MOLECULAR BIOLOGY (5)

Author: ARIZ LÓPEZ DE CASTRO USUE.
Year: 2005.
University: PAÍS VASCO [www.ehu.es].
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: PROGENIKA BIOPHARMA S.A. Y DEPARTAMENTO NEUROCIENCIAS (UPV).

Summary: Multiple sclerosis is an inflammatory demyelinating disease of the CNS and the most common neurological disorder in young adults. For a better understanding of the processes involved in the development of demyelinating diseases like this, it is important to use mock-ups in which they can lead the process of desmielinizaicón and allow the breeding of new molecular targets of interest. The model used in this work consisted of isolation and cultivation of oligodendrocytes from perinatal rat optic nerve (P12). These cultures were stimulated with receptor agonists glutamatérgicos (AMPA or kainato) at concentrations apoptotic simulating the process of oligodendroglial death that occurs in the disease. In order to obtain an overview of the changes that occur in the cell was conducted an analysis of differential gene expression chips with expression (Affymetrix ®) in crops and crop stimulated control. The results were validated by real-time quantitative PCR obtained a list of genes whose expression varies following the implementation of incentives. One of them, Dusp6 proved to be involved in the process of death exitottóxia since inhibition of synthesis through its antisense oligonucleotides resulted in an increase in cell survival following the implementation of incentives excitotóxicos, which is a starting point for design new treatments.

Author: JUSTO FERNÁNDEZ ALFREDO.
Year: 2005.
University: VIGO [www.uvigo.es].
Place of defense: FACULTAD DE BIOLOGÍA.
Place of preparation: FACULTAD DE CIENCIAS.

Summary: In this thesis has studied the taxonomy, ecology, corología and phenology of genres amanita, limacella, torrendia, pluteus and volvariella in Iberia and Balearic Islands. It has obtained a list of 94 taxa distributed the following way: amanita (43), lamacella (5), torrendia (1), pluteus (33) and volvariella (12). It proposes 2 new species for science and cited 2 taxa for the first time on the European continent.

Author: SEPÚLVEDA JUSTO MARIA DEL ROSARIO.
Year: 2005.
University: EXTREMADURA [www.unex.es].
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: FACULTAD DE CIENCIAS.

Author: SEGRELLES HUELGA CARMEN.
Year: 2005.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FAACULTAD DE BIOLOGÍA.
Place of preparation: CIEMAT (CENTRO DE INVESTIGACIONES ENERGÉTICAS MEDIOAMBIENTALES Y TECNOLÓGICAS).

Summary: Cancers of epithelial origin constitute over 80% of all cancers diagnosed in humans. There are many studies being conducted to try to establish the cause of this disturbance neoplastic transformation. In many cases, this process is a result of alterations in oncogenes and / or tumor suppressor genes coding for proteins that are part of signal transduction pathways involved in cell homeostasis. Much of these studies are carried out through animal models. In this sense, the model of chemical carcinogenesis in mouse skin (DMBA / TPA), is one of the best-known models induction carinogénesis chemistry. In this model, data shown in this thesis show that the signaling mediated by Akt kinase (PI3K/Akt) is a key mediator in the development of carcinogéneiss in mouse skin. In addition, studies of tumorigenesis in vivo demonstrate that overexpression of Akt in the line of keratinocytes PB increases their ability tumorigéncia. The tumors obtained for microinjection of these keratinocytes transformed kinetic show a rapid onset of a larger size and a higher degree of malignancy. This transformation mediated Akt translates into an increase of stabilizing beta-catenin in the cytoplasm, an increase in transcription, translation and localization ciclina D1 and an increase in transcription, translation and stability of the c-myc oncogene, it produces in the cell deregulation in the process of proliferation and apoptosis. Furthermore, the activation of Akt in keratinocytes leads to firing mechanisms angiogenic, via a post-regulation of VEGF, thereby allowing greater tumor growth. Finally transgenic animals expressing a permanently active Akt develop spontaneous tumors which confirms in akt role in the development of epithelial tumorigenesis.

Author: GARCÍA-ARANDA JIMENEZ CRISTINA.
Year: 2005.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE FARMACIA.
Place of preparation: FACULTAD DE FARMACIA.

Summary: In recent years there has developed a growing interest by basic and clinical researchers to work together on an issue of enormous social importance, as is cancer. While figures on the incidence of cancer are alarmente, these numbers obscure the fact that, at the cellular level, the malignant transformation is extremely rare. In fact we are effectively protected from tumorigenesis by a complex set of defense mechanisms and tumor development is only possible when inactivated these mechanisms. As you know the routes through which controls the tumor removal was revealed different points of regulation that critics are often altered during malignant progression. The shortening of the telomeres and the suppression of telomerase in most human somatic tissues, is a major barrier to protect us against cancer. By contrast, the stabilization of telomeres, generally through the activation of telomerase, is a critical step in cell proliferation indefinitely, and therefore to the formation of tumors. This has led to a great interest in identifying the role of telomeres and telomerase in the tumorigenic process, with the ultimate aim of their use as therapeutic targets to mimic the exponential growth of transformed cells. The investigation of the dynamics of telomeres provides us with an excellent opportunity to understand the mechanisms of control of cell proliferation. This thesis is an elaboration of the understanding of the context and the mechanisms by which the telomeres and telomerase contribute to the development of different types of human tumors, such as colorectal cancer, gastric cancer and cancer not microcítico lung. The analysis purposes forecasts for various parameters of telomeric role in these tumors has revealed that in the case of cancer, colorectal cancer and non microcítico lung tumors not reactivated telomerase confer a more favorable prognosis than those in which detects activity of the enzyme, whereas gastric tumors in the reactivation of telomerase appears to have no impact prognosis. In all three types of tumors, our results showed no significant association between telomerase activity and the telomeric length, and that the mechanisms for maintaining telomeres alternative enzyme constitute an event of very little importance. Analysis of the telomeric length in the three pathologies revealed the importance of this study for the purpose forecasts, as well as a dual role of telomeres in the processes tumorigénicos. In the case of colorectal cancer, we have been able to demonstrate in vivo the role of protein TRF1 as a negative regulator of the length of teloméros. TRF1 is sobreexpresada in colorectal tumors, and their levels were significantly higher in cases that show telomérico shortening. His analysis provides a select group of patients with a more favorable prognosis in cases reactivated telomerase. We found that alterations in these genes are common in colorectal cancer and have important implications for the loss of growth control, own tumor cells. We believe that this study may be useful in human clinical oncology for the selection of patients with different clinical outcome after curative surgery, in order to establish treatment protocols tailored to each patient group.

Author: FERNÁNDEZ MILLÁN ELISA.
Year: 2005.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE FARMACIA DE LA UNIVERSIDAD COMPLUTENSE DE MADRID.
Place of preparation: FACULTAD DE FARMACIA DE LA UNIVERSIDAD COMPLUTENSE DE MADRID.

Summary: This thesis has been carried out in an animal model of undernourishment where protein has been shown previously that a restriction of 65% of the intake for the mother during the third week of gestation increases in the fetuses to term, mass of beta cells and the response insulino-secretora to glucose and amino acids. When undernourishment continues during the postnatal period through adulthood, it appears the opposite situation: a reduction of the mass of beta cells in parallel with an injury in the response insulino-secretora. The study of metabolic and molecular processes responsible for these disturbances caused by malnutrition globalha been the main objective of this thesis. As a result have reached the following conclusions: 1-Restricting intake for the mother during the last third of gestation, resulting in fetuses to term an increase in the insulin response to glucose, due to a higher content pancreatic insulin and an increase in the expression of the gene that encodes. Moreover, the greater the oxidative metabolism of glucose of these animals could be enhancing the activation of p38/SAPK2 and, consequently, PDX-1. 2 - The chronic undernourishment, from the fetal stage until the 70 days of life, results in a decline in insulin release in response to glucose, as a result of a minor pancreatic insulin content and the expression of mRNA of the hormone. That decline appears to be directly related to low levels of pancreatic PDX-1 found in adult rats undernourished. Furthermore, the activation of the transcription factor might also be reduced as a result of lower oxidative metabolism of glucose observed in these animals. 3-Increasing the mass of beta cells, present in fetuses at term from undernourished mothers, appears to be related to an increase in the replication of these cells due to high levels pancreatic of IGF-1. This fact is favored by the largest number of recipients of IGF-1 presented by fetal islets, and the high expression in the pancreas of the carrier protein IGFBP-2. Therefore, the local action of IGF-1 seems to protect fetal pancreatic endocrine Impact of maternal undernutrition in a key moment for the development and growth of beta cells. 4 - The maternal undernourishment caused in the fetuses to term an increase in the content islet IRS-2 and the activation of the path IRS-2/PI3K/Akt, both at baseline and after stimulation with glucose and IGF- 1. All these molecular changes appear to be responsible for the higher beta cell replication found in fetuses undernourished, and therefore could be contributing to an increase in mass of beta cells. 5, - beta cells of adult rats undernourished retain the ability to regenerate spontaneously after the stimulus of pancreotomía part of the 90%. In this process actively involved IGF-1, whose overexpression in pancreas, in response to the pancreotomía promotes rats undernourished in the regeneration of beta cells via stimulation, in the short term, from neogénesis, and longer-term the replication, with superior efficacy to that seen in the control population. We believe, therefore, that our animal model of intrauterine and chronic undernourishment is a very useful tool to analyze, in vivo and in vitro, the effects that different factors may have on development, growth and regeneration of beta cells pancreáti 8 Cass.

Author: RODRÍGUEZ HERNÁNDEZ CARLOS JAVIER.
Year: 2006.
University: POLITÉCNICA DE VALENCIA [www.upv.es].
Place of defense: Dep. Biotecnologia.
Place of preparation: Universidad de la Laguna.

Summary: The immunosuppressant FK506 (Tacrolimus, Prograf) has increased the survival rate of organ transplantation. FK506 exerts its immunosuppressive action by inhibiting the phosphatase calcineurin in activated T cells. Unfortunately, therapy FK506 is associated with therapeutic effects were not unwanted, among them diabetes, involving various other targets of calcineurin. To identify these targets we studied the cellular toxicity of FK506 in yeast gemación Saccharomyces cerevisiae. FK506 increased sensitivity of yeast to osmotic stress in a manner independent of calcineurin and proteins binding to FK506. FK506 also prompted a strong and fast amino acid activation of the path of overall control of nutrients (GCN). The prototrofía of tryptophan or excess tryptophan removed the toxicity of FK506, which shows that the fast tryptophan half of this effect. The mutation of genes GCN3 and 4 relieved partially toxicity of FK506, suggesting that the activation of the path GCN by FK506 is also involved in the osmotic tolerance. FK506 enhanced phosphorylation of the kinase Hog1p dependent on osmotic stress but without induction of a reporter dependent Hog1p. Interestingly, the disruption of the gene GCN2 abolished hiperfosforilación of Hog1p dependent FK506 and restored the activity of the reporter dependent Hog1p. Conversely, the interruption of the gene HOG1 affected the activation of Gcn2p and translation of a reporter GCN4-lacZ dependent FK506. This shows the existence of a functional interaction between kinases Gcn2p and Hog1p. Taken together these data show that both the amino acid fast as the activation of the path GCN induced FK506 contribute to the cellular sensitivity to osmotic stress and show a positive regulator loop routes between the GCN and HOG.

Author: TÁRRAGA HERRERO SUSANA.
Year: 2006.
University: POLITÉCNICA DE VALENCIA [www.upv.es].
Place of defense: Dep. Biotecnologia.
Place of preparation: Universidad Politécnica de Valencia.

Summary: The plants, over evolution, have developed a number of mechanisms to protect themselves from attack by pathogens. Some of these mechanisms are constituent, being established on the ground before the arrival of the disease, while others lead after the perception of pathogenic signals arising from the attack and can provide protection not only at the site of infection but systemically, along the entire plant. Numerous studies indicate that the salicylic acid (SA) plays a crucial role in setting the defensive responses. The most immediate is a very significant increase in their levels of accumulation versus infections necrotizantes both at the site of infection as in the distal areas of the plant. Moreover, the application of exogenous SA induced systemic resistance, which means, among other things, an accumulation of defensive proteins, such as proteins related to the pathogenesis (PRs). By studying the possible involvement of other phenolic compounds on the road signaling the defensive response in plants, has been described in our laboratory, which the acid 2,5-dihidroxibenzoico or acid gentísico (GA), shows a significant increase in their levels accumulation in infections not necrotizantes. This compound, derived metabolic HS, being exogenously applied in tomato plants, induces a series of PR proteins different from those induces SA, which may have a complementary role to that of SA in the face of pathogens signaling in plants . Both the SA and the GA, as is the case with many other secondary metabolites, are accumulated in the plant in the form of glycoconjugates. Reactions glicoconjugación consist of the union of one or more molecules of sugar to a metabolite in question and are catalyzed by enzymes belonging to the family of glicosiltransferasas. This conjugation alter the structure, stability and other properties

Author: CAPARRÓS MARTÍN JOSÉ ANTONIO.
Year: 2006.
University: POLITÉCNICA DE VALENCIA [www.upv.es].
Place of defense: Universidad Politécnica de Valencia.
Place of preparation: Universidad Politécnica de Valencia.

Summary: We have isolated two genes from Arabidopsis thaliana, AtGpp1 and AtGpp2 by homology with phosphatases yeast low molecular weight Gpp1 and Gpp2, which show specificity DL-glicerol-3-fosfato and also share homology with DOG1 and DOG2 that defosforilan 2-deoxiglucosa-6-fosfato. Using an approximation of comparative genomics, genes were identified as putativas protein hidrolasas similar to those dehalogenasas haloácidas. AtGpp1 (gi 18416631) and AtGpp2 (gi 18423981), encode proteins that share a 95% identity with a predecida MW of 33 and 27 KDa and a pI of 7.8 and 5.6 respectively. Both isoforms have high specificity DL-glicerol-3-fosfato, pH optimum at 7.0 and one km in the range of 3.5-5.2 mM. AtGpp1 and AtGpp2 expressed over the development in all organs of the plant, mostly in siliques, and the expression is not affected by osmotic stress, or oxidative ion. Coupled with the N-terminal end of AtGpp1 there is a putative peptide transit to chloroplast cTP, AtGpp2 devoid of this signal is predicted as cytosolic; this compartmentalization was confirmed by a subcellular fractionation. The study of immunohistochemical localization using antibodies anti-AtGpp2 indicates that proteins AtGpp are mainly located in meristemos flower immature and vascular elements of the root, stem, leaves, silícuas and the developing embryo, specifically in the cytosol as well as in chloroplasts of different cell types of Arabidopsis, with striking location on the particular cells accompanying tissue floemático. Transgenic plants of Arabidopsis, which sobreexpresan AtGpp2 have an altered phosphatase activity and an improved tolerance to salt stress, and oxidative osmotic.

Author: MARCO MARÍN CLARA.
Year: 2006.
University: VALENCIA [www.uv.es].
Place of defense: FACULTAD DE MEDICINA - UNIVERSITAT DE VALÈNCIA.
Place of preparation: INSTITUTO DE BIOMEDICINA DE VALENCIA.

Summary: During my working thesis, I have dealt with four enzymes whose specific function synthesis and release anhydrides phosphate, which are crucial for the biosynthesis of amino acids ornithine / arginine, proline, isoleucine and lysine, and for the synthesis microbial of nucleátidos of pyrimidine. Prior to my inclusion in the laboratory, it had identified a folding characteristic common to the novel and carbamate kinase enzymes (Navy, A. et al., 1999; Ramón-Maiques, S. et al., 2000) and acetylglutamate kinase (Ramon - Maiques, S. et al., 2002). Both enzymes belong to the group of Enzyme Commission EC 2.7.2, transferidores a fosforilo a carboxylate group, and therefore, with the specific task of synthesizing anhydrides mixed carboxflico-fosfárico, routes biosynthesis of amino acids. Carbamato kinase kinase and acetylglutamate belong to the family structural amino acid kinase (Database PFAM, family PFO0696; http. / / Www.sanger.ac.uk / Software / Pfam), family by amino acid sequence similarity, also includes , enzymes aspartatoquinasa, glutamate 5-quinasa, UMP bacterial kinase and N-acetil L-glutamato synthase. Given the sequence similarity between the amino acid enzymes of the family kinase, the laboratory suggested that the folding of carbamate acetylglutamate kinase kinase and would be common to the rest of the family of enzymes and pilot testing of this hypothesis has been one of the main objectives of the lab and my work. Part of my initial work focuses on acetylglutamate kinase (NAGK), the enzyme whose structure had already been given to high resolution (Ramon-Maiques, et al., 2002), by other members of the group that I developed my work . My work under this enzyme was corroborated by directed mutagenesis, inferences derived from the functional study structural advance. Given the similarity between acetylglutamate kinase and aspartoquinasa (AK), another enzyme family amino acid kinase, a key in the synthesis of threonine, methionine, isoleucine and lysine, and with great potential in terms of biotech, we use the structural information of NAGK, to perform work directed mutagenesis of aspartoquinasa, whose results have enabled us to construct a structural model of AK, which is so far the best approach to the structure of this enzyme. The third enzyme which I studied, the UMP kinase, plays a key role in the synthesis of nucleátidos of pyrimidine and has the special feature of enzymes within the kinase family amino acid to synthesize the formation of an acetic fosfárico-fosfárico. My job with this enzyme has focused on determining its structure using x-ray diffraction Finally I have studied the enzyme glutamate 5 - kinase, a key in the synthesis of proline in microorganisms and plants, which is also key to the synthesis of ornithine in animals. I have determined the three-dimensional structure of the glutamate 5-quinasa E. Coli by difraccian X-ray The scope of study with the four enzymes aim of this thesis has always been the structural looking connect and structural features associated with functional behavior. The work that I have done, has led wings following publications: -Site-directed mutagenesis of Escherichia coli acetylglutamate kinase and aspartokinase III probes the catalytic and substrate-binding mechanisms of these amino acid kinase family enzymes and allows three-dimensional modeling of aspartokinase. Clara Marco-Marfn, James Ramón-Maiques, Sandra Tavarez and Vincent Rubio. Journal of Molecular Biology (2003) 334, 459-476. - First-time crystallization and preliminary X-ray crystallographic analysis of a bacterial-archaeal type UMP kinase, a key enzyme in microbial pyrimidine biosynthesis. Clara Marco-Marín, Juan Manuel Escamilla-Honrubia and Vincent Rubio. Biochim. Biophys. Minutes. (2005) 1747, 271-275. - The crystal structure of Pyrococcus furiosus UMP kinase provides insight into catalysis and regulation in microbial pyrimidine nucleotide biosynthesis / Clara Marco-Marin Fernando Gil-Ortiz and Vincent Rubio. Journal of Molecular Biology (2005) 352 8, 438-45 3e7 4. - A novel two-domain architecture within the amino acid kinase enzyme family revealed by the crystal structure of Escherichia coli glutamate 5-kinase. Clara Marco-Marin Fernando Gil-Ortiz, Isabel perez-Arellano Javier Cervera, Ignacio Fita and Vincent Rubio. Accepted the Journal of Molecular Biology.

Author: BOURGON BAQUEDANO LUCRECIA CATALINA.
Year: 2006.
University: POLITÉCNICA DE VALENCIA [www.upv.es].
Place of defense: UNIVERSIDAD POLITÉCNICA DE VALENCIA.
Place of preparation: UNIVERSIDAD POLITÉCNICA DE VALENCIA.

Summary: Previously in our laboratory and as a result of functional screening of a cDNA library of Arabidopsis was isolated cDNA corresponding to the gene SRL1 by the phenotype of conferring salt tolerance expression in the yeast Saccharomyces cerevisiae. The protein encoded in the cDNA appeared to be involved in the processing of pre-mRNA. That entailed a new relationship between salt-tolerant cells ecuarióticas and a process of great importance in cellular metabolism as is the processing of pre-mRNA. The purpose of this Ph.D. Thesis has focused on biochemical analysis and functional SRL1 and study the mechanism of inhibition of splicing in the presence of salt. It conducted the study of expression of the gene SRL1 in Arabidopsis thaliana plants under various stress conditions. These studies indicated that the gene was activated transcripcionalmente in salt stress conditions, high temperatures and low water stress and treatment with acid abscísico. As a further study bioinformático the promoter sequences of the gene SRL1 indicated the presence in the same sequence homologous to those in other genes whose expression was triggered under the same stress conditions. Moreover, in the quantitative study of phenotype of drought and salt tolerance in transgenic Arabidopsis plants that sobreexpresaban SRL1, it was observed that the transgenic plants obtained further development both vegetative and reproductive compared to the unprocessed for stress conditions assayed. By RT-PCR technique Amended got confirmation that the prosecution of intron splicing was a target or toxicity of salt in both plant and in yeast. Similarly note that the plants overexpression of SRL1 getting counteract such inhibition. The use of polyclonal antibodies recognizing protein SRL1 allowed inmunolocalizar subcelularmente this protein in Arabidopsis thaliana roots and Allium cepa both in normal and treated with salt. Its provision in all cases was consistent with their status as splicing factor, as it was located in the nucleus in diffuse form in the absence of salt to pass grouped in the form of spots in salt stress conditions.

Author: JAVIER BELLIDO SANCHEZ.
Year: 2006.
University: CANTABRIA [www.unican.es].
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: FACULTAD DE MEDICINA.

Summary: The microtubule cytoskeleton is composed of microtubules and accessory proteins. The microtubules are polymers of tubulin, a heterodímero consisting of a molecule of alpha and another betatubulina. Both the synthesis of the monomers and the formation of heterodímero are complex processes and finely regulated because it is necessary to ensure the proper folding of this so you can join the microtúbulo. In these processes besides prefoidina and CST (who also sit on the folding of actin), involving up to five cofactors (p14oCoA, CoB, CoC, C and CoE) and protein Arl2.P14, C and Arl2 involved in the folding of the beta tubulin. The main aim of the thesis is to study the role of Arl2. It has been cloned the human gene in expression vectors making it possible to purify the recombinant protein. There have also been specific polyclonal antibodies that will be used to determine the localiación at histochemical and intracellular Arl2 in different tissues and cell lines using techniques inmunocitoquímicas. Also will consider the role of p14 in mammalian cells by inhibiting their test synthesis using genetic techniques silencing. Objectives: to Arl2 in cloning vectors suitable for expression in bacterial cells and ecucarióticas, both as' mild type 'as a' taG 'histidinas for ease of purification. Production and purification of recombinant protein. Study biochemist, immunocytochemical and immunohistochemical the expression of Arl2 in different tissues and cell lines. Production of complex Arl2-Cod through coinfection cell Sf9 with recombinant baculovirus. Production and purification of p14 according to the methodology described. Study of the effect of inhibiting the synthesis of p14 and Arl2 by siRNA in cell lines appropriate under investigation.

Author: BOSCH PRESEGUE EULALIA.
Year: 2006.
University: POLITÉCNICA DE CATALUÑA [www.upc.edu].
Place of defense: SALA DE CONFERÈNCIES, EUETIT.
Place of preparation: ETSEIB, PAVELLÓ G Campus SUD.

Summary: Rhodopsin (Rho) is the visual photoreceptor responsible for dim light vision. This receptor, that is located in the rod cell of the retina, has seven transmembrane helices and is a prototypical member of the G protein coupled receptors (GPCR) superfamily, being the only member of this superfamily whose structure has been resolved by X-ray chrystallography. The study of GPCR is of outstanding pharmacological interest because they are involved in a wide variety of physiological and pathophysiological processes. Structural and functional studies of Rho should provide clues about common structural motifs in GPCR and allow us to elucidate the molecular bases of a proposed common activation mechanism. Besides, the study of Rho mutants associated with retinal diseases, such as retinitis pigmentosa (RP) provides information about the molecular mechanism of this pathology. The main goal of this work is unraveling the structural details underlying the molecular mechanism of Rho activation and its interaction with transducin. To this aim we have analyzed the structural and functional role of helices I and II of Rho, as well as the role of retinal in the photoactivation process of the receptor. We have also tried to determine the amino acids involved in the specificity of G protein recognition. We have studied the adRP mutants G51A, G51V and G89D, as well as non-natural mutations at these positions, in order to determine the role of helices I and II in Rho structure and conformational changes upon light activation. The detailed characterization of mutations at 51 position shows that this position is very sensitive to the size of the introduced side chain. The increase volume of side chain of the introduced amino acid can be correlated with dark and active conformation (MetaII) stability and eventually with the capacity to activate the G-protein transducin. The study of mutations at 89 position suggests that the charge effect is more important for mutations in this position. Additionally, G51V and G89D mutants showed an abnormal photobleaching, with formation of an altered photointermediate which is in equilibrium to MetaII. We suggest that G51V and G89D mutants alter the proposed equilibrium between MetaIa, MetaIb and MetaII to favour the MetaIb photointermediate, and this fact together with the observed MetaII instability could be the molecular defects that caused RP. Wt Rho was regenerated with 11-cis-7-methylretinal (7-methyl-Rho), to gain insights into the opsin-retinal coupling process and to determine the role of retinal in the photoactivation process. 7-methyl-Rho showed chromophore formation similar to Rho but an abnormal photobleaching behaviour with formation of an altered photointermediate which is in equilibrium to MetaII photointermediate. The methyl group at C7 of retinal would introduce a structural change at the vicinity of Met-207 that would result in receptor trapping at the MetaIa conformation during the photoactivation process. MetaIa, MetaIb and MetaII equilibrium would be controlled by a network of complex interactions between helices I, II and VII of rhodopsin and also by opsin and retinal interactions. In another approach, we constructed and expressed Rho-m3 mutants at intracellular loops C-II and C-III. These mutants showed chromophore formation and photobleaching behaviour similar to Wt Rho. Single point mutants at the C-II loop showed transducin activation like Wt Rho with the exception of V138S mutation that caused a reduction in transducin activation like mutants at C-III loop and conjugated mutants at C-II and C-III loops. None of these mutants showed Giáq activation with regard to Wt protein. The results suggest that Val-138, Val-227, Val-250, Val-254 and Ile-255 amino acids of Rho participate in transducin binding and/or activation, and that hydrophobic interactions involving these residues would be an important factor mediating Rho-transducin interaction.

Author: VÁZQUEZ MARTÍN ALEJANDRO.
Year: 2006.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE BIOLOGICAS.
Place of preparation: FACULTAD DE BIOLOGÍA.

Summary: The fatty-acid synthase is the main enzyme involved in the synthesis of fatty acids. This protein is sobreexpresada in most human cancers and its inhibition induces apoptosis of tumor cells. In this study we evaluated the combination of treatments antineoplásticos inhibitor of the fatty acid synthase in cell lines of breast cancer. We noticed that the joint power the effects of anthracyclines, cisplatin and 5-fluorouracil, is an additive in combination with faslodez and antagonist in combination congefitinib and tamoxifen. We concluded that the fatty acid synthase modulates the sensitivity of tumor cells to chemotherapeutic agents and treatments is a new target in the treatment of breast cancer.

Author: SÁNCHEZ MOLINA SARA.
Year: 2006.
University: BARCELONA [www.ub.es].
Place of defense: UNIVERSIDAD DE BARCELONA.
Place of preparation: UNIVERSIDAD DE BARCELONA.

Summary: Mutations turned off at Ras oncogene in a constitutively active with the capacity to transform the cell. One of the potential targets in this process of transformation is the chromatin. In this context we study modifications of histones and --- modifying histones in the cell transformation induced by Ras.

Author: MARTÍN JAULAR LORENA.
Year: 2006.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAD DE BIOLOGÍA.
Place of preparation: FACULTAD DE BIOLOGÍA.

Summary: This thesis has been characterized arginine transport in primary macrophages derived from fetal liver and bone marrow, as well as in alveolar macrophages. Our results indicate that in basal conditions, the flow of arginine in the plasma membrane of these primary macrophages occurs primarily by the transport system and + L (increased 75%), with a small contribution and System + (mayor10%) . The activity and + L may be due to LAT and + and + LAT-2. The mRNA of + LAT1 is the most abundant of all transporters for arginine. The system activity and + is due to the expression of CAT-1 with contribution of CAT-2 dependent on the strain of animals used for the study. The activities of the systems b0, + and B0 + are not present in these macrophages. The treatment of primary macrophages with incentives and alternative classical activation induces a significant increase in the transport mediated by the system and +. E contrast, the proliferation produces only a modest increase in activity and +. For activation, but not for the proliferation, this increase is mediated by CAT-2B. Indeed, the lack of CAT-2 undertakes transport and metabolism of arginine induced activation classic and alternative without compromising proliferation. The metabolism of arginine macrófago detectivos for CTA-2 is a response to situations of macrófago high demand for arginine. In response to treatment with GM-CSF increases the primary macrophages arginine transport through CATA-2B and metabolism at the enzyme Arginasa I. In the absence of CATA-2 there is a partial compensation for CAT-1 of the effect of GM *- CSF. In macrophages and for defective + LAT-1 is not committed transportation and arginine metabolism in response to the activation or treatment with GM-CSF. However, there is an increase in the intracellular content of this particularly when amino acid metabolism through arginase is inhibited. These data suggest that the output of arginian through and + LAT-1 is involved in regulating the intracellular content of this amino acid.

Author: MIRONES AGUILAR ISABEL.
Year: 2006.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE BIOLOGÍA.
Place of preparation: CENTRO DE INVESTIGACIONES ENERGÉTICAS MEDIOAMBIENTALES Y TECNOLÓGICAS (CIEMAT).

Summary: The angiogenesis consise in the formation of blood vessels from preexisting others. SE is a complex process that requires the coordinated interaction of multiple cell types. The factor of growth Endotelio Vascular (VEGF) is essential for angiogenesis skin, however, has not yet been fully characterized the role of other vascular specific signaling molecules, in part due to a possible effect on the part of VEGF that mask this function. In this study we have isolated and characterized a cell line of mouse keratinocytes, spontaneously immortalized, and deficient in VEGF by adenoviral gene transfer of recombinasa Cre, subcultures serial under controlled conditions and cell cloning. The Southern blot analysis and ELISA confirmed the complete loss of VEGF gene and protein level, respectively, in clones of keratinocytes treated with the recombinasa. We tested the tumorigénicas of cells, as well as their characteristics of growth and differentiation in vitro and in vivo. Subcutaneous injection into immunodeficient mice identified the absence of tumorigenic potential of the cell line stands in the VEGF. The trial of differentiation in vitro, under conditions of high concentration of calcium, found that these cells retain their ability to differentiate normal. Moreover, two models were tested transplant crop organotípico to immunodeficient mice (cameras silicone and transplantation ortotópico). In both csdos, keratinocytes deficient in VEGF were able to form a normal skin from the point of view of architecture and organization epidermal skin. Many studies described the establishment of cell lines keratinocytes inmortalizadas by oncogenes however, in this work we have created a cell line deficient mouse keratinocytes in angiogénico main factor (VEGF), which keeps intact the characteristics of this cell type, thus constituting an important tool for studying routes angiogénicas regardless of the strong influence of VGF.

Author: ANDREU SOMAVILLA NURIA.
Year: 2006.
University: BARCELONA [www.ub.es].
Place of preparation: UNIVERSIDAD DE BARCELONA.

Summary: This thesis has been divided into two chapters for the work done within two research groups where he has done. The objective of the first part of the thesis was to identify new genes following an approximation of human cloning in silico using the databases of ESTs and the collection of clones IMAGE available. We identified a new gene was named PALML (Uparalemmin-like) because of its similarity to the human protein PALM (paralemmin), and mapó in 1p21. PALML is expressed mainly as a transcribed from 2.4 kb, and has a ubiquitous expression. To determine the involvement of this gene in non-syndromic primary vesicoureteric reflux (VUR) is developed the analysis technique mutacional SSCP-HD for that gene, found only a change nucleotídico that it was silent in 1 of 5 individuals with VUR studied. The refining of the chromosomal location of PALML confirmed its location in 1p21, excluding their involvement in VUR. The study continued with the determination of the subcellular localization of the protein PALML, in the cytoplasm of cells. The second part of the thesis has focused on the study of the syndrome Wiskott-Aldrich. The gene responsible for the syndrome Wiskott-Aldrich (WAS) is called WASP, is located on the short arm of chromosome X. This gene encodes for a protein that is expressed exclusively in hematopoietic cells, and is involved in the reorganization of the actin cytoskeleton and in various signal transduction pathways. We have studied 35 families with Spanish syndrome Wiskott-Aldrich and thrombocytopenia linked to the X chromosome, which has identified 18 mutations different. In the course of this thesis we have identified 4 mutations new which had not been previously described as causing disease. This disease has a recessive inheritance linked to the X chromosome, so that in principle, women heterozigotas for a gene mutation WASP are asymptomatic carriers. We have studied the cases of 2 Spanish women heterozigotas for a mutation in the gene WASP who showed symptoms of thrombocytopenia. The analysis of the pattern of X chromosome inactivation in these women has enabled us to correlate the presence of clinical symptoms with a change in the pattern of X chromosome inactivation (hematopoietic cells of these women have preferred the inactive X chromosome carrier WASP wild allele ). The analysis of cellular dysfunction associated with the actin cytoskeleton in B lymphocytes allowed us to determine: B cell lines derived from patients with WAS flaws in the reorganization of the actin cytoskeleton in response to stimulation with bradykinin, as well as in training of pseudopodios. The training capacity of pseudopodios in cells B-EBV, as well as the normal regulation of actin cytoskeleton in response to stimulation with brdiquinina, is restored by gene transfer using WASP retroviral vectors.

Author: FERRER ORTA CRISTINA.
Year: 2006.
University: BARCELONA [www.ub.es].
Place of defense: UNIVERSIDAD DE BARCELONA.
Place of preparation: INSTITUTO DE BIOLOGÍA MOLECULAR DE BARCELONA (CSIC).

Summary: The FMD virus is a prototype of the genre Aphthovirus belonging to the family Picornaviridae. This group of viruses is one of the smallest and is characterized by having a single molecule of RNA chain simple and positive polarity as genome. The FMD is a contagious disease affecting the aritodáctiles (cloven-hoofed animals). The main causes of the difficulty in controlling pests of FMD are the high variability in the manifestations pathogenic, the antigenic diversity easy transmission, the ability to establish persistent infections and asymptomatic, and the limited efficacy of existing vaccines so far . Many of these causes stems from the limited copying and the absence of mechanisms for correction presented by the RNA-dependent RNA polymerases responsible for replicating its genome. The RpdR are the central components of the life cycle of the virus RNA. In all structures resolved so far you can see the structure in the form of his right hand, with subdominos of the fingers, palm and the thumb. These domains are similar to those of other types of polymerases described so far. The RNA polymerase is an important therapeutic target, and that if they increase the rate of mutations of these polymerases can cause this virus is extinguished by excess mutations. The initial objective of this thesis was to study the structure of the polymerase 3D of the FMD virus and how it works, this will raise the resolution of the structure of the polymerase as free and forming complexes with RNA, nucleotides, analogues the nucleotide and protein initiator of replication VPg. It has been possible to solve the structure of the polymerase 3D VFA, which provides an overall folding in the form of his right hand closed characteristic of RpdR, where you can differentiate the subdomains of the fingers, palm and the thumb. In different complexes made with RNA were able to identify the nucleotide RNA that interacts with both those responsible for directing the chain mold RNA to the center assets, as lso carried out the reaction of nucleotide addition. There have also been identified amino acids responsible for the selection of the incoming nucleotide. In complexes polymerase 3D protein VPg has described the complex initiation of uridilación, there have been no dramatic conformational changes in the polymerase 3D, but have now been identified amino acids responsible for the process uridilación and has noticed the presence of two ions in the active center of the protein. All these data may provide a basis for the design of new antiviral therapies that carry the virus to extinction by accumulation of mutations.

Author: MERCHAN STEPHANIE EMILIE.
Year: 2006.
University: POLITÉCNICA DE VALENCIA [www.upv.es].
Place of defense: Dep. Biotecnologia.
Place of preparation: Universidad Politécnica de Valencia.

Summary: The mechanisms of K + homeostasis and H +, although very well studied, remain partially known processes. In the case of yeast, Saccharomyces cerevisiae, the output of protons of the cell is regulated by the H +-ATPase Pma1p, whose functioning depends on the entry of potassium through transporters high affinity Trk1 and Trk2. Genetic studies have established that the conveyor Trk1 is activated by the kinases Hal4/Sat4 and Hal5 and inhibited by phosphatases Ppz1 and Ppz2. In relation to the integrity of cells, we showed that the strains that lack the gene PPZ1 and PPZ2 show an increase in the turgencia due to excessive accumulation of potassium, which affects both cell size and the tolerance to high concentrations of this ion in the external environment. It also demonstrated that the increase in turgencia produces a constant stress in the cell wall of these strains. These phenotypes are dependent on the presence of genes encoding transporters potassium high affinity, TRK1 and TRK2 and can be rescued by the presence of an osmotic stabilizer. In a second part of the work, we have established that Trk1 is located in the plasma membrane subdomains called "rafts." Furthermore, we demonstrate that Ppz1 is also located on the plasma membrane, its subcellular localization is modulated according to the levels of expression of Trk1 and Ppz1 and Trk1 co-inmunoprecipitan in vivo. On the other hand, Trk1 appears phosphorylated both in vivo and in vitro and in vivo phosphorylation is increased in mutant ppz1 ppz2. Finally, noting cell cycle progression, we found that the mutant ppz1ppz2 are more sensitive to agents that damage DNA, presents a delay in the development of the S phase of the cell cycle, and have a higher frequency of mutations that a strain . In addition, we confirm that the combination of the loss of function of genes PPZ1 and CDC7 is lethal. CDC7 is a kinase needed during the S phase for the resumption of the progression of replication forks arrested. This phenotype, as previously described, is dependent on the presence of genes TRK1 and TRK2. These data, seen together, strongly suggest the existence of a defect in the machinery of DNA replication in terms of high pH and high concentration of intracellular potassium.