http://hdl.handle.net/10366/4604 2026-05-02T04:00:41Z http://hdl.handle.net/10366/146279 [EN]Expression of the nerve growth factor (NGF) receptors TrkA and p75NTR was found to vary at the surface of PC12 cells in a cell cycle phase-specific manner. This was evidenced by using flow cytometric and microscopic analysis of cell populations labeled with antibodies to the extracellular domains of both receptors. Differential expression of these receptors also was evidenced by biotinylation of surface proteins and Western analysis, using antibodies specific for the extracellular domains of TrkA and p75NTR. TrkA is expressed most strongly at the cell surface in M and early G1 phases, whereas p75NTR is expressed mainly in late G1, S, and G2 phases. This expression reflects the molecular and cellular responses to NGF in specific phases of the cell cycle; in the G1 phase NGF elicits both the anti-mitogenic effect, i.e., inhibition of the G1 to S transition, and the differentiation response whereas a survival effect is provoked elsewhere in the cell cycle. A model is proposed relating these responses to the surface expression of the two receptors. These observations open the way for novel approaches to the investigation of the mechanism of NGF signal transduction. 1998-01-01T00:00:00Z http://hdl.handle.net/10366/141137 [EN] Polyphenols are considered to be responsible for some of the health benefits derived from the consumption of red wine. These protective effects might probably be explained in the context of the xenohormesis theory that considers plant metabolites as interspecific chemical signals. However, the complexity of the polyphenolic constituents of different wines makes it difficult to clarify the specific contribution of polyphenols to such effects. In the present work, we fractionated the polyphenols of a red wine and evaluated the effect of each polyphenolic fraction on the growth pattern of the yeast Saccharomyces cerevisiae. We observed a different contribution of the phenolic fractions to the xenohormetic response of S. cerevisiae, the fractions that were enriched with red pigments being the most protective against oxidative insults. Moreover, we found that red wine phenolic fractions exert their biological activity through the activation of the Yap1 and Msn2 stress-responsive regulators. Above all, the anthocyanins delphinidin 3- glucoside and petunidin 3-glucoside were found to improve significantly the growth rate of S. cerevisiae in an Msn2-, Msn4-dependent manner, indicating that the stress regulators Msn2 and Msn4 participate in the xenohormetic activity of the wine polyphenols delphinidin and petunidin. 2010-01-01T00:00:00Z http://hdl.handle.net/10366/125259 [EN]Schizosaccharomyces pombe contains four putative (1,3)β-D-glucan synthase (GS) catalytic subunits, Bgs1p to Bgs4p. In this work, we cloned bgs4+ and show that Bgs4p is the only subunit 1) essential for maintaining cell integrity during both cytokinesis and polarized growth, and 2) found to be part of the GS enzyme. Here we show that bgs4+, cwg1+ (cwg1-1 shows reduced cell-wall β-glucan and GS catalytic activity) and orb11+ (orb11-59 is defective in cell morphogenesis) are the same gene. bgs4+ is essential during spore germination. bgs4+ shut-off produces cell lysis at growing poles and mainly at the septum prior to cytokinesis, suggesting that Bgs4p is essential for cell wall growth and for compensating an excess of cell wall degradation during cytokinesis. Shut-off and overexpression analysis suggest that 1) Bgs4p forms part of a GS catalytic multiprotein complex, and 2) Bgs4p-promoted cell-wall β-glucan alterations induce compensatory mechanisms from other Bgs subunits and (1,3)α-D-glucan synthase. Physiological localization studies showed that Bgs4p localizes to the growing ends, the medial ring and septum, and in each process of wall synthesis or remodeling that occurs during sexual differentiation: mating, zygote and spore formation, and spore germination. Bgs4p timing and requirements for proper positioning during cytokinesis and its localization pattern during spore maturation differ from those of Bgs1p. Bgs4p localizes overlapping the contractile ring once Bgs1p is present and a Calcofluor white-stained septum material is detected, suggesting that Bgs4p is involved in a late process of secondary or general septum synthesis. Unlike Bgs1p, Bgs4p needs the medial ring but not the Septation Initiation Network proteins to localize with the other septation components. Furthermore, Bgs4p localization depends on the polarity establishment proteins. Finally, F-actin is necessary for Bgs4p delocalization from and relocalization to the growing regions, but it is not needed for its stable maintenance at the growing sites, poles and septum. All these data show for the first time an essential role for a Bgs subunit in the synthesis of a (1,3)β-D-glucan necessary to preserve cell integrity when cell wall synthesis or repair are needed. 2005-01-01T00:00:00Z http://hdl.handle.net/10366/125258 [EN]Schizosaccharomyces pombe Bgs1p/Cps1p has been identified as a putative (1,3)β-Dglucan synthase (GS) catalytic subunit with a possible function during cytokinesis and polarized growth. To study this possibility, double mutants of cps1-12 and cdc septation mutants, were made. The double mutants displayed several hypersensitive phenotypes and altered actin distribution. Epistasis analysis showed mutations prior to septum synthesis were dominant over cps1-12, while cps1-12 was dominant over the end of septation mutant cdc16-116, suggesting Bgs1p is involved in septum cell-wall (1,3)β-D-glucan synthesis at cytokinesis. We have studied the in vivo physiological localization of Bgs1p in a bgs1Δ strain containing a functional GFP-bgs1+ gene (integrated single copy and expressed under its own promoter). During vegetative growth, Bgs1p always localizes to the growing zones: one or both ends during cell growth, and contractile ring and septum during cytokinesis. Bgs1p localization in cdc septation mutants indicates that Bgs1p needs the medial ring and septation initiation network (SIN) proteins to localize properly with the rest of septation components. Bgs1p localization in the actin mutant cps8-188 shows it depends on actin localization. In addition, Bgs1p remains polarized in the mislocalized growing poles and septa of tea1-1 and tea2-1 mutants. During the meiotic process of the life cycle, Bgs1p localizes to the mating projection, to the cell-to-cell contact zone during cell fusion and to the neck area during zygote formation. Also, Bgs1p localization suggests it collaborates in forespore and spore wall synthesis. During spore germination, Bgs1p localizes first around the spore during isotropic growth, then to the zone of polarized growth and finally, to the medial ring and septum. At the end of spore-cell division, the Bgs1p displacement to the old end only occurs in the new cell. All these data shows Bgs1p is localized to the areas of polarized cell wall growth and according to that, we propose it might be involved in synthesizing the lineal (1,3)β-D-glucan of the primary septum, as well as a similar lineal (1,3)β-D-glucan when other processes of cell wall growth or repair are needed. 2002-08-01T00:00:00Z http://hdl.handle.net/10366/125257 [EN]A series of thiamine-repressible shuttle vectors has been constructed to allow a more efficient DNA manipulation in Schizosaccharomyces pombe. These high-copy-number vectors with regulatable expression (pJR) are based on the backbone of the pREP-3X, pREP-41X and pREP-81X plasmids. The pJR vectors are all uniform in structure, containing i) sequences for replication (ori) and selection (AmpR) in Escherichia coli, ii) the f1 ori sequence of the phage f1 for packaging of ssDNA, making them suitable for site-directed mutagenesis, and iii) the ars1 sequence for replication in S. pombe. The pJR vectors differ among them in i) the selectable marker (Saccharomyces cerevisiae LEU 2 gene, that complements S. pombe leu1- gene, and S. pombe ura4+ and his3+ genes); ii) the thiaminerepressible nmt1 promoter (3X, 41X and 81X with extremely high, moderate or low transcription efficiency, respectively); and iii) the multiple cloning site (two multiple cloning sites, with twelve restriction sites each). The expression level of the pJR vectors has been analyzed using the β-galactosidase gene as reporter. Three levels of expression for each nmt1 promoter version, with any selectable marker and for either repressed or induced conditions, have been found. The expression is dependent on the distance to the initiation codon, varying from 0.001 to 15 times the activity characterized for the pREP plasmids. Also, the gene expression has been found to be extremely sensitive to the nucleotide sequence prior to the initiation codon, being up to 50 fold higher with an A/T sequence than with a G/C sequence. Finally, the β-galactosidase mRNA levels were found to be similar in each nmt1 series, suggesting a translational effect on gene expression. As a result, any of these eighteen new vectors allow performing gene expression in fission yeast as well as a more versatile cloning, sequencing and mutagenesis directly in the plasmid without the need for subcloning into intermediary vectors. 2000-02-17T00:00:00Z http://hdl.handle.net/10366/125256 [EN]Cytokinesis is a crucial event in the cell cycle of all living cells. In fungal cells, it requires coordinated contraction of an actomyosin ring and synthesis of both plasmatic membrane and a septum structure that will constitute the new cell wall end. Schizosaccharomyces pombe contains four essential putative (1,3)β-D-glucan synthase catalytic subunits, Bgs1p to Bgs4p. Here we examined the function of Bgs1p in septation by studying the lethal phenotypes of bgs1 + shut-off and bgs1 Δ cells and demonstrated that Bgs1p is responsible and essential for linear (1,3)β-D-glucan and primary septum formation. bgs1 + shut-off generates a more than 300-fold Bgs1p reduction, but the septa still present large amounts of disorganized linear (1,3)β-D-glucan and partial primary septa. Conversely, both structures are absent in bgs1 Δ cells, where there is no Bgs1p. The septum analysis of bgs1+-repressed cells indicates that linear (1,3)β-D-glucan is necessary but not sufficient for primary septum formation. Linear (1,3)β-D-glucan is the polysaccharide that specifically interacts with the fluorochrome Calcofluor white in fission yeast. We also show that in the absence of Bgs1p abnormal septa are formed, but the cells cannot separate and eventually die. 2007-07-01T00:00:00Z http://hdl.handle.net/10366/76539 [ES] En esta tesis se identifican nuevas proteínas efectoras o reguladoras de las vías de señalización de las GTPasas de la familia Rho. Para ello se plantea el estudio de la ORF SPBC4F6.12, que codifica una proteína homóloga a Pxl1 de S. cerevisiae y a la paxilina de células animales, se determina la posible relación de SpPxl1 con las GTPasas de la familia Rho, Rho1 y Cdc42 y se estudian las funciones de Pxl1 en S. pombe.; [EN] In this thesis we identify new effector or regulatory proteins of the signaling pathways of Rho family GTPases. It does so by studying the SPBC4F6.12 ORF, which encodes a protein homologous to S. Pxl1 paxilina cerevisiae and animal cells, determine the possible relationship SpPxl1 with Rho family GTPases, Rho1 and Cdc42 and functions are studied in S. Pxl1 pombe. 2010-01-01T00:00:00Z http://hdl.handle.net/10366/22505 2009-01-19T00:00:00Z http://hdl.handle.net/10366/22504 Las modificaciones epigenéticas tales como acetilación, fosforilación, ubiquitinación y ADP-ribosilación de las histonas influyen en la genética potencial del DNA. El nivel de acetilación de las histonas está controlado por las acetilatransferasas de histonas (HAT) y las Desacetilasas de histonas (HDAC). El patógeno humano Candida albicans puede crecer en al menos cuatro diferentes morfologías: levaduras, pseudohifas, hifas y clamidosporas. Las pseudohifas y las hifas son formas elongadas y se engloban bajo la denominación de formas filamentosas. El cambio entre todas las morfologías es resultado de compleja interacción entre factores internos y externos y está coordinado in parte por proteínas que regulan polaridad y que están conservadas en las células eucariotas. En C. albicans existen múltiples rutas de regulación que controlan la transición levadura-hifa. Estas rutas controlan la transcripción de un set de genes específicos de hifa, la mayoría de ellos codifican conocidos factores de virulencia. Efg1p es el mayor regulador morfogenético de C. albicans. Sin3p, un componente del complejo multiproteico de Desacetilasas de histonas Sin3p-Rpd3p en Saccharomyces cerevisiae, se une al promotor del gen EFG1. Con todos los datos obtenidos se propuso un modelo que explica la relación entre Efg1p-Sin3p y las HDAC en la morfogénesis de C. albicans. En esta levadura existen al menos cinco HDACs. El objetivo de este trabajo fue entender el papel de dos de ellas, Hos3p y Rpd3p en la regulación de la morfogénesis de la levadura patógena C. albicans.En este trabajo, se interrumpieron tres genes que codifican las proteínas Hos3p y Rpd3p (dos de ellos) en C. albicans y se analizó el papel de los mismos en la morfogénesis, en especial en la transición levadura-hifa.Las células carentes del gen HOS3 mostraron una morfología similar a la del silvestre tanto en medio sólido como en medio líquido, incluso durante la transición dimórfica. También fueron capaces de formar clamidosporas. Se analizó el perfil global de transcripción y se obtuvieron 233 genes cuya expresión se veía afectada por la interrupción del gen HOS3 tras la comparación del mutante ?hos3 y la cepa silvestre. Estos genes pertenecían a diferentes categorías funcionales, principalmente metabolismo, transporte celular y comunicación celular y transducción de la señal.C. albicans tiene dos genes homólogos al gen que codifica la HDAC Rpd3p en S. cerevisiae. Ambos genes fueron interrumpidos y el doble mutante presentaba alteraciones en la transición levadura-hifa y la formación de clamidosporas. Los mutantes sencillos presentaron fenotipo similar a la cepa silvestre. El doble mutante además presentó alterada la expresión de los genes implicados en el cambio dimórfico EFG1, CPH1, YWP1 y HWP.; Epigenetic modifications, such as acetylation, phosphorylation, methylation, ubiquitination, and ADP ribosylation, of the highly conserved core histones, H2A, H2B, H3, and H4, influence the genetic potential of DNA. The enormous regulatory potential of histone modification is illustrated in the vast array of epigenetic markers found throughout the genome. The histone acetylation level is balanced by histone acetyltrasferases (HAT) and histone deacetylases (HDAC). The human fungal pathogen, Candida albicans can grow in at least four different morphologies: yeast, pseudohyphae, hyphae and chlamydospore. Pseudohyphae and hyphae are both elongated and sometimes there has been little attempt to distinguish between them, as both are "filamentous forms" of the fungus. The switch between these forms is the result of a complex interplay of external and internal factors and is coordinated in part by polarity-regulating proteins that are conserved among eukaryotic cells. C. albicans uses a network of multiple signaling pathways to control the yeast-->hypha transition. These pathways control the transcription of a common set of hypha-specific genes, many of which encode known virulence factors. Efg1p is the mayor regulator of morphogenesis in C. albicans. Sin3p, a component of a specific histone deacetylase complex Sin3p-Rpd3p in S. cerevisiae, was shown to bind to the EFG1 promoter. A proposed model explains the relationship between Efg1p-Sin3p and HDAC in morphogenesis. C. albicans has at least five HDACs. The focus of our work was on understanding the role of two of them, Hos3p and Rpd3p in the regulation of the morphogenesis in the pathogen yeast C. albicans.In this work, we have disrupted three genes which codified to Hos3p and Rpd3p (two of them) in C. albicans and have analyzed their role in morphogenesis and especially in the yeast-hypha transition.Cells lacking of HOS3 gene could grow normally on solid or in liquid media, even during the yeast-hypha transition. ?hos3 cells were able to form normal chlamydospores. We have analyzed global transcription profile and we obtained 233 differentially expressed genes after comparison of the hos3 mutant with the C. albicans wild type strain. These genes belong to several functional categories mainly those involved in metabolism, cellular transport and cellular communication/signal transduction. C. albicans has two genes homologous to ScRpd3p. We have disrupted both genes and the double mutant showed an altered behaviour during the dimorphic switch and chlamydospores formation. Single mutants presented wild type phenotype. The double mutant has also altered the expression of EFG1, CPH1, YWP1 and HWP1, genes implicated in morphogenesis. 2009-01-01T00:00:00Z http://hdl.handle.net/10366/22497 2009-01-01T00:00:00Z http://hdl.handle.net/10366/22503 La formación de la pared celular de Schizosaccharomyces pombe requiere la actividad coordinada de enzimas involucradas en la biosíntesis y modificación de sus componentes, entre los que destacan por su abundancia los glucanos. El complejo enzimático ?-glucán-sintasa sintetiza ?-1,3-glucanos lineales, que permanecen desorganizados y solubles en álcali hasta que se establecen enlaces covalentes entre los ?-1,3-glucanos y otros componentes de la pared celular. Las transferasas de la pared celular cuyos sustratos son los ?-1,3-glucanos podrían estar desempeñando importantes funciones en el ensamblaje y reordenamiento de la pared celular. Diversas proteínas pertenecientes a la familia 72 de las glicosil-hidrolasas (GH72) con actividad ?-1,3-glucanosil-transferasa han sido caracterizadas en otros organismos (Gas1p, Gas2p y Gas4p de Saccharomyces cerevisiae, o Gel1p de Aspergillus fumigatus). La secuenciación del genoma de S. pombe ha permitido identificar cuatro genes (gas1+, gas2+, gas4+ y gas5+) que codifican cuatro hipotéticas proteínas pertenecientes a la familia GH72.Desde el punto de vista funcional, gas1p es esencial para la viabilidad celular durante el crecimiento vegetativo, ya que los mutantes carentes de este gen sólo son capaces de crecer en medios estabilizados osmóticamente. Por su parte, gas4p desempeña un papel básico en la construcción de la pared celular de las esporas. Finalmente, las proteínas gas2p y gas5p deben ejercer un papel minoritario en la síntesis de la pared celular vegetativa, ya que mutantes carentes de estos genes (o el doble mutante) no presentan defectos morfológicos significativos.Bioquímicamente, todas ellas poseen actividad ?-1,3-glucanosil-transferasa, aunque se diferencian por la especificidad de longitud de sustrato, el punto de corte y los productos generados. Todo ello, junto con las diferencias de expresión a lo largo del ciclo biológico, sugiere que las distintas proteínas de la familia GH72 presentes en S. pombe podrían estar realizando funciones complementarias, no solapantes, en la levadura de fisión.; The formation of the Schizosaccharomyces pombe cell wall requires the co-ordinated activity of enzymes involved in the biosynthesis and modification of its components, such as glucans. The ?-glucan-synthase complex synthesizes linear ?-1,3-glucans, which remain unorganized and alkali-soluble until covalent linkages are set up between ?-1,3-glucans and other cell wall components. Cell wall transferases using ?-1,3-glucans as substrates could perform important functions in the assembly and organization of the cell wall. Several proteins belonging to the glycoside hydrolase family 72 (GH72) with ?-1,3-glucanosyltransferase activity have been described in other organisms, such as the Saccharomyces cerevisiae Gas1p or the Aspergillus fumigatus Gel1p. At least four genes encoding putative ?-1,3-glucanosyl-transferases gas1+, gas2+, gas4+ y gas5+- have been identified in the S. pombe sequencing project. In this work, we report the characterization of gas1p, gas2p, gas4p and gas5p during the different stages of the life cycle and their catalytic activity.From the point of view of their functionality, gas1p is essential for cellular viability during vegetative growth, as gas1? null mutants are only able to grow in media supplemented with an osmotic stabilizer. gas4p plays an essential role during the construction of the ascospore wall, being necessary for spore germination. Finally, gas2p and gas5p seem to play a minoritary role in the synthesis of the cell wall, as gas2? and gas5? null mutants or gas2? gas5? double mutant do not show significant morphologic defects.From the biochemical point of view, all of them display ?-1,3-glucanosyl-transferase activity, although they differ in their specificity for the substrate length, the split point and the range of the generated products. Taking all the data into account, together with the differences in their expression profiles during the life cycle, it suggests that the GH72 proteins present in S. pombe may accomplish complementary, non-overlapping, functions in the fission yeast. 2008-01-01T00:00:00Z