YEAST

Author: CIUDAD SÁNCHEZ ANTONIA.
Year: 2002.
University: EXTREMADURA [www.unex.es].
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: DPTO. MICROBIOLOGÍA FAC. DE CIENCIAS.

Summary: The yeast Candida albicans is a fungus disease, which causes systemic infection in AIDS patients and patients undergoing treatment of the immune system. This has been identified in a fungus counterpart protein Rad52 of Sacchatomyces cerevisae, Schizosaccharomyces pombe and humans. The absence of CaRad52p in the cell results in defects in the mechanisms of homologous recombination processes reflected in the integration of genetic fragments líneales DNA plasmid maintenance, as well as greater genomic instability, manifested for instance in loss chromosomes with high frequency. The absence of CaRad52p in the cell also causes fungus growth pseudofilamentoso's in a position where it is not induced growth --. Finally, cells lacking CaRad52p are more invasive and less virulent.

Author: FRASÉS CARVAJAL SUSANA.
Year: 2003.
University: MIGUEL HERNÁNDEZ DE ELCHE [www.umh.es].
Place of defense: MEDICINA.
Place of preparation: UNIVERSIDAD MIGUEL HERNÁNDEZ.

Summary: The cryptococcal disease is a fungal disease, a serious and often opportunistic, often affect patients with deterioration in the immune system. The disease is not among those considered to be notifiable in any country in the world so there is an unequal information about its impact on the general population and specific risk groups such as those with AIDS, cancer patients or individuals receivers organ transplants. The different ecology, geographical distribution, epidemiology and virulence of its two varieties (Cnvar. Neoformans and Cnvar.gattii) has assumed an interesting stimulus for the study of many physiological and molecular aspects of this yeast. Studies seeking molecular typing serotypes and varieties Cruptococcus, have sought to interpret terms edpidemiológico looking for patterns that show a certain relation to the origin of the isolates. The molecular markers proposed so far include elements repeated the genome (the so-called sequences minsatélites), the pattern digetión of certain genes, the sequence of espciadores intergénicos between rDNA genes and others. Some of the proposed markers have come to establish a particular distribution pattern according to geographical areas, but the results are not yet conclusive, and it is of great interest to expand the sample value. In this paper we have analyzed 110 strains of the Constitution from various sources (environmental, clinical and veterinary clinic human). The characterization of isolates has been carried out both from the point of view phenotypic and genotypic. On the one hand, have been analyzed variables such as phenotypic variety, auxonotipo and serotype C.neoformans. The genotypic characterization has been made on the basis of the valuation of 5 markers. Two of them are located in the region of rDNA (5.8S-ITS and ISM), another is for the gene URA5 and the remaining two are minisatellite sequences (M13 and (GACA) 4). The results of this study show that the study of 110 strains of C.neoformans reveal the existence of 99 profiles genotypic and 23 profiles different phenotypic, which demonstrates the high variability intraespecie in this yeast. The phenotypic analysis of the yeast reveals that the serotype A is the most prevalent in our pious, this variable is the key factor in the phenotypic clusters, there being no block of homology to submit several strains of different serotypes grouped. Genotypic analysis of the yeast shows a useful molecular marker differential according to the employee. This fomra, the survey marker 5.8Sy their spacers inergénicos (STI) shows its usefulness as a good target for molecular diagnosis and as a good marker for typing interespecie. For his part, RFLP of genes conserved, 5S and IRA5 brings together isolated with more than one trait in common recognizable phenotype (serotype most origin). Also present clonal stability. These markers are reproducible between and within-. However, there are good markers for studying strains very close because they are unable to differentiate between them. The analysis of the patterns obtained with the marker minisatellite M13 aggregates variety isolated by offering a large number of patterns in the range neoformans. This marker combined with the information given by the serotype is a useful tool for application and 8 pidemiol 82b ógica. As for the minisatellite (GACA) 4 is the one that offers greater variability intraespecie, being very useful because it is capable of identifying and primoinfecciones relapses, as well as characterize the mimso isolated obtained from two different biological samples. The analysis of the marker minisatellite (GACA) 4 in isolated from the outbreak of cryptococcosis goats, highlights differences between the strains, which kept most other markers fail to embrace. In any case, the characterization of isolates of this study showed evidence that endorse the existence of the third variety of Cruptococcus neoformans (Cnvar grubii). In our study, all molecular markers tested discriminate clearly between the two varieties classic (and gattii neoformans). The joint analysis of the profiles found with each molecular marker and the potential equivalence with traits of behavior, ecology, etc. of the isolates, shows that the molecular tools are a useful proposal for additional epidemiological study and specific aspects of phenotype among those who highlights serotype. This study also highlights the discovery of the first cryptococcus var.gattii serotype B in human pathology in Spain. It is suspected that this may be native variety in our region but in a minority. The final demonstration of its existence as an indigenous strain in Spain will be obtained when isolated in the wild. The molecular epidemiology of cryptococcal disease requires analysis of a larger number of isolates, especially varieties and serotypes minority, to endorse the use of molecular markers.

Author: GARCERÁ TERUEL ANA.
Year: 2003.
University: VALENCIA [www.uv.es].
Place of defense: FACULTAD DE FARMACIA.
Place of preparation: FACULTAD DE MARCIA.

Summary: Following the partial annotation of the genome of Candida albicans, a study was conducted in silico from the database of C.albicans. Two genes were identified with characteristics of cell wall proteins, the IPF 3054 with a 62% homology with the gene ScSSR1 (Stress Response Secretory protein) from Saccharomyces cerevisiae, which was called CaSSR1 and PEI 564. We performed the interruption of the two genes in order to study the involvement in the construction of the cell wall. Disruption of gene Ca SSR1 caused greater sensitivity to the parental strain to calcofluor white and red Congo substances distor-sionan polymers of the cell wall. The study of the material released by the enzyme zimiliasa with activity mostly B-1 ,3-glucanasa showed the disappearance of a species of 70 Kda protein. Over gene CaSSR1 in strain mutant gene CaSRR1 led to the emergence of the species de novo protein in the extract zimoliasa of the strain sobreexpresante gene CaSSR1. To locate proteins CaSSR1 and Ca564 are labeled with the c-myc epitope, introduced after the court to peptidasa signal, the location in C.albicans could not be performed with this epitope, while in S.cerevisiae were detected these protein extracts in SDS and the membrane fraction mixed. A study of the site using GPI anchor the cell wall of the protein CaSsr1, a number of buildings that were eliminated in the region hidrobfóbica corresponding to the amino acids 217 and 234, has been implicated in anchoring the region and w, w + 1, w +2, corresponding to the amino acids 199 to 201, described as necessary for the anchor to the cell wall. Different versions were introduced in the mutant strain for the gene CaSSR1 and is considered the anchor to parede of different versions, in the case of the version in which the site was removed w, w +1, w +2, it was not detected in the extract zimiliasa, but was detected a new species protein released into the culture medium, which the amino acids 199 to 201 are required for the incorporation of the protein to the cell wall. In the case of the removal of the queue for hydrophobic amino acids 217 to 234, paroteína result was found in the extract zimoliasa. The study of gene expression CaSSR1 was studied in yeast and morphology mycelial, speaking in both stadiums alike. The overall expression of the mutant gene CaSSR1 is to carried out through DNA microarrays, they are represented in the 6039 genes C.alicans well as a series of genes controls, the transcriptional profile of this mutant was compared with its parent strain, while a total of 39 genes that changed expression due to the interruption of the gene CaSSR1 under the conditions studied. Of the total number of genes 18 are of unknown function. We found seven genes sobreexpresados a total of 27 repressed.

Author: QUIRÓS ASENSIO MANUEL.
Year: 2004.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE CIENCIAS BIOLÓGICAS.
Place of preparation: FACULTAD DE CIENCIAS BIOLÓGICAS.

Summary: Yeast Debaryomyces hansenii can isolate itself in a variety of habitats including processed foods. In some of them, like cheese, it has been found beneficial in his speech matured from them. Lately, it has been suggested that could be added to crops initiators of sausages to improve their organoleptic characteristics. But in this thesis is found on one side the physiological capacity (increased metabolic performance) to develop environmental conditions in the ripening chambers and on the other hand, the ability to deteriorate through gas production increased in the presence of additives are commonly used for the processing of sausages. In this thesis, moreover, is designed and evaluated the quality of half cromogénico, selective and differential for this yeast based on the detection of the enzyme beta-glucuronidasa. Similarly, it develops a method for detecting differential against other species of yeast that often contaminate food based on the study of polymorphism of restriction fragments in the region IGSE of Dna ribosomal (ISM rDNA PCR-RFLP). This technique is also applicable to other species of the genus Debaryomyces where they can check their taxonomic value.

Author: ROMÁN GONZÁLEZ ELVIRA.
Year: 2004.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE FARMACIA.
Place of preparation: DPTO. MICROBIOLOGÍA II (FACULTAD DE FARMACIA , UCM.).

Summary: The signal transduction pathways mediated by MAP kinases are one of the main systems that allow organisms to adapt to changes in the environment around them, in many cases regulate processes essential for the cell. In this regard, and with the increase in the incidence and mortality rate of pathogenic fungus Candida albicans, studies in yeast that has increased considerably in recent years. In our working group we have cloned and characterized some of the elements of these routes in the fungus pathogen C.albicans. Specifically, we have established that the route HOG is involved in adaptation to different types of osmotic stress and oxidativo- stimuli that induce activation of MAP kinase in the path hog1, as well as in regulating the transition dimórfica and virulence in this fungus. This study has addressed the study of protein Sho1 in physiology C.albicans, from several aspects. First, we have characterized the response of the mutant sho1 to stimuli that trigger the route (saline and oxidative) and analyzed by obtaining double mutants sho1 hog1, epistasis possible relationships between genes SHO1 and HOG1. In addition, studies in mutant ssk1 have enabled us to establish the branch that is mediated Ssk1-y not mediated by Sho1-primarily responsible for the transmission of the stimulus activator MAP kinase Hog1 in response to hydrogen peroxide. Secondly, it has analyzed the role of Sho1 in the biogenesis of the fungal cell wall, establishing the existence of a possible route SVG in C. Albicans in vegetative growth that would regulate and control the activation of MAP kinase Cek1 which requires activation of the protein Sho1. Finally, it has studied the relationship between SHO1 and transition dimórfica in this fungus.

Author: VESES JIMÉNEZ VERÓNICA.
Year: 2004.
University: VALENCIA [www.uv.es].
Place of defense: FACULTAD DE FARMACIA.
Place of preparation: FACULTAD DE FARMACIA.

Summary: By inmunorrastreo a genoteca expression of Candida albicans with a polyclonal antibody specific to the form of that mycelial fungus (PAb anti-gt) has been isolated, cloned and characterized a new gene of this microorganism. This gene is contained in the contig 5-3205 of the draft genome sequencing of C. Albicans, and contains an open reading frame that encodes for a putative polypeptide of 288 amino acids with a molecular weight of approximately 32,921 Da. The suppression of isolated gene resulted in a severe defect in the morphogenesis of C. Albicans, characterized by the formation of yeast chains, so the gene was named ABG1 (English, "altered budding growth"), based on the phenotype for growth gemación altered observed in the mutant strains for that gene. Trials Immunoblotting of subcellular fractions with a polyclonal antibody versus Abg1p and fusion studies with green fluorescent protein (GFP) from Aequorea victoria, revealed that Abg1p was located in the vacuolar membrane of C. Albicans. The pattern of fluorescence protein Abg1p-GFP colocalizó with that seen after staining with FM4-64, agent stains and vacuolar membrane vesicles endocíticas. In order to establish the role of ABG1 in the biology of C. Albicans proceeded to the construction of a null mutant, which could not take place despite using two different techniques of gene disruption. It proceeded to the construction of a conditional mutant, in which the first copy of ABG1 was interrupted with the cassette URA-blaster and the second copy was placed under the control of the promoter of MET3. By suppressing the expression of ABG1, by the repressive action of methionine and cysteine on the promoter of MET3, the mutant was unable to grow, which established the essential character of ABG1 for C. Albicans. The phenotypic analysis of conditional mutant revealed abnormalities in the morphology, size and number of vacuolas present in the cells. The suppression of ABG1 in C. Albicans caused, in addition, defects in cell division. In cells levaduriformes, alterations in the process of citocinesis, and the cells miceliales was detected a greater number of ramifications in the hyphae. The suppression gene ABG1 resulted in a series of pleiotrópicos effects such as increased susceptibility to agents that interfere in the biogenesis of the cell wall, as Calcofluor White and Red congo, and greater resistance to SDS, which suggests a possible alteration of the structure and / or composition of the cell wall. It also detected an increase in the sensitivity of the mutant strain antifungal agents to the family of azoles. That could indicate a change in one of the mechanisms of resistance to these drugs, such as flow pumps, some of which have been described that are located in the vacuolar membrane. The search for proteins that interact in vivo with Abg1p using a technique of marking with Protein A, revealed the possible interaction of Abg1p a aminopeptidasa (Ape2p) and a protein of the family of heat stress proteins (Ssa1p). From these observations one can suggest a role model for Abg1p in the vacuolar membrane of C. Albicans, in which the product of the gene ABG1 act as receiver Ape2p, to be transported in the cytosol to the vacuolar membrane through interaction with Ssa1p. This paper presents for the first time, evidence of the existence of a gene product, Abg1p, which intersects with biology vacuolar cell morphogenesis, biogenesis of the cell wall and cell cycle progression, processes all of them intimately associated with the virulence of C. Albicans. In addition, Abg1p is the first essential vacuolar protein described in this fungus, and has no peer in mammalian cells, which represent 8 to a di 2ca amy potential for the development of new therapeutic strategies.

Author: GONZALEZ-NOVO SANCHEZ ALBERTO.
Year: 2004.
University: SALAMANCA [www.usal.es].
Place of defense: FACULTAD DE FARMACIA.
Place of preparation: FACULTAD DE BIOLOGIA.

Summary: Candida albicans is a fungus opportunistic pathogen man with a growing medical interest. For C. Albicans can develop its infectivity, it is very important the ability to make the transition between different morphologies constitute its life cycle. There is a family of proteins, called septinas very important fungal morphogenesis. In the yeast model S. Cerevisiae, known seven septinas, of which five are in a ring neck between mother and daughter cells. In order to ascertain whether in C. Albicans the septinas are involved in morphogenesis, we proceeded to delecionar one, CaCDC10, and analyzed the phenotype of the strains generated. Thus, it was found that although neither the capacity nor the cellular morphology of filamentation be affected to a great extent, yes there was a severe disruption of the structure of the cell wall and the septa, and the process of cell separation. By testing in animal models, it was found that CaCDC10 is necessary to develop the infection, proper adherence to epithelial cells efficiently and colonize different tissues. We through testing location of different proteins in mutant strains of C. Albicans, that there is a hierarchy in the assembly of the various elements of the structure septinas. Finally, in order to deepen the analysis of the molecular structure of septinas of C. Albicans, we analyze the state of phosphorylation of various septinas. Thus, we see that the septina CaShs1 is fosforilada, and that this phosphorylation occurs in the neck. In addition, this protein undergoes an increase in quantity and in the filamentation phosphorylation, which are unique among septinas. These changes depend on the ciclina specific filament Hgc1 and phosphatases CaCdc14 and Ypa1.

Author: PABLO SÁNCHEZ GUADALUPE.
Year: 2004.
University: EXTREMADURA [www.unex.es].
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: FACULTAD DE CIENCIAS - UNIVERSIDAD DE EXTREMADURA.

Author: AHUATZI CHACON DEIFILIA.
Year: 2005.
University: OVIEDO [www.uniovi.es].
Place of defense: DPTO DE BIOQUIMICA,BIOLOGIA MOLECULAR.
Place of preparation: DEPARTAMENTO DE BIOQUIMICA Y BIOLOGIA MOLECULAR. FACULTAD DE MEDICINA.

Summary: Two of the most important proteins involved in the repression by glucose in Saccharomyces cerevisiae are Mig1 and Hxk2. The mechanism through which operates protein Hxk2 is not precisely known but the party dependent Mig1 is known in great detail. In this paper we show that the protein Hxk2 has a nuclear localization is regulated by glucose and Mig1, a transcriptional repressor responsible for glucose repression of many genes are required to retain the protein Hxk2 in the nucleus. Mig1 and Hxk2 interact in vivo in a trial of double hybrid and in vitro experiments inmunoprecipitación and coprecipitación type "GST-pull down." We have found that the decapeptide K6-M15 of Hxk2, it is necessary for nuclear localization of the protein is also essential for the interaction with Mig1. Our results demonstrate that the interaction Hxk2-Mig1 is physiologically significant because both proteins were detected in a complex with DNA fragments containing the site MIG1 promoter's SUC2 both in vivo and in vitro. We have also found that the S311 - Mig1 is essential for the interaction with the protein Hxk2 and location dependent Snf1 of Mig1. We conclude that Hxk2 operates by interacting with Mig1, inhibiting its phosphorylation during growth on the high glucose and generating a repressor complex to be located in the promoter of the genes of S. Cerevisiae regulated by Mig1.

Author: FERNÁNDEZ LOPEZ ALMUDENA.
Year: 2005.
University: CÓRDOBA [www.uco.es].
Place of defense: E.T.S.I. AGRONOMOS Y MONTES.
Place of preparation: E.T.S.I. AGRONOMOS Y MONTES.

Summary: The work has been developed in isolation, identification and characterization of the yeast responsible for the production of fine wines aging of the PDO "Montilla-Moriles." They have isolated strains of yeast in a cellar in the area of Montilla, in the different stages of the development of fine wine (ALVEAR Cellar). It deals with the study, using taxonomic criteria, physiological and molecular, while applying a mathematical model on the behavior and evolution of these yeasts, for different parameters wine. Lastly, there has been a comparative study with strains equivalent of the wine area of Jerez, chosen by the similarity in the process of developing and fine wine for their geographic proximity.

Author: MONTIEL DIAZ VERA.
Year: 2005.
University: CÓRDOBA [www.uco.es].
Place of defense: E.T.S.I. AGRONOMOS Y MONTES.
Place of preparation: E.T.S.I. AGRONOMOS Y MONTES.

Summary: Yeast Debaryomyces hansenii is an organism that is found in environments with high concentration saline and / or low activity as saline water or fruit juice. The mechanisms by which this body is able to develop in these environments are unknown, but suspect they are different. This paper discusses some of the factors that can determine the nature halotolerante of D.hansenii through studies of biochemical and molecular biology. In a first section discusses the characteristics of fluidity and composition of the plasma membrane of this body to various stress conditions saline and pH. In the second section identifies and explores the role of a potential target sodium Debarymyces, Hal2p. This protein is involved in the processes of salt tolerance in yeast, plants and animals. The third section discusses the distribution of intracellular sodium and potassium in this yeast and identifies a gene involved in the distribution process. Nhx1 is a conveyor vacuolar involved in the regulation of the cation concentration and pH in space citoplasmático.

Author: MARTÍNEZ-SOLANO GONZÁLEZ LAURA.
Year: 2005.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE FARMACIA.
Place of preparation: FACULTAD DE FARMACIA, UNIVERSIDAD COMPLUTENSE DE MADRID.

Summary: Macrophages are the immune system cells whose role in the fight off the systemic candidiasis is essential. On the one hand, and acted as fagocíticas cells secrete cytokines that are capable of activating other cells and directing the immune response. They can also act as antigen presenting cells. In order to deepen the role of these cells, we have done a proteomic study of the response of a cell line demacrófagos murine (RAW 264.7) compared with cells from a strain of Candida albicans (SC5314) both live as inactivated by heat as has been described to exert different effects on the activation of macrophages and epithelial cells. It has been studied phagocytosis on both cell types to 45 min. And there has been a condensation F-actina around the yeast alive. At the same time interaction has been studied the differential expression of proteins macrófago, and have been many differences of expression, not only between the baseline of macrophages control (2D map reference), and those confronted, either the cells inactivated by heat or from living cells, but also have noticed many differences in protein expression between the two stimuli. We have been able to identify 50 proteins, of which 30 are of differential expression. These 30 proteins are involved in various: morphogenesis, signal transduction, cell fate, protein synthesis, metabolism and iron homeostasis. If we attend functions differential expression of these proteins, as a whole, we can say that, after 45 minutes of interaction, both living cells and inactivated C.albicans bring out macrófago an increase in protein involved in the reorganization of the cytoskeleton and some possibly involved in protection from apoptosis. However, cells inactivated for heat stimulate the metabolic pathways as glicólisis or route acids tricarboxílicos. In addition, living cells provoke a reaction mainly pro-inflamatoria, unlike the inactivated by heat, as opposed to which the answer is basically anti-inflamatoria. In order to deepen the reaction of macrophages compared with C.alicans, we have to point the methodology for conducting the survey of four subcellular fractions: cytosol, organelle, the core and cytoskeleton and increased our time interaction to 3h. The study of differential expression of proteins of different subproteomas being done with technology DIGE. We have studied three of them observed that the fraction of the cytoskeleton is appearing more profoundly altered wings 3h interaction. This work opens up broad prospects in the study of the interaction macrófago-C.albicans may enable the discovery of new strategies in the treatment of systemic candidiasis and new virulence factors of yeast, and therefore, anti new targets fungal.

Author: PALOMINO MARTINEZ CARLOS AARON.
Year: 2005.
University: OVIEDO [www.uniovi.es].
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: FACULTAD DE MEDICINA.

Summary: During the four years of scholarship FICYT I worked on the protein Rgt1, a transcription factor that suppresses the expression of certain genes in the absence of glucose, and participating in the expression of others in the presence of the same. By Northern tests, and RT-PCR analysis of the expression beta-galactosidasa have shown that RGT1 is expressed constitutively. In addition, thanks to experiments with GFP localization using mergers have verified that constitutively localized in the nucleus. Analyzing the expression of the promoter HXK2 merged into reading the pattern of gene indicator beta-galactosidasa in mutants rgt1 (obtained through interruption genomics) and different carbon sources have shown that Rgt1 regulates gene expression HXK2 in S.cerevisiae, inhibiéndola media with non-fermentable carbon sources. But a mutant rgt1 not have altered gene expression SUC2 (inhibited by a protein complex which Hxk2 and Mig1 part), which encodes for the Invertase. These data are consistent with other findings showing that Mig and Hxk2 not have altered their pattern of cellular localization in mutants rgt1. To know how it works Rgt1 on promoter HKK2 were conducted tests delay mobility electroforética used as probes regulatory sequences in the promoter of the gene HXK2 marked radiolabelled. Different complex ADN-proteina depending on the source of carbon. To demonstrate that Rgt1 binds directly to the promoter HXK2 were repeated these tests with a protein Rgt1 recombinant purified, and conducted tests inmunoprecipitación of chromatin (CglP). Tests have been conducted on double hybrid coinmunopreciptación and coprecipitación GST -pull-down to demonstrate the interaction in vivo and in vitro between Rgt1 and proteins Hxk2 and Med8 in different growing conditions. We have also identified the specific region of Hxk2 that interacts with Rgt1 using buildings with different versions delecionadas gene HXH2. We conducted a second study to identify potential kinases and phosphatases that regulate the binding of Rgt1 DNA. Through an analysis of Western and testing delayed mobility electroforética with nuclear extracts of a battery of mutant strains and using a protein Rgt1 labeled with the epítopo HAS we find that the subunit Tpk3 of PKA is the most important for the kinase the state of phosphorylation of Rgt1 able fermentativas and Snf1 is a kinase crucial to the proper state of phosphorylation of Rgt1 in culture media not fermentativos. As for phosphatases, the subunits Bmh not participate in the elimination of phosphates Rgt1; protein Reg1, trasloca to phosphatase Glc7 to the kernel, does not seem to participate directly on Rgt1, but if you have an influence indirectly through its effect on Snf1 . In addition Tpk3 is not necessary for the union of Rgt1 DNA in growth media with glucose and Snf1 for binding to DNA in culture media not fermentativos. In mutants Glc7 Rgt1 is linked to the DNA so establishing (since Snf1 is constitutively activated). We have also identified an interaction entrelasproteínasRgt1 and Med8 (both necessary for the suppression of gene expression HXK2 in the absence of glucose) growth media not fermentativos through trials Dual Hybrid and coprecipitación GST -pull down. To provide a physiological explanation for such interaction, it was an experiment of Chromatin Immunoprecipitation of the two pairs of oligos, flanking the plea RGT1 and two flanking the plea MED8 gene HXK2 respectively. It was considered a PCr using as a mold the eluído procedent 8 and the AD 59th N ChlP and two pairs of oligos, flanking the plea RGT1 and two flanking the plea MED8 gene HXK2 respectively. Additions appeared with the two pairs (286 and 266 bp, respectively), but not the amplified fragment encompassing the two regions (854 pb) which demonstrates the formation of a blucle level of DNA, because without this structure is impossible explain that to amplify the two small fragments and not the entire piece. This test, in addition to highlighting again the interaction of Rgt1 and Med8 showed that the inhibition of gene expression HXK2 media growth with low glucose is due to the formation of a loop in chromatin produced by the interaction between Rgt1 and Med8.

Author: Valdivieso Ugarte Magdalena.
Year: 2005.
University: GRANADA [www.ugr.es].
Place of defense: Facultad de Ciencias.
Place of preparation: Puleva Biotech S.A..

Summary: With the exception of those called anaerobic organisms, which are adapted

Author: GARCIA LLORENTE IGNACIO.
Year: 2005.
University: SALAMANCA [www.usal.es].
Place of defense: FACULTAD DE BIOLOGIA.
Place of preparation: DEPARTAMENTO DE MICROBIOLOGIA Y GENETICA.

Summary: The (1.3) -glucano is a polymer essential in the cell wall of Schizosaccharomyces pombe. The thesis focuses on the study of biosynthesis and degradation of the polymer. The biosynthesis of (1.3) -glucano is carried out by Mok1p during vegetative growth. There has been detected the presence of activity (1.3) -glucanasa in S. Pombe, however in the yeast genome of this there are two ORFs encoding two proteins, Agn1p and Agn2p with high identity with enzymes described (1.3) -glucanasas other fungi. In part that analyzes degradation (1.3) -glucano demonstrates that Agn1p and Agn2p are enzyme activity (1.3) -glucanasa. Agn1p is necessary for cell separation occurs properly. Agn1 + cyclically and expressed protein is located in the septum of separation, and the enzyme responsible for the degradation of (1.3) -glucano ring cell wall in the region of the septum. In part that analyzes the biosynthesis of (1.3) -glucano characterized proteins Mok11p, Mok12p, Mok13p and Mok14p, these proteins have high identity and similarity with the (1.3) -glucán synthase Mok1p. Mok12p, Mok13p and Mok14p are necessary for the process of forming the ascosporas happen normally. Mok12p is located in the membranes of ascosporas and synthesizes a (1.3) -glucano necessary for the ascosporas will pack correctly, and viable. Mok13p is located in the membranes of ascosporas early in the process of training them and synthesizes a (1.3) -glucano necessary for the ascosporas are resistant to various stress conditions. Mok14p synthesizes (1.4) -glucano giving the spores characteristic brown color in the presence of Iodine vapors. This polymer is involved in the resistance of spores to different types of stress. Mok14p and (1.4) -glucano are located both partners to the wall of asca as the ascosporas.

Author: MARTIN CUADRADO ANA BELEN.
Year: 2005.
University: SALAMANCA [www.usal.es].
Place of defense: CENTRO DE INVESTIGACION DEL CANCER.
Place of preparation: INSTITUTO MICROBIOLOGIA - BIOQUIMICA.

Summary: The cell wall is a structure present in yeast and other fungi that completely surrounds the cell serving protection. Enzymes involved in the dynamics degradativa of beta-glucano, majority component of the cell wall, are beta-glucanasas, which catalyze the hydrolysis of liaison beta-O-glicosídico of the carbohydrate chain. The first part of the thesis focused on the structural study of (1.3) beta-endoglucanasas the family GHF81 of the yeast Schizosaccharomyces pombe, SpEng1 and SpEng2 and Saccharomyces cerevisiae ScEng2. It proceeded to purify these proteins and analyze different biochemical variables. By HPLC-PED was studied its mechanism hydrolytic demonstrating its ability to degrade at least one laminari-pentasacárido, in a manner similar to other family members GHF81. We identified the potential catalytic domain of ScEng2 at its carboxyl end, which was then purified and analyzed. By directed mutagenesis were identified several key amino acids in its enzymatic activity. In addition, it has been shown that SpEng1 has a domain binding glucanos at its carboxyl end, it was found to be purified and specificity compared to insoluble substrate with link (1.3) beta. Subsequently, we studied the involvement of Eng1 and Agn1 ((1.3) alfa-endoglucanasa) in the separation process and the cellular mechanisms of transport and in areas where action is required. We note that Eng1 is necessary to degrade the primary septum that separates the two daughter cells after mitosis, and Agn1 the cylinder wall that surrounds this septum of separation. Both proteins are located in a ring around the septum cells in the wild. The study of its location in mutants defective in cell separation, as the complex exocisto, mid2 and spn, concluded that in S. Pombe, the ring of septinas function as a marker for the correct positional secretion and location of Eng1 and Agn1, which would be transported by vesicles towards the ends of the septum through the complex exocisto.

Author: CONDE LUQUE RAUL.
Year: 2005.
University: EXTREMADURA [www.unex.es].
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: FACULTAD DE CIENCIAS.

Author: GARCIA SALCEDO RAUL.
Year: 2006.
University: CÓRDOBA [www.uco.es].
Place of defense: ETSIAM.
Place of preparation: ETSIAM.

Summary: Agriculture extensive arid and semi-arid areas of the world depends on the irrigation and enfrena with a serious dilemma: to increase or at least maintain crop productivity as well as soil and water are becoming increasingly saline. Although the problem of salinization affecting mostly agricultural plants of interest, these are not the only systems models used for research in this area. Saccharomyces cervisis is an experimental model simple and user-friendly that it is frequently used in the study of salinity. In the present work is proposed as a model organism for this kind of research to Debarymoyces hansenii, a yeast more tolerant to salt S.cerevisiae, and for this reason offers certain advantages for identifying new genetic factors involved in the mechanisms of salt tolerance. D.hansenii is a yeast navy also dwells in other environments brackish and sausages why has been defined as a yeast osmo-, cero- and halotolerante. In this paper we have addressed thesis studying the halotolerancia and tolerance to high pH D.hansenii by finding new genes (GZF3 and TDH1) involved in these processes as well as through the study of a gene previously described in S. cerevisiae (KHA1) under the mechanism halotolerancia. The gene DhGZF3 coding for the first transcription factor GATA family of the identified D.hansenii. The analysis of this gene based on its overexpression in S.cerevisiae suggests that like ScGZF3 regulates negatively the expression of genes sensitive to catabolite regulation of nitrogen (RCN), but that was different from the gene S.cerevisiae for their participation in certain phenomena such as tolerance to alkaline pH and salt. The expression of DhGZF3 gives S.cerevisiae a phenotype of tolerance of high pH and certain drugs such as rapamycin phenotype as well as a sensitivity to toxic cations such as lithium and sodium. This second phenotype is explained on the basis of the decrease in the levels of expression of the gene ENA1 into cells carrying the gene D.hansenii. The transcriptional analysis of cells that sobreexpresan DhGZF3 reveals the suppression of a large number of genes, many of which are involved in the metabolism of nitrogen, among these striking inhibition of the expression of all genes involved in the synthesis of Lys from La-aminoadipato. Among the carriers described in S.cerevisiae related ionic homeostasis is the encoded by the gene KHA1. The work on this gene agree to assign a role in the transport of potassium and in the regulation of intracellular pH, but disagree on other aspects such as their location cellular barajándose as the plasma membrane potential or orgánulo intracellular. The genome sequence of D.hansenii there is very similar to coding gene KHA1 of S.cerevisiae. The results of the study suggest that this sequence encodes a protein involved in the transport of sodium, located at some orgánulo intracellular possibly the Golgi apparatus. The study of halotolerancia in D.hansenii through proteomics methodology has allowed the identification of several proteins whose expression is altered as part of the answer adapatativa this yeast to salt. The gene that encodes one of these proteins has a high degree of genes THD1-3 of S.cerevisiae which encode three isoforms of the enzyme gliceraldehido-3-P dehydrogenase (GAPDH). In the presence of sodium induces the expression of the protein DhTdh1 and consequently under these conditions increases the activity GAPDH.