MOLECULAR BIOLOGY (3)

Author: HERNÁNDEZ LÓPEZ MARÍA JOSÉ.
Year: 2005.
University: VALENCIA [www.uv.es].
Place of defense: INSTITUTO DE AGROQUÍMICA Y TECNOLOGÍA DE ALIMENTOS.
Place of preparation: INSTITUTO DE AGROQUÍMICA Y TECNOLOGÍA DE ALIMENTOS.

Summary: In recent years the use of mass for frozen bakery and pastry has experienced a marked increase. The callidad of these products is far from that obtained with fresh masses because of the sensitivity of the commercial strains of S. Ceremisiae the process of freezing and to the high pressure osmóticas. In the first chapter of this thesis explores the possible use of strains Torulaspora delbrueckii able to ferment a mass panaria and having intrinsic characteristics of criorresistencia and osmotolerancia. The results obtained in this chapter show that a strain of this species, strain Pycc5321 shows a high tolerance to osmotic stress and ionic therefore propose to this strain as a model for study of stress tolerance in yeast baking. In Chapter II describes the production of the molecular tools needed to be able to work with this strain in the laboratory. In Chapter III of this paper describes the isolation of the protein Tdhog1 of this strain, as well as the characterization of the route H0g, the main route implicated in resistance to osmotic stress EUS. Cerevisiae in this species. This chapter also shows the isolation of genes T. Delbrueckii TdENA1 and Tdcrz1, counterparts, respectively, a gene that encodes for Atp-0sa Ena1 S. Cerevisiae and one that encodes for a transcription factor, CRZI involved in regulating ENA1, and the characterization of the route of Calcineurina-CRZ1p in T. Delbrueckii.

Author: MARTÍNEZ BONO BÁRBARA.
Year: 2005.
University: VALENCIA [www.uv.es].
Place of defense: BIBLIOTECA CAMPUS DE BURJASSOT.
Place of preparation: FACULTAD DE CIENCIAS BIOLÓGICAS.

Summary: The route of the Protein kinase C in Saccharomyces cerevisiae desemperia essential roles in the control of cellular integrity, and in addition it has been suggested that participating in the coordination between growth and cell division. This paper has studied the molecular mechanism of regulation of gene expression mediated by PKC route, modeled on gene FKS1 coding for the catalytic subunit of the glucan synthase. It has been marked that the minimal sequence responsible for the regulation mediated by PKC route in the gene FKS1 correspond to a sequence of binding of factor Rlm1. Union also has been detected in vivo Rlm1 the promoter gene FKS1 and that the binding capacity is not regulated by PKC route. On the other hand, I found that the MAP kinase SIt2 the route PKC binds to the DNA in the area near the ATG gene FKS1. It detects that the RNA polymerase II binds to both IGT area as at the end of the gene FKS1 regardless of the role of PKC route. On the other hand, there has been a change post-traduccional of protein Paf1 (member of the complex Paf1 C) dependent SIt2, consistent with a possible phosphorylation of Paf1 by SIt2. We note that although the route does not regulate the ability of the union alive in gene FKS1 of Paf1 in the area if it appears IGT controlling the displacement of Paf1 to 10 throughout the gene FKS1. This thesis has done a comprehensive study of expression of a mutant pkc1-8 heat sensitive, the study has identified 65 genes whose expression was significantly modified to inactivate the function Pkc1. In the same vein we have identified proteins associated with Pkc1 and Sit2 using the TAP method of purification of proteins. A highlight proteins Rpc34, Ssk22 and Apd 1 that were detected associated with Pkc1. And proteins Mot1, Sec31 and Kap123 associated with SIt2. Studies have found a relationship between the carioferina Kap123 the route PKC. Kap123 could participate in the route PKC imported to the kernel to any of the proteins downstream in the path PKC. It has also identified a relationship between the gene route PKC and cíclina Cln2, in which the deletion of the gene CLN2 suppresses the growth of defects in mutants of PKC route, possibly due to the delay in the process gemación that supane inactivation the cíclina Cln2.

Author: RODRÍGUEZ PIÑEIRO ANA M..
Year: 2005.
University: VIGO [www.uvigo.es].
Place of defense: FACULTAD DE BIOLOGÍA.
Place of preparation: FACULTAD DE BIOLOGÍA.

Summary: Colorectal cancer (CRC) is one of the most common malignancies. However, at present there are no good markers for tumor diagnosis and prognosis. Therefore, in this paper we have used biochemical methodologies proteómicas and, in order to detect serum tumor markers useful. First, they used a method prefraccinamiento by affinity chromatography through Concanavalina In analyzing fraction N-glicoproteínas, as they have many alterations in carcinogenesis. Using two-dimensional electrophoresis, protein patterns were studied using three different approaches. The first detected 72 proteins with altered levels in RCC, 37 plus. Among them were included proteins associated with apoptosis (clusterina) and signal transduction (LRG), of great interest as potential markers for CRC. The second approach is to use two-dimensional protein patterns as a diagnostic tool. Using analysis and principal components analysis discrimianate, could establish a pattern of differential expression, which enables the diagnosis of an individual. The third approach is the detection of changes in the relative position of proteins in two-dimensional maps, which reflect changes in the isoelectric point and molecular mass. Through analysis of deformation related, were detected significant changes in the position of N-glicoproteínas serum of patients with RCC, permmitiendo also discriminate between healthy individuals and patients, and the detection of the contribution of each protein. Since proteins with altered levels are selected clusterina, in order to study its various forms serum, and compare their distribution and levels in the serum of healthy individuals and patients with CRC. The most remarkable discovery was the detection, exclusively in patients, a form of clusterina eluted in the first chromatographic fraction (EF CLU), consisting of at least 5 isoforms, with a mass of 85 kDa and native of 40 kDa subunits. His desglicosilación revealed that it was a N-glicoforma, probably with an aberrant glycosylation. In relation to its clinical utility, it was determined that the clusterina total serum has a higher efficiency than the diagnostic marker for clinical use. AEC. The most important clinical finding was significant correlation of how EF CLU with metastasis. This fraction clusterina behaves as a prognostic marker for the RAC.

Author: ACOSTA COBACHO JUAN CARLOS.
Year: 2005.
University: CANTABRIA [www.unican.es].
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: FACULTAD DE MEDICINA.

Summary: During the doctoral thesis were established lines transfectantes stable from K562 with inducible expression of p27. In ectopic induction of p27 was found that the protein induced a stop in proliferative phase G1 cell cycle, accompanied by inhibiting the activity of regulators phase G1. Also, it was observed that p27 induced a process of differentiation toward specific erythroid lineage. To investigate the functional interaction of the protein p27 and MYC was established lines transfectantes stable from K562 with a conditional expression of both p27 and MYC. Analysis of these cell lines, it was concluded that MYC is able to block the erythroid differentiation induced by p27, in a process independent of its action on the cell cycle. In addition, it was found that the blockade of differentiation mediated by MYC is done primarily through the repression of key genes in controlling such erythroid differentiation. Moreover, it was found that MYC had an effect on the cell cycle dependent on the amount of p27 in that system. Finally, it was found that there was an inverse relationship in the expression of MYC high and low p27 in B lymphocytes from patients with chronic lymphocytic leukemia.

Author: DANEL KORTAZAR ZABALA.
Year: 2005.
University: CANTABRIA [www.unican.es].
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: FACULTAD DE MEDICINA.

Summary: Following the decoding of the human genome, and the fact that every day there are more diseases resulting from a folding abnormal proteins (approximately 50% of the pathologies), is now a need to know how to fold polypeptide to develop its role . One of the more complex models - completos- folding is the process that leads to the formation of heterodímero of tubulin, subunit microtubule. The microtubules are polymers of tubulin, ubiquitous, involved in all key functions varied in the cell, from mitosis (and meiosis), the cytoplasmic transport and maintenance of the cellular architecture. The functional and structural subunit of microtubules is heterodímero of alfa-beta-tubulina that, united with one another in a linear form the protofilaments, thirteen of them partners in a circle forming a microtúbulo. The microtubules are dynamic structures citosqueleto tremendously, and precisely on the ability of these dynamics in tbsp cell (tissue) depend on his / her specific functions. The de novo synthesis of heterodímeros from polypeptides of alpha and beta-tubulina is a complex process which starts with the interaction of newly synthesized polypeptides with CST and prefoldina. Once released the polipeptidos of chaperonina are processed by the cofactors of tubulin: TBCA, TBCB, TBCC, TBCD, TBCE and Arl2. Following the association of tubulinas with them forming different complexes, finally, after the hydrolysis of GTP, it frees hterodímero of alfa-beta-tubulina responsible for the polymerization into microtubules. To date the role of these factors in vivo is not known, although it is known that these proetinas are necessary for life and that mutations in their genes produces síndormes, some of them very important. Recent studies indicate that, apart from being involved in the folding of tubulin, these cofactors may be involved in the dynamics microtubular, because when they are sobreexpresados in cells despolimeriza completely citosqueleto microtubular. In fact, contrary to what was thought, tubulinas are not proteins unstable and heterodímeros not dissociate or are associated in the absence of cofactors of folding. Thus, the cofactors of tubulin might have missions in vivo as modulate the dynamism with a view to promoting the exchange of subunits of tubulin, for example, tubulinas with different postraduccionales isotipos or different. Both TBCE and TBCB join the alfa-tubulina. Studies along this thesis show that sobrexpresión of TBCB, like that of TBCE, is capable of despolimerizar the microtubules in the cell. This finding is part of the driving force behind the studies in the thesis. Understanding how TBCB is capable of despolimerizar polymers tubulin when in vitro studies demonstrate that does not interact with tubulin in its native form (as heterodímero) is the body of the investigation. On one side it shows that TBCB not participating in the de novo synthesis of active tubulin and not form complexes with heterodímeros of tubulin tubulin, although it is capable of joining alpha tubulin during the process of synthesis in vitro (ie, TBCB recombinant studied is functional). These results lead the authors to question whether TBCB could interacionar with TBCE to unite and decouple heterodímero of tubulin. Experiments conducted with both cofactors produced and purified recombinant form, incubated in vitro alone or in various combinations thereof with / without tubulin finally demonstrate that TBCE and TBCB act together, amplificándose the effect it produces TBCE only. These findings are confirmed by in vivo experiments co-transfección. Subsequent studies of high resolution electrophoresis gel filtration accompanied by denaturants not finally demonstrate that TBCE and TB 8 CB inter 49th acionan physically with a estequiometría 1:1, and in turn to the alpha tubulin. Finally this thesis proposes a model of interaction between the two cofactors through one of its domains, the domain UBL both share but in both, in agreement with published structural models, may be seen up structural zones that would encourage interaction between the two proteins, which in turn would promote the dissociation of dínero of tubulin and the union of the alpha-tubulin complex.

Author: RAMON PORTES EVA.
Year: 2005.
University: POLITÉCNICA DE CATALUÑA [www.upc.edu].
Place of defense: ETSEIB, EDIFICI H PLANTA 4 Campus SUD.
Place of preparation: ETSEIB, EDIFICI H PLANTA 4 Campus SUD.

Author: VAQUE DIEZ JOSE PEDRO.
Year: 2005.
University: CANTABRIA [www.unican.es].
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: FACULTAD DE MEDICINA.

Summary: This study analyzed the functional interference between oncogenes Myc and Ras in two models where the cellular activity of Ras is antriproliferativa and distinguish. In the first cellular model, derived from human chronic myelogenous leukemia (cell K562), it is shown that overexpression of the three genes both wild and oncogenic Ras inhibits the growth of this cell line. This finding is consistent with the absence of Ras gene mutations detected in the disease. The mechanism involves activation of the protein p21, a cell cycle inhibitor. This paper shows that Ras active p21 through activation RAF-ERK in a manner independent of p53. The transcription factor oncogenic Myc, it is able to inhibit the activity of Ras on the activation of p21 at a point between the union of Sp1 the promoter of p21 and the kinase activity of ERK. This mechanism is different from any other mechanism proposed to explain how Myc inhibits transcription although there have been analyzed and ruled out other options in this system. Finally it is shown that Myc reverts stop proliferative caused by Ras in these cells. The second model cell (cell UR61) differs in response to the activity of Ras. It has been found that cells UR61-Myc no different to activate the route Ras-RAF-ERK. Myc in this system is able to inhibit the transcription of the gene of c-Jun. This inhibition is responsible for the lack of differentiation of the cells UR61-Myc compared to control cells. The fact that this model has no cellular protein Max (considered essential for transcriptional activity of Myc), gives this study of particular interest, because it shows the ability of Myc to join the DNA and regulate processes in a way transcripcionales independent Max, unpublished until this work. In addition, it was demonstrated that inhibition of differentiation Myc is decoupled from possible effects on the activation of proliferation. This information is also of interest because many authors believe that the ability to increase the proliferation is responsible to inhibit differentiation processes terminal Myc. This work shows that the specific inhibition of transcription of genes involved in differentiation is used by Myc to inhibit differentiation in the model cell UR61.

Author: SAURÍ PERIS ANA.
Year: 2005.
University: VALENCIA [www.uv.es].
Place of defense: BIBLIOTECA DEL CAMPUS DE BURJASSOT.
Place of preparation: FACULTAD DE CIENCIAS BIOLÓGICAS - UNIVERSITAT DE VALÈNCIA.

Summary: The propagaci6n a infecci6n viral plants this mediated proteins encoded by the virus known as protein movement (MP). These proteins bind the viral genome cooperatively and without specific sequence, forming complex RNA-MP, which is associated with the endoplasmic reticulum (ER) to be transported through the cytoskeleton toward plasmodesmos channels membranosos interconnecting the cell plant . Once these complex reaches plasmodesmos, RNA is translocado to adjacent cells through a mechanism is still unknown. The combination of these complexes RNA-MP ER proves to be essential for this transportation celula-a-celula RNA virus takes place. So, the central objective of this thesis has been based on the study and characterization of the association of these proteins in the membrane of this orgánulo. The virus has been the subject of study virus Moteado of Carnation, which encodes two MPs, p7, a soluble protein capable of uniting the viral genome and p9, introducing two regions hidrof6bicas likely to interact with cell membranes. First, through experiments transcription / translation in vitro has been shown to p9 is an integral membrane protein that contains two transmembrane fragments and adopts an orientation N-/C-terminal cytoplasmic. Secondly, it has been characterized the mechanism for integrating p9 in cell membranes. The results obtained show that the insertion of p9 takes place so co-traduccional and is dependent SRP. Addition, the virus exploits's cell machinery responsible for the integration of cell membrane proteins. The complex Sec61 and TRAM protein involved in the process of inserting p9 in membrane IR. Finally, there has been a topological study of the determinants of the protein that determine the orientation of viral protein in the membrane. We have explored a wide range of parameters, including the presence of charged residues in the sequence of the protein, hydrophobicity of the transmembrane fragments, the length of domain extramembranosos and / or the presence of aromatic residues. The results show that the protein viral p9 Reporting topological distributed to 10 throughout the sequence of the protein, so the possible emergence of a mutation not conservative, very common in viral genomes, does not alter the orientation of the protein in membrane yen definitive its biological function.

Author: MARTÍNEZ JIMÉNEZ CELIA PILAR.
Year: 2005.
University: VALENCIA [www.uv.es].
Place of defense: FACULTAD DE MEDICINA DE LA UNIVERSITAT DE VALÈNCIA.
Place of preparation: FACULTAD DE MEDICINA.

Summary: At present has substantially increased demand for liver cells for functional prediction metabolism and toxicity of new drugs as well as for clinical applications such as cell therapy and the study of liver diseases, so the development of different human cell models has become an important issue for both basic and applied science. However, the use of models in vitro human hepatocellular has serious deventajas as are processes desdiferenciación and loss of hepatic phenotype. First have characterized various human hepatoma to establish their degree of desdiferenciación and its loss of phenotype. Secondly, we have studied the molecular mechanisms associated with these processes desdiferenciación focusing on the transcription factors liver HNF4? C / EBP, and their relationship in the transcriptional control of cytochrome P450. And finally has studied the role of coactivadores PGC1? And SRC1 in maintaining the hepatic phenotype and in the regulation of cytochrome P450. C / EBP has two isoforms: LAP and LIP, participating in a coordinated way in the process of inflammation by regulating the expression of CYP3A4 and thus playing an important role in interindividual variability of the expression of this cytochrome. After studying the molecular mechanisms involved in the transcriptional regulation CYP3A4 we saw that the role of the transcription factor hepatic HNF4? It is limited by two coactivadores keys in the process of gene regulation of genes involved in the maintenance of the liver phenotype are PGC1 and SRC-1. These coactivadores play a crucial role in maintaining the role of transcription factors closely related to the development and differentiation of liver cells as in the case of Factor HNF4. We have studied as PGC1, and to a lesser extent SRC-1, are essential for maintaining the functionality factor HNF4 in genes involved in both the hepatic metabolism of endogenous compounds, as well as exogenous compounds would like genes cytochrome P450 metabolising xenobiotics .

Author: TORRANO MOYA VERONICA.
Year: 2005.
University: CANTABRIA [www.unican.es].
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: FACULTAD DE MEDICINA.

Summary: CTCF is a transcription factor described as a repressor of c-MYC. In its structure are dominated by binding to DNA containing zinc finger eleven. CTCF is a nuclear protein expressed ubícuamente and highly conserved. Thanks to the combination of its eleven zinc fingers, CTCF is capable of joining different DNA sequences, with no consensus sequence binding. Thanks to this capability multipotente binding to the DNA, CTCF it is viewed as a multipurpose transcription factor. Among the genes regulated by CTCF are at the MYC oncogene, the gene of the chicken lysozyme, the gene APPB and gene telomerase. CTCF controls a large number of sequences insulating chromatin, is the only protein described cfapaz joining these sequences. CTCF is modified by phosphorylation and poly (ADP-ribosil) ation. Previous studies in this work indicate the ability of CTCF as an inhibitor of cell growth. And there are econtrado loss heterocigosidad in the locus where mapped the gene CTCF. They also point mutations were found in their zinc fingers in some tumors. Therefore, CTCF is a good candidate tumor suppressor gene. The importance of CTCF in the regulation of cell proliferation has been described previously, however its possible role in cell differentiation has been less studied. Preliminary results showed a differential expression and phosphorylation of CTCF during myeloid differentiation. One objective of this work was to analyze the role of CTCF in proliferation and differentiation of cells mielodies K562. The results showed that overexpression of CTCF cells K562 leads to a delay in cell growth, without leading to an increase in apoptosis. As for differentiation, on the expression of CTCF induces an increase in the expression of markers of erythroid differentiation, without affected differentiation megacariocítica. One of the proposed mechanisms by which CTCF might mediate their effects is the inhibition of MYC. This study has shown that CTCF is located in the nucleolus cell K562 differentiated into neurons and post-mitóticas. It has been shown that the domain responsible for this location is the domain of binding to DNA, that localization is dynamic, dependent rDNA transcription and protein synthesis. It also shows that the nucleolar localization of CTCF inhibits transcrición nucleolar through a mechanism dependent on the activity poly (ADP-ribosil) polymerase.

Author: MASIÁ ADALID SUSANA.
Year: 2005.
University: VALENCIA [www.uv.es].
Place of defense: FACULTAD DE MEDICINA DE LA UNIVERSITAT DE VALÈNCIA.
Place of preparation: INSTITUTO DE BIOMEDICINA DE VALENCIA-CSIC.

Summary: The study of the actions of retinoic acid (RA) in neuroblastoma cells has two tracks of interest. First, the RA is a serial that plays a central role in embryonic development and the generation of various organs and systems, including the nervous system, and 10 so there is a scientific interest in knowing the molecular mechanisms by which these exercises shares. Second, the RA and its synthetic derivatives are being tested in neoplastic therapy, because of its ability to regulate the growth, differentiation and cell survival, clinical outcomes with very relevant, as in the case of neuroblastoma. However, the molecular mechanisms by which the RA exerts its therapeutic actions have not been clearly established. The aim of this thesis has been to decipher the molecular mechanisms by which the RA induces differentiation of neural cells, using as a model system of neuroblastoma cells. Preliminary laboratory results indicate that, in neuroblastoma cells, the response to the administration of RA not only occurs at transcriptional level, but also leads to the activation of the signaling path of PI3K/AKT (López-Carballo, G. 2002 . J Biol Chem 277, 25297-25304). This activation is required for induction of differentiation by RA and think that occupies a central place in the control exercised by RA on growth, differentiation and cell survival, so we lIevado out their characters with more detail. Through studying the mechanistic aspects of the activation of the road to serializacion of PI3K/AKT by RA, we determined that the activation of pathways PI3K/AKT and MAPK / ERK by RA occurs through a mechanism not genomic it does not require new or transcription of genes or protein synthesis. Furthermore, we have shown that this activation is not unique to neuroblastoma cells, but is a more general process. It has established the participation of Nuclear Receptor activation of RAR in the path of serialization of PI3K/AKT, and what we have characterized domain receptor RAR is involucrads in the activation of the path of PI3K/AKT. We have also shown that the intracellular localization of RAR plays an important role in the activation of PI3K, and have described the physical interaction between the receiver and RAR components of the transduction channel signal PI3K/AKT and of the union of the RA receiver RAR regulates these interactions. Finally, we have analyzed the consequences of these actions not transcripcionales genomic of RA in neuroblastoma cells by using DNA microarrays. The findings raise a new model for the activation of the via PI3K/AKT by RA in which the binding of ligand to receptor nuclear RAR plays a central role in regulating the intracellular localization of RAR and interaction with RAR the subunit of PI3K.

Author: MARTÍNEZ HERNÁNDEZ CRISTINA.
Year: 2005.
University: POLITÉCNICA DE VALENCIA [www.upv.es].
Place of defense: UNIVERSIDAD POLITÉCNICA DE VALENCIA.
Place of preparation: UNIVERSIDAD POLITÉCNICA DE VALENCIA.

Summary: It has traditionally been considered to -oxidación as a process essential for seed germination, mobilizing lipids stored at the plant and providing enough energy for its development. However, it also is involved in the processing of complex molecules that lead to simpler molecules, such as acids jasmónico (BOA) and salicylic (SA) with important roles in the development of the plant and on the responses of defense against some stressors. The present work has been carried out the molecular and functional analysis of genes ACXs Arabidopsis thaliana, responsible for the first step of the -oxidación. Of the six genes ACXs described, it was found that only ACX1 responds to stressors nature of biotic and abiotic, representing a node activation in response to different stressors. By generating antisense transgenic lines for AXC1 showed that this gene encodes a protein of the -oxidación involved in the synthesis of BOA in response to the wound in Arabidopsis. This function ACX1 connects -oxidación with the activation of responses to defend the plant against a wide range of stressors. However, the role of ACX1 is not limiting for activating defenses dependent JA or SA, since T-DNA lines void for ACX1 trigger a full expression of marker genes both signaling pathways in response to factors as the wound or stress inoculation with pathogens biotrofos. It was also found that the reduction or cancellation of transcribed from ACX1 does not result in changes in processes of development in which the BOA or -oxidación are involved as germination, the transition to the stage or reproductive senescence, and it does alter the baseline resistance versus pathogens biotrofos. However, the analysis transcriptómico, conducted in the present study suggests that the reduced expression of ACX1 leads to numerous changes molecular expressed by the reduced expression of many genes whose function is related directly or indirectly to defense. Therefore, alterations in the -oxidación not yet leading to a readily detectable phenotype can be instrumental in organizing the activation and maintenance of different responses in defense plants under various stress situations.

Author: AGORIO NORSTRÒM ASTRID MARÍA.
Year: 2005.
University: POLITÉCNICA DE VALENCIA [www.upv.es].
Place of defense: UNIVERSIDAD POLITÉCNICA DE VALENCIA.
Place of preparation: UNIVERSIDAD POLITÉCNICA DE VALENCIA.

Summary: The gene EP5C codifies an extracellular peroxidase cationic and was identified with Lycopersicon esculentum, which noted that its transcription is induced in pathogenic interactions compatible and incompatible. The H202 is the signaling molecule that activates transcription of EP5C, while other signaling molecules in response to pathogens such as salicylic acid (SA), acid jasmónico (BOA), or ethylene (ET) do not. Based on this observation, that argument was used in the gene EP5C as a marker to study the route of signal transduction mediated by H2O2 in the defensive response versus pathogens. To address this study we used a strategy of molecular biology studies complemented by reverse genetics, and a strategy of using direct genetic mutants ocp (overrexpressor of cationic peroxidase) that activate the transcription of the transgene pEP5C:: GUS of constitutively in Arabidopsis thaliana. A functional analysis of promoter EPSC in A.thaliana was protected an area promoter EP5C, 51 pb, called R1 and required for the activation of transcription of the gene compared with Pseudomonas syringae pv.tomato DC3000 (PST DC3000) or H2O2. A tracking in a simple hybrid yeast transcription factors led to the identification of A.thaliana who join R1, which belong to the family R2R3 factors MYB or family WIP. After characterization of the union to R1 of transcription factors MYB46, MYB112 and WIP2 showed that the first two of the three required guanine element present in the cis MBSII contained in A1 to join the latter, on the contrary, Studies resistance to pathogens Pst DC3000 noted that none of the three factors is necessary for the proper establishment of the defenses in front of this bacteria. While studies of the regulation of transcription of pEP5C:: GUS in A.thaliana showed that the absence of MYB46 favors induction of transcription of this gene in pathogenic infections, and that MYB112 is necessary for the constitutive expression of pEP5C: GUS in the mutant ocp3. In a second phase to be studied mutant ocp11 established that it is a mutation semidominante that constitutively expresses the transgen pEP5C:: GUS. Analysis of resistance showed that ocp11 is hipersusceptible the pathogen biotrofo Pst DC3000 both compatible and incompatible interactions, susceptible to the bacterium "non-host" P.tabaci and hipersusceptible to fungi necrotrofos Botrytis cinerea and Plectosphaerella cucumerina. Studies indicate that molecular markers probably inhibition of resistance that shows the mutant ocp11 not depend on the routes defenses classic mediated by SA or JA / ET. Finally clonó mutation ocp11, which helped pinpoint the gene OCP11 as the gene AGO4 and turn the mutation ocp11 in allele ago4-2. It was demonstrated that the mutant ago4-2 have reduced methylation of islands CpNpGp in ellocus AtSN1, deducted a loss of function of the mutant protein AGO4 in this regard to the methylation of AtSN1. In this work associates for the first time mutants ago4 involved in the methylation of DNA and histones, to the defense of the plants in front of bacterial and fungal pathogens.

Author: ORPINELL MERCADÉ MERITXELL.
Year: 2005.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAT DE BIOLOGÍA.
Place of preparation: FACULTAT DE BIOLOGIA.

Summary: This project addressed the involvement of the gene DOR (Diabetes and Obesity Related Gene) in the pathophysiology of diabetes mellitus type 2 through an analysis of molecular biology (1) and cellular (2) of the protein encoded by this gene. 1-Molecular Analysis of the pattern of distribution and function through the characterization of interactions with other proteins. Based on preliminary evidence suggesting a role of DOR as a transcriptional regulator of the activity, has been established DOR potential interaction with proteins involved in gene expression mechanisms, such as nuclear hormone receptors (TR, GR, PPAR), coactivadores (SRC-1), histone acetilasas (CBP, p300), and desacetilasas (SirT1) through tests inmunoprecipitación protein or chromatin (ChlP). It has also been described as the interaction of two proteins citoplasmáticas previously identified by the technique of Double Hybrid in Bread. The analysis of the interaction of DOR with Translokina allowed for a possible regulatory mechanism of this in the translation of DOR's core and their levels of expression and, consequently, its regulatory activity. Moreover, the interaction of DOR with TXNIP allowed for a possible involvement of DOR in the mechanisms responsible for maintaining the redox potential at the cellular level. 2 - The characterization of the pattern of expression of cell and tissue protein DOR, both endogenous as ectopic, had identified mechanisms involved in the localization and expression of it, such as differentiation miogénica, the transcriptional activity, traduccional and proteasomal, stability citoesqueto, the state of phosphorylation, as well as the energy state or cellular redox potential.

Author: PUJADAS PUIGDOMÈNECH LLUÍS.
Year: 2005.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAT DE BIOLOGÍA DE LA UNIVERSITAT DE BARCELONA.
Place of preparation: FACULTAT DE MEDICINA - FACULTAT DE BIOLOGÍA.

Author: VALLS JORDANA ESTER.
Year: 2005.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAT DE BIOLOGÍA.
Place of preparation: FACULTAT DE BIOLOGÍA.

Author: MORENO GONÇALVES ANA BEATRIZ.
Year: 2005.
University: BARCELONA [www.ub.es].
Place of defense: IBMB-CSIC.
Place of preparation: IBMB-CSIC.

Summary: The plants are constantly subjected to different environmental stress. Fungi cause a large number of diseases in plants, being responsible for major economic losses. Currently, control of these diseases is carried out through the use of chemical compounds, with a consequent negative impact on the environment and on health. An alternative to chemical control is to obtain transgenic plants resistant to fungus. At first, most of transgenes used for this purpose came delas own plants (genes AB). Currently, given the reduced effectiveness of this strategy, are working on the identification of genes from other defense agencies, such as bacteria, insects, animals and even other fungi. In this paper, has evaluated the usefulness of the protein AFP (antifungal protein), produced by the soil fungus Aspergillus gigantesus, to act as antifungal agent versus pathogens of plants, more specifically geranium and rice. The protein AIA is a small protein with a very basic and compact structure, which is secreted extracellular space. Previous studies had shown its antifungal activity in front of various filopatógenos (The chain et al, 1995; Vila et al 2001). Thus, the first objective of this thesis was to study the antifungal activity of this protein in front of the fungus Botrytis cinerea. This fungus is responsible for the gray rot disease in many plants, including ornamental. Our results reveal that the protein AFP presents a strong antifungal activity in front of different strains of Botrytis cinerea, inhibiting both the development of higas as germination of spores. When used in combination with the protein cecropina A lepidopteron, there is an antifungal additive effect between the two, which can be useful for development of a strategy for simultaneous expression of both genes in transgenic plants. In addition, the AFP inhibits the growth of B.cinerea both in vitro and in vivo, geranium plants. Moreover, the fungus Magnaporthe grisea, is responsible for the disease known as piriculariosis in rice plants. The antifungal activity of the protein AFP address this fitopatógeno had been previously described both in vitro and in vivo (Vila et al, 2001). This paper has developed a strategy to express the gene afp of inducible and controlled manner, thus avoiding possible negative effects of a constitutive expression, both metabolic spending by the plant, and its acceptance by the consumer end . The results showed that the promoter of a gene PR maize gene ZmPR4, is functional and induced by the fungus M.grisea in rice plants. This promoter is capable of controlling gene expression afp and confer resistance to infection by the fungus Magnaporthe grisea in transgenic plants. Their use has an additional advantage because this promoter is not active in the seed endosperm of the rice (organ intended for consumption), which prevents the proceeds of trangén to accumulate in the tissue. Finally, given the great potential of gene afp for biotechnology application, it was necessary to determine its mechanism of action against fungi, as well as its potential impact on plant or animal cells. In the latter part of this thesis has been made different studies using as a model the fungus M.grisea. By confocal microscopy using different fluorescent marking systems, and transmission electron microscopy, it has been observed that the protein AIA is able to form pores in the membrane of the fungus. The AFP protein penetrates the cell of the fungus and accumulates in the nucleus. Further, it is owned interact with nucleic acids, either DNA or RNA. These results which suggest that the mechanism of action of this protein is based on a combination of activities: formation of pores in the membrane and interaction with nucleic acids, leading to cell death. We also testing plant cells (protoplasts rice) and human (HeLa cells), which h 8 an allow 3ae tido determine that the protein AFP does not exercise any significant adverse effect on these cells. Overall, the results of this work suggest that the gene afp is a good candidate to be used as a transgene to protect geranium plants and rice from disease caused by the fungus Botrytis cinerea and Magnaporthe grisea, respectively. The promoter gene ZmPR4 also represents a good choice to head the antifungal gene expression in transgenic rice plants.

Author: GAS PASCUAL ELISABET.
Year: 2005.
University: BARCELONA [www.ub.es].
Place of defense: CENTRO DE INVESTIGACIÓN Y DESARROLLO (CSIC).
Place of preparation: LABORATORI DE GENÉTICA MOLECULAR VEGETAL - INSTITU DE BIOLOGÍA MOLECULAR DE BARCELONA - CONSORCI CSIC-IRTA.

Summary: In most grains, protein reserve majority of the seed are called prolamins and accumulate in the endosperm. The genes that encode specific tissue and are presented efficient transcription, suggesting a narrow gene regulation. In corn, our species model, prolamins are called zeínas and accumulate in some vesicles derived IR called protein bodies, while other proteins reserve as globlulinas or oleosinas that accumulate in the aleurona and the embryo. The interest of our group focuses on gamma-zeína, a protein specific reserve endosperm, which is the 15% protein content of grain, despite the fact that its gene (gamma-Z) is present in only one or two copies per haploid genome. The aim of this study was to characterize new transcription factors implacados in the expression of specific proteins reserve seed, linked to the development of grain, especially those involved in the expression of the gene gammaZ. The specific objectives and the results are briefly described below: 1-functional characterization of PBF as positive regulator gene gamma Z. PBF is a transcriptional activator, whose domain activator is located at its C-terminal region. The PBF activity depends on its ability to binding to DNA. 2-New transcription factors involved in gene expression gammaZ: cloning of the gene GAMYB corn and studies inmunolcoalización. Characterization of this factor as a transcriptional regulator of gene gammaZ. ZmGAMYB encodes for a transcription factor-type Myb R2R3 that binds specifically to the cashier AACA Z and the promoter active gamma gene expression gammaZ from its sponsor. ZmGAMYB is expressed specifically in developing maize endosperm, the same way they do other regulators gamma gene Z. The factor protein accumulates in the surface layers of the endosperm (aleurona and subaleurona). 3-New transcription factors associated with the expression of proteins reserve seed: cloning of the gene FUSCA3 corn, studies inmunolocalización and characterization of this factor. ZmFISCA3 encodes for a transcription factor-type B3 which expresses mayoritáriamente in developing embryo. The factor protein accumulates in large numbers in the aleurona, as well as in the embryo, suggesting a role in the accumulation of reserves substances in these two tissues. FUSCA3 joins the reasons RY-like promoter's gamma Z, so unspecific, and is unable to activate the expression from this developer.

Author: AGUILERA XIOL CRISTINA.
Year: 2005.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAD DE FARMACIA.
Place of preparation: INSTITU DE RECERCA ONCOLOGICA -IDIBELL.

Summary: The overall objective raised in this thesis has been studying the mechanisms that regulate pathways NFkB and Notch and how these two pathways interact. The first chapter shows that the 14-3-3 regulate the classical pathway of NFkB favoring exported nuclear complexes p65/IkB alpha after activation by TNF-alpha. We show that p65 contains at least two domains binding to 14-3-3 including amino acids 38-44 and 278-283, and mapamos the binding site of IkB alpha to 14-3-3 in the amino acids 60 - 65. The mutation of these domains redistributed p65 and IkB alpha to the nucleus of cells suggesting that 14-3-3 is involved in the export of these proteins in the nucleus. Treatment with TNF-alpha promotes the recruitment of 14-3-3 and IkB alpha to the promoters of target genes of NFkB and the union of p65 to 14-3-3. By inhibiting the activity NFkB with a dominant negative of 14-3-3, the complex IkBalfa/p65 accumulate in the nucleus of cells. Under these conditions there is a constituent association of p65 to its target genes resulting in a lack of response of these genes to the activation induced by TNF alpha. So we showed that the 14-3-3 is necessary for the proper regulation of NFkB and the way that facilitates exporting nuclear complexes IkBalfa/p65. The second chapter shows how inhibitor route of NFkB, IkB-alpha plays a role in the nuclear chromatin associated gene target of Notch as hes1 i herp2, this being a new element in the mechanisms linking route Notch and the NFkB. We propose that IkB alpha is involved in transcriptional repression recruiting elements corepresores to specific promoters. We show as IkB alpha interacts with elements repressors nuclear HDACs. By immunoprecipitaciones chromatin demonstrates that IkBalfa are recruited in the promoter of hes1 conjunction with HDAC1 and HDAC5. IkBalfa is released temporarily promoter hes1 when cells are treated with TNFalfa, that correlates with an increase in the acetylation of histones and the transcriptional activity of the gene. The release of IkBalfa of hes1 and its transcriptional activation coincides with the recruitment of IKKalfa and IKKbeta in this promoter. Moreover, the release of IkBalta of hes1 as the increase in the acetylation of histones induced TNFalfa are affected in fibroblasts deficient IKKalfa.

Author: NAPAL BELMONTE LAURA.
Year: 2005.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAT DE FARMACIA.
Place of preparation: UNVIERSITAT DE BARCELONA.