significant change in the heart rate (HR) of urethane-anesthetized rats. The hypotensive effects of ovokinin(2-7) were five orders of magnitude lower compared to that of bradykinin and were largely prevented by pretreatment with the bradykinin B2 receptor antagonist HOE140 (81.6±18.4%) and moderately affected by the B1 receptor antagonist [des-Arg10]-HOE140 (26.3±15.5%). Intracellular Ca2+ levels, as measured by Fur 2-AM, were significantly elevated in cultured aorta smooth muscle cells by ovokinin(2-7). The increases were abolished by HOE140 and unaffected by [des-Arg10]-HOE140. The elevation of intracellular Ca2+ by ovokinin(2-7) was dependent on Ca2+ entry from extracellular space as it was reduced in a Ca2+-free solution. Pretreatment of the cells with the phospholipase C inhibitor U73122 (2 μM) eliminated the Ca 2+ increase by the peptide. PA phosphohydrolase and phospholipase A2 inhibitors significantly reduced the responses as well. Our results show that ovokinin(2-7) modulates cardiovascular activity by interacting with B2 bradykinin receptors.

Historically, Australia has been important in the study of, and the development of hypotheses aimed at understanding, the evolution of viviparity in amniote vertebrates. Part of the importance of Australia in the field results from a rich fauna of skinks, including one of the broadest ranges of diversity of placental structures within one geographic region. During the last decade, we have focussed our studies on one lineage, the Eugongylus group of skinks of the subfamily Lygosominae because it contains oviparous species and some that exhibit complex placentae. Our specific objective has been to attempt to understand the fundamental steps required when viviparity, and ultimately complex placentae, evolve from oviparous ancestors. We have taken a three-prong approach: (1) detailed study of the morphology and ontogeny of the placentae of key species at the light microscope level; (2) study of changes in the uterus associated with pregnancy, or the plasma membrane transformation; and (3) measures of the net exchange of nutrients across the placenta or eggshell of key species. In turn, we have found that: (1) details of the morphology and ontogeny of placentae are more complex that originally envisaged, and that the early conclusions about a sequence in the evolution of complex placentae was naïve; (2) a plasma membrane transformation occurs in viviparous, but not oviparous lizards, and thus may be a fundamental feature of the evolution of viviparity in amniotes; and (3) species with more complex chorioallantoic placentae tend to transport more nutrients across the placenta during pregnancy than those with simpler chorioallantoic placentae but, because the correlation is not tight, the importance of the omphaloplacenta in transporting nutrients may have been overlooked. Also, the composition of yolk of highly matrotrophic species is broadly similar, but not identical, to the yolk of oviparous species. Some of the interpretation of our data within the context of our specific objective is not yet possible, pending the publication of a robust phylogeny of Eugongylus group skinks. Once such a phylogeny is available, we are in a position to propose specific hypotheses about the evolution of viviparity that can be tested using another lineage of amniotes, possibly Mabuya group skinks. © 2002 Elsevier Science Inc. All rights reserved.

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