MOLECULAR BIOLOGY

Author: BEYER KATRIN.
Year: 2002.
University: AUTÓNOMA DE BARCELONA.
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: UNIVERSIDAD DE A CORUÑA.

Summary: The disease Alzehimer (AD) is genetically heterogeneous. Cases can be classified by family background in family EA and EA sporadic, and by the age of onset of illness in EA early with a debut before the age of 65 and EA with a belated debut from the 65-year-old. At approximately 1% of all cases are detected family background, and 1% of them have a dominant pattern of inheritance of Greenland. The cause of 80% latter form of the EA are point mutations in the genes of precursor protein beta-amiloide (PDB) and the presenilins (PS1 and PS2). In the EA sporadic APOE allele e4 gene acts as a major risk factor that explains about 50% of cases of sporadic AD and late. The allele E4 confers risk, but its presence is not necessary nor sufficient to cause the disease. Other genetic factors interact with the apolipoprotein E and the result of this interaction is the emergence of the disease. We are investigating genes that might be somehow relates to the emergence of neuropathological changes in AD. In some of these genes have been described polymorphisms that confer risk to suffer from AS. In this paper we have analyzed several genetic factors to determine their role in the development of different types of EA and determined a strategy for detecting locio effect lower for the development of AD. The study of the genes that cause the EA identified the association of a new mutation (V148I) gene of the PS2 in a patient with a family history of late debut, while mutations PDB and PS1 were not detected. The analysis of gene APOE allele confirmed that the APOE * 4 is a major risk factor in all patients with AD to 80 years and is the most important risk factor for patients who debuts between 60 t and 64 years. In addition, the effect of APOE allele * 4 is precipitating dose genotype APOE4 / 4 elcomienzo of EE. The detection of the presence of minors loci is possible only by dividing the population into subgroups separated by intervals of five years. The genes of the cistatina C, PS 1, butyrylcholinesterase (BChE) and catepsina D (catD) are loci of lesser effect. Specifically, the allele CST3-A is fun risk factor for individuals younger than 65 years, the genotype PS1 1 / 1 acts in synergy with the APOE allele * 4 in patients debuts before the 69 years and the alleles BChE-Ky catD-T are markers that confer risk patients debuts between 75 and 79 years. The polymorphism Th1/E47 promoter APOE also can be seen as a locus of lesser effect. The allele Th1/E47-G presents a risk factor for patients with AD which debuts after the 80 years. In addition to risk factors that relate to suffer EE two alleles were detected in the protective promoter of APOE. The allele -491AT-T is accumulated in control individuals under 65 years compared with patients and controls of other age groups, thus turning into protective factor. The allele Th1/E47-T is accumulated in individuals control 75 years and older compared with patients and controls of the other age groups. Therefore, it may be considered that the allele Th1/E47-T is a factor of protection for people of 75 years and older.

Author: YUSTE PARRA MARÍA.
Year: 2002.
University: COMPLUTENSE DE MADRID.
Place of defense: FACULTAD DE VETERINARIA.
Place of preparation: FACULTAD DE VETERINARIA.

Author: GODINO GÓMEZ JAVIER.
Year: 2003.
University: COMPLUTENSE DE MADRID.
Place of defense: FACULTAD DE FARMACIA.
Place of preparation: HOSPITAL CLÍNICO SAN CARLOS LAB. ONCOLOGIA MOLECULAR.

Summary: To achieve this thesis were selected 82 Spanish families suspected of suffering from the syndrome of hereditary cancer not polipósico (HNPCC) in total were studied 239 patients. The molecular basis of this syndrome are defects in the repair of errors that occur during DNA replication. A 90% of cases are due to mutations in the genes MLH1 or MSH2. We studied in these families microsatellite instability MSI by PCR and sequencing and Immunohistochemistry IH as evidence of identification of families carrying mutation in MLH1 or MSH2. In view of our results suggest the MSI as evidence initial selection and IH as a secondary test to determine which of the 2 genes is likely to be mutated. Amsterdam criteria I or II, only 12% are in families with the lowest family aggregation. In the 54% of families that meet the criteria of Amsterdam I or II was found mutation compared with only about 7% in families that do not comply. The mutations in MSH2 were mayoritariamentes Gross deletions to change the reading frame and production of a truncated protein. In MLH1 instead predomian changes nucleotide generation with a colon stop either one amino acid change, the pathogenicity of these amino acid changes were studied by LOH and segregation with the disease. It is observed relationships genotipo - fenotipo clear although the presence of mutations in MSH2 was associated with a lower average age of diagnosis of the tumors (48 years versus 50.4 in mutation carriers in MLH1) and proportion of tumors extracolónicos (42% vs. with 34%) in any case these differences reached statistical significance.

Author: ARRIBAS GARCÍA MÓNICA.
Year: 2003.
University: COMPLUTENSE DE MADRID.
Place of defense: FACULTAD DE FARMACIA.
Place of preparation: FACULTAD DE FARMACIA.

Summary: The study of Diabetes Type II is of great importance, since it provides solutions to a serious problem of the aging society of western developed countries, where the disease has a high prevalence. In this paper, we have attempted to elucidate the roles of three proteins IRS in a target tissue of insulin Specifically, the brown adipose tissue. In adipocitos brown expressed predominantly two of these proteins, IRS-1 and IRS-2. A significantly lower levels, also expressed IRS-3 and IRS-4. The work of this thesis provides results that ensure the leading role of IRS-1 in mediating insulin's role as inducing of brown adipocyte differentiation. Thus, the lack of IRS-1 in brown adipocitos carries some significant shortcomings in the phenotype adipogénico and thermogenic of these cells. With regard to the first, the poor adipocitos IRS-1 presents lower lipid content in their cytoplasm and reduced expression of fatty acid synthase and the transcription factor ADD-1/SREBP1c in response to insulin. As regards the phenotype of thermogenic brown adipocitos lacking IRS-1, there has been a total lack of response to insulin to induce the synthesis at the transcriptional, protein descoplante type 1 (UCP-1), while other proteins descoplantes are not altered. Furthermore, the expression and activity of transcriptional factor C / EBP alpha in response to the hormone are diminished. The loss of IRS-1 in brown adipocyte causes certain alterations in intracellular signaling of insulin. First, it is lost completely activity PI3-K associated with IRS-1 and see a partial defect in the activiación of this enzyme associated with fosfotirosinas. Secondly, the lack of IRS-1 entails an almost total inability of the cell to activate the Akt protein. It has been found that the lack of IRS-1 blocks the activation of Akt isoforms all in response to insulin. These alterations of insulin signaling caused by deficiencies mentioned in the phenotype of adipocitos poor IRS-1. The IRS-2 is the other substrate for insulin receptor expressed mainly in the adipocitos brown. We have found that this substrate is not completely replace the IRS-1 in the above functions. However, the IRS-2 exerts other metabolic functions. In the complete absence of IRS-2, adipocitos brown lose their ability to transport glucose into the cell response to insulin, and the mechanism of translocation of glucose transporter GLUT 4 to the plasma membrane, which is the first trial possible. In this paper, we have investigated the defects produced by the lack of insulin signaling through IRS-2. The adipocitos lacking that no substrate activity PI3-K associated with IRS-2 and show an activity PI3-K overall lower. These alterations are completed with the loss of PKC activity * in response to insulin. Our results relate to the loss of insulin signaling through IRS-2 and PKC- * with the decline in the uptake of glucose by adipocitos brown. Finally, it has studied the ability of IRS-3 as an alternative molecule to the IRS-1 in the insulin signaling in the adipocyte brown. The adipocitos lacking IRS-1 that sobreexpesan IRS-3 recovered some intracellular signaling in response to insulin as the trigger PI3-Ky of Akt. It has been found that the activation of this route of insulin signaling by IRS-3 is instrumental in the recovery of the response to insulin in terms of expressing markers adipogénicos as FAS and ADD-1/SREBP1c in response to insulin. However, the presence of IRS-3 is not restored expre 8 sion 2ff UCP-1 or factor C / EBP alpha in these cells.

Author: GUAL GARCÍA LORENA.
Year: 2003.
University: COMPLUTENSE DE MADRID.
Place of defense: FACULTAD DE FARMACIA.
Place of preparation: FACULTAD DE FARMACIA.

Summary: Deficiency Immunoglobulina A (IgA D) is the imnunodeficiencia primary more common in populations Caucasian (1 / 600 individuals). Its manifestations vary, and range from symptoms until infeciones recurring in the respiratory and gastrointestinal tracts with diferntes degrees of affectation. Its etiology is unknown, but suggests that defects may exist in the stages of differentiation of B cells producing IgA membrane of B cells producing plasma IgA secreted. Sometimes it has been noted some family aggregation in this disease, which may exist for determining genetic susceptibility D IgA, These genes etarían located within the Main Histocompatibility Complex (MHC), because it is l azona genome in which have seen a greater partnership to D IgA. Earlier have been associated prositivamente to illness the haplotipor bearers of the following alleles of the gene DRB1 (located in the MHC class II) DR3, DR1 (subtype DRB10102) and DR7. Our purpose is to finally locate the susceptibility genes within this haplotypes, using tools such as study DTT and commparación of haplotypes found in the Spanish population with those of other populations norteeuropeas. Another objective was to find out whether there haplotypes protection for the D IgA, as had been described in other studies that the allele DR2 was negatively associated with the disease. Finally, it wanted to study the possible intercciones géneticas among haplotypes susceptibility and protection to D IgA. Our results show the existence of different susceptibility genes in different MHC haplotypes, some located in the region of Class II (around locus DR-DQ) and others inthe region Class III. We also found out that the subtype DRB1 1501 allelic DR2 is a marker of a protective gene for the disease, and that this gene is present in all haplotypes carriers of this allele. Finally, it was found that the combined presence of two susceptibility alleles conferred any greater risk to suffer D IgA, while the emergence of a joint susceptibility allele with the susceptibility allele removing protective provided by the first.

Author: FERNÁNDEZ FRANCO LAURA.
Year: 2003.
University: COMPLUTENSE DE MADRID.
Place of defense: FACULTAD DE FARMACIA.
Place of preparation: FACULTAD DE FARMACIA.

Summary: The Crohn's disease and ulcerative colitis are characterized by a chronic bowel inflammation of unknown origin that affects 6-8 individuals and every 100,000 in Spain. Celiac disease is a condition in which gluten from the diet is not tolerated, causing chronic inflammation of the small intestine, and that affects 1 in every 300 people in Spain. The epidemiology has provided considerable information on the factors of susceptibility to these digestive diseases. The description of geographic gradients prevalence and migration studies support the existence of environmental factors. However, the uneven distribution of the prevalence according to racial background, and especially the increased frequency of the disease among relatives of patients and particularly among monozigotos twins are strong arguments in favor of a genetic susceptibility, according clearly implies several interacting genes. Over the past few decades have been assessed in studies of association and linkage many candidate genes as the idea of a pathogenesis mediated by the immune system, and recent studies of the whole genome linkage crawling with anonymous markers. The results available, with discrepancies between studies due to doferencias metológicas and a misclassification of patients, underscoring the role of the region cmplejo greater histocompatibility (MHC), located on the short arm of chromosome 6, as the main determinant for genetic these diseases: However, it is postulated that contribute to the susceptibility of these diseases than other genes confer sen not located the MHC might be important, or even more. OBJECTIVES 1-Studying the possible existence of association and linkage of MICA gene (located within the MHC) with celiac disease. This study was conducted using a case-control analysis and subsequent analysis in families (DTT). 2-Identify possible relationships genotipo - fenotipo in Crohn's disease by studying genes of MHC class II. 3-Locate in the MHC, using the technique of mapping microsatellites, the location of susceptibility genes for ulcerative colitis. 4-Evaluate the role of polymorphisms of the anti-inflammatory cytokine IL-10 in the susceptibility to suffer from inflammatory bowel disease. CONCLUSIONS 1, - allele MICA5.1 (MHC Class III) is a marker within the haplotype ancestral 8.1 (HAS 8.1), a second susceptibility gene in celiac disease. 2 - The shape of ileal Crohn's disease is characterized by genetically found positively associated with the allele DRB1 * 07 and with mutations in the gene NOD2/CARD15, but negatively with the allele DRB1 * 0103. 3 - The form of colonic Crohn's disease is characterized by the absence of genetically partnership with the alleles DRB1 * 07 and mutations in the gene NOD2/CARD15, but is strongly asocaida the allele dRB1 * 0103, susceptibility allele that shares with ulcerative colitis. 4-In the MHC, the combination of the following alleles: DRB1 * 0103, d6s273-5, bat2-8, tnfA11B4C1D3E3, ikbl +378 (C) and MICA5.1 appears to be the biggest determinant genetic suscpetibilidad and outreach our population of ulcerative colitis. 5, - allele 14 microsatellite IL-10G and allele -1082G the promoter of the gene for IL-10, especially when they appear together in an individual, are markers of susceptibility to Crohn's disease in our population.

Author: CROOKE ÁLVAREZ ALMUDENA.
Year: 2003.
University: COMPLUTENSE DE MADRID.
Place of defense: FACULTAD DE FARMACIA.
Place of preparation: FACULTAD DE VETERINARIA.

Summary: The difficulties in obtaining a vaccine effective against falciparum and capaciedad of parasite to develop resistance to drugs antimalários suggest, as a strategy for research, exploration of new targets functional allowing the development of new approaches terapérutocos frenta wing malaria. Validation of a gene, or its product, as a target requere know the function of the same within the biology parásito.En Here, have developed various systems for functional analysis of genes of P. Falciparum as the inactivation of gene recombination, systems or antisense RNA silencing through RNA interferente.Los objective through this research work have been: the selection of genes model P.falciparum which could represent specific targets pair designing new antimalarial agents, explore different sistemaas of gene silencing in P.falciparum, which has necessitated the construction of a collection of vextores, the design of short oligoribonucleotides and optimizqación systems transfection, and for developing systems molecualres of last analysis efecrto functional silencing in the life cycle of the parasite. The introduction and expression of exogenous genetic material in Plasmodium falciparum is temporarily efficient, but the drop segragación plasmids pair is a serious impediment to long-term expression and the establishment of clones parásito.El silencing through disruption by homologous recombination for analysis of the depending on how genes with significant drawbacks experimentales.Es possible partial gene silencing through interferenca with RNA, as well as RNA antisense RNA or double chain paqueño size, which seem to inhibit translation by a mechanism shared The silencing of genes maedante RNA small double chain is the system of choice. At present, in Pfalciparun to assess the function of individual genes of interest. It has been desarrolladdo a method which allows to quantify the expression of RNAm using minimal amounts of crop P. Falciparum, which has resulted in a significant increase in sensitivity and allows its climbed for the analysis of expression of a large number of genes in multiple experimental conditions of cultivation. With systems in silencing P. Falciparum we have seen that there is a high correlation between levels of RNAm and its product in the two genes studied Pfg6pd and Pfrab5a.El analysis silencing of the Rab5A demonstrates the dispensabilidad their role perhaps as a result of the existence of isoforms in the genome the parásito.El analysis silenciamientos of G6PG has highlighted the importance of self G6PD and system tioredoxina and particulasr of tioredoxin reductase in the cascade of antioxidant parasite, and thus in his supervivencia.El difente size protein PfG6PD, obsevado through inmunodetección, depending on the estadoi parasite indicates the existence of a mechanism for processing of this particular protein bifuncional structure unique in the genus Plasmodium.

Author: ALVAREZ DE FRUTOS CRISTINA.
Year: 2003.
University: COMPLUTENSE DE MADRID.
Place of defense: CIENCIAS QUÍMICAS.
Place of preparation: INSTITUTO CAJAL,CSIC.

Summary: In previous laboratory work has shown that Snail family gene is involved in the induction of the transition epitelio-mesénquima (TEM) both during embryonic development (Nieto and col.1994; Del Barrio and Nieto, 2002) and in the progression tumoral (Cano y col.,2000;Blanco y col., 2000).Esta función se realiza ,al menos en parte , por la represión directa de la transcripción del gen de la cadherina E, si bien se piensa que sebe tener dianas adicionales . Functional studies in the mouse embryo have shown that Snail is essential for TEM, as the mutant mice die enetapas temperatures training mesodermo due to its inability to carry out this transition (caver and col.2001). Infeasibility This makes it impossible the study of the role of Snail in development processes later (formation of neural crest or cartilage etc.) or pathological processes in adulto.Por that reason, the purpose of this project was to develop a system inducible expression of Snail in the mouse. This paper describes a system of conditional expression of Snail transcription factor, consisting of a building containing the plasma region coding of this transcription factor coupled with a mastery of binding to estrogen, previously mutated, which presents a high affinity for estrogen synthetic, 4-hidroxi-tamoxifeno.Tras his administration, estrogen is able to induce the activity of the protein translocation Snail for his tumor. We induced activity Snail in three stages of development of the mice. In preimplantation embryos stadiums, the activation of Snail accompanied by the gradual loss of cadherina E and the consequent descompactación of oocytes from mice trasgénicos.Este phenotype should be deleted by oppressors of Snail, we are proposing the cultivation of embryos preimplantation system as returning officers for potential oppressors. The introduction of the Snail activity in mice post-natales has caused an increase in vasculación of long bones, and the colonization of the same by excess osteoclastos.El increase in this population of cells should be responsible for the disqualification bone of these mice, suggesting that activation of pathologic Snail in the adult could lead to this phenotype degenerativo.Asimismo, induction of Snail leads a phenotype of dwarfism, accompanied by a decline in the growth plate, probably caused by inhibition the proliferation of cellular phenotype which has been confirmed in mouse embryos late.

Author: RODRIGUEZ DE LA CONCEPCIÓN M. LUISA.
Year: 2004.
University: BARCELONA.
Place of defense: FACULTAD DE BIOLOGÍA.
Place of preparation: FACULTAD BIOLOGÍA.

Summary: The results presented in this thesis fall into three blocks of study: The Implication of brown adipocyte in lipomatosis Induced by the high activity of antiretroviral therapy (HAART), the study of new factors regulating mitochondrial function in the adipocyte brown and effect of antiretroviral drugs in adipocitos in culture. The analysis of gene expression of adipocyte marker brown UCP-l in lipomas number of patients infected with HIV and antiretroviral therapy involves adipocyte brown in etiopatología of lipomatosis associated with HAART therapy. The study of the regulation of mitochondrial biogenesis at the molecular level in adipocitos brown, led us to note that the brown adipocyte differentiation is associated with the induction of the mitochondrial transcription factors Bl and B2 And co-activador PGC-la, association with an increased mitochondrial biogenesis. The gene expression of these factors is positively regulated by agonist PPARy and insulin. By contrast, norepinephrine suppresses the expression of the transcription factors mitocondrlal Bl and B2 by mechanisms dependent transcription and independent of protein synthesis. This leads to a reduction in the expression of genes encoded by the mitochondrial genome as a subunit II of the cytochrome c oxidase. This effect is dependent on the degree of brown adipocyte differentiation and is favored by the presence of activators PPARyo insulin. The expression of co-activador PGC-la is induced by norepinephrine, RXR agonists PPARyy agonists in adipocitos brown differentiated, but not preadipocitos. These inductions are given by independent mechanisms of protein synthesis. The induction PGC-la by noradrenaline is facilitated by the presence of activators PPARy. The induction by RXR agonists PPARy and is blocked by an antagonist PPARy and is independent of the activation of adrenergic receptors. It describes, therefore, a new way of regulating PPARyjRXR-dependiente gene PGC-la. We studied the effect of lithium, a drug used to treat schizophrenia and bipolar disorder, brown adipocyte differentiation. It was noted that litlo inhibits so dependent on the length of exposure and dose morphological differentiation of cultured adipocitos browns, as well as gene expression in the preferential expression of adipocyte brown. We studied the effect of antiretroviral drugs reverse transcriptase inhibitors on the brown adipocyte by analyzing their differentiation and gene expression in relation to mitochondrial function. The results indicated that we inhibitors, nucleoside reverse transcriptase no-análogos to have an opposite effect on the differentiation of brown adipocyte: efavirenz blocks the accumulation of iípidos while nevirapine has a positive effect on the overall differentiation adipocitaria, mitochondrial biogenesis and expression of UCP-1. The reverse transcriptase inhibitors, non-nucleoside analogues affect brown adipocyte differentiation. The stavudine, zidovudine and didanosine cause a decrease in the content of mtDNA, but the expression of the mitochondrial genome is only diminished in the cells treated with didanosine. The compensation to the depletion of mtDNA in cells with stavudine could be explained by the increase in the expression of the mitochondrial transcription factors. In addition, stavudine induces the expression of a specific gene UCP-l through mechanisms that involve the path of retinoic acid. Finally, we develop a model of cellular adipocitos human primary culture and what we use in the analysis of the effect of the drug to 8 ntiretro 2cf viral didanosine. The results showed us that preadipocitos human primary culture can differentiate into adipocitos with morphological characteristics, gene expression and functional characteristic of human adipocyte white. The dldanosina effects consistent with a blockage in mitochondrial function.

Author: SORIANO ZARAGOZA FRANCESC XAVIER.
Year: 2004.
University: BARCELONA.
Place of defense: FAC.BIOLOGIA.
Place of preparation: FAC.BIOLOGIA U.BARCELONA.

Summary: SUMMARY: Mitofusina 2 (Mfn2) is a mitochondrial protein fusion. The experimental evidence demonstrate a lower gene expression Mfn2 in skeletal muscle of obese subjects and patients with diabetes type 2. By inhibiting the expression of Mfn2 cells in culture reduces the oxygen consumption, the mitochondrial membrane potential and oxidation of glucose, indicating an important role of Mfn2 in mitochondrial biology and in the pathophysiology of obesity and / or diabetes Type 2. In addition, there are related mutations in the gene Mfn2 with neuropathy of Charcot-Marie-Tooth type 2 and has been described antiproliferative a role in vascular disorders. Consequently, the study of the regulation of gene expression Mfn2 is of considerable interest. In this thesis, has been donated by the developer human Mfn2 and it has been determined that no TATA box and is located in a CpG island, which makes it capable of being regulated by methylation. In skeletal muscle possesses at least 6 early transcription in a window of 171 base pairs. Studies with genes reporters have determined that Spl could have a leading role to the basal transcription machinery to form the complex preiniciación. The expression of Mfn2 is higher in tissues with high energy requirements. Trials with reporters have identified gene transcription factors that could be responsible for the expression of tissue-dependent. It has been studied in detail induction Mfn2 in skeletal muscle and adipose tissue brown with exposure to cold and treatment receptor agonists B3-adrenérgicos conditions both caracterizadaspor a high energy expenditure. Increased expression of Mfn2 under these conditions is mediated by PGC-1a which coercive to ERRa that joins the promoter Mfn2 in the form of monomer between bases -413 and -408. The increase in the mitochondrial membrane potential associated with the expression of PGC-1a is inhibited by the suppression of the expression of Mfn2. Mfn2 could be a crucial effector activation induced mitochondrial PGC-1 a. It has brought new evidence of the relevance of Mfn2 in controlling the mitochondrial oxidation of substrates to show a greater expression of Mfn2 in oxidative muscle fibers in fiber glucolíticas. The transcript of Mfn2 increases in response to increases in the concentration of intracellular calcium. Our results suggest that MEF2, which binds to and active promoter of Mfn2, could be an effector of the signaling cascade initiated by the cytosolic calcium. The data obtained indicate that the expression of Mfn2 in skeletal muscle are increased under conditions of high energy expenditure which stimulates oxidation of substrates, causing increases in mitochondrial membrane potential. Depending on the demand of cellular ATP, the mitochondrial membrane potential is dissipated to produce ATP or generate heat.

Author: ABDEEN ASHRAF M. A..
Year: 2004.
University: BARCELONA.
Place of defense: FACULTAD DE FARMÁCIA.
Place of preparation: DE BARCELONA -CSIC.

Summary: The gene leucine aminopeptidasa (LAP) is induced by injury and jasmonato. Studies linker-scan mutagenesis promoter LAP identified two new cis-elements required for the expression induced by JA. These elements include a box MYB and why T / G-box. Two transcription factors type bHLH (JAMYC2 and JAMYClO) who join this gene were identified. The silencing of genes JAMYC2 and JAMYCIO by VIGS, indicating a possible role for these redundant transcription factors with other factors MYC in the same family. The removal stable through RNAi of the entire family of genes resulted in a blockade in the levels of expression of the gene LAP, demonstrating a function of these transcription factors as positive regulators of these genes. The microarray analysis of the lines silenced for the family JAMYC revealed that these factors differentially regulate two branches in the path of sefialización JA. On the one hand, JAMYC activates the expression of genes induced in response to wound and JA, such as pin2, pinl and LAP, while negatively regulate the expression of genes induced in response to JA and ET, such as PR-I, PR -5 And b-CHL and induced during the reaction of defense against pathogens. Factors JAMYC well would have a regulatory function similar to the gene AtMYC2 Arabidopsis, both transcription factors playing a key role in the activation of different sets of genes of defense against attack by insects and pathogens infection. The silencing by VIGS gene LeMYB24 tomato reduced gene expression PR-I in response to treatment by SA or viral infection. Genes AtMYB24 and AtMYB57 Arabidopsis have high homology with LeMYB24 and are induced by the treatment of BOA. Mutants insertion in the gene AtMYB24 showed at times early induction, low levels of expression of the gene PR-I in response to treatment by SA. The silencing by RNAi gene AtMYB57 in the background mutant atmyb24 resulted in a blockade on the expression of the gene PR-I. The overexpression of AtMYB24 in Arabidopsis led to the constitutive activation of this gene. The protein AtMYB15 joins the box MYB present in the promoter LAP and is able to activate the expression of this gene in transient expression experiments on cells BY2 of snuff. The co-transfección of AtMYBl5 and factors JAMYC results in an increase in levels of activation of the building reporter -317LAP, indicating a cooperative interaction between AtMYB15 and proteins JAMYC. Mutants insertion in the gene AtMYBl5 show altered expression of genes regulated by BOA and JAlET. These mutants showed reduced levels of messenger VSP in response to JA, and higher levels of messenger for genes regulated by JAlET- such as PDFI.2 and PR-4, suggesting a key role of AtMYB 15 in the activation of genes JA and dependent on the repression of genes dependent JAlET-. This function is probably exercised through a cooperative interaction with AtMYC2 or through a direct regulation of gene AtMYC2.

Author: HERRERO RODRÍGUEZ LAURA.
Year: 2004.
University: BARCELONA.
Place of defense: FACULTAD DE FARMACIA.
Place of preparation: FACULTAD DE FARMACIA.

Author: PEDRAZA GONZALEZ NIEVES.
Year: 2004.
University: BARCELONA.
Place of defense: FACULTAD DE BIOLOGÍA.
Place of preparation: FACULTAD DE BIOLOGIA.

Author: FERNANDEZ FERNANDEZ CRISTINA.
Year: 2004.
University: COMPLUTENSE DE MADRID.
Place of defense: FACULTAD DE CIENCIAS BIOLOGÍCAS.
Place of preparation: CENTRO INVESTIGACIONES BIOLÓGICAS CCSIC.

Summary: The objective in this work has been advancing and deepening the knowledge of the path of degradation Phenylacetic acid (PA) in Escherichia coli W First has characterized the second stage in the degradation of PA, the transformation of AP in fenilacetil - CoA (PA-CoA). It has been shown on the one hand, that genes paaABCDE are all necessary for the hydroxylation of the aromatic ring of PA-CoA first intermediary path, and secondly, that this is dependent hydroxylation of oxygen. Secondly have studied the gene paaX, which encodes a repressor that controls the expression of genes catabólicos. This work has confirmed that the protein PaaX also is involved in the suppression of its expression, and it has been observed that PA-CoA is the inducer system. The protein PaaX is able to compete and displace the binding of RNA polymerase to the promoter region. It has been observed that the promoters of the path of degradation PA can not be activated under anaerobic, because it can be synthesized PA-CoA since the PA-CoA ligasa quickly loses its activity in the absence of oxygen. Thirdly has studied the role of gene paaY which is adjacent to the gene paaX The absence of the gene paaY delay significantly the growth of E. PA coli is present but only when the repressive PaaX. Moreover cells deficient in this gene are not able to activate an effective route paa used only when the bacterium PA as a source of energy. The protein PaaY has hiperexpresado and purified Check is a metaloenzima trimérica introducing activity tioesterasa on some compounds derived from CoA.

Author: LÓPEZ BARRAGÁN M. JOSÉ.
Year: 2004.
University: COMPLUTENSE DE MADRID.
Place of defense: FACULTAD DE CIENCIAS BIOLÓGICAS.
Place of preparation: CENTRO DE INVESTIGACIONES BIOLÓGICAS CSIC.

Summary: Studies in this thesis has allowed the identification and characterization of potential catabólico of Azoarcus sp. IBC, which can degrade anaerobically (conditions desnitrificantes) aromatic compounds such corno toluene and m-xileno. For the first time in the genre Azoarcus has proposed a path central anaerobic degradation of aromatic compounds via benzoil-CoA and have identified the genes responsible for that route biochemistry, which are grouped in one cluster, called bzd organized in a regulatory operon , consisting of the gene bzdR, controlled by the promoter and another PR operon catabólico, bzdNOPQMSTUVWXYZ, controlled by the promoter PN. It has designed a cassette carrying the genes bzd into a vector mobilized broad spectrum of host with the aim of exploring the potential of engineering anaerobic routes, as this is the first cassette as described in the anaerobic catabolism of aromatic compounds. The gene bzdA encodes a benzoato-CoA ligasa involved in the initial reaction of catabolism anaerobic benzoate and recognizes corno substrates greater variety of aromatic compounds than others benzoato-CoA ligasas described so far, so their guests hiperproducción in heterologous shows interest biotech for the synthesis of derivatives benzoil-CoA. The protein regulatory BzdR is the first transcriptional repressor that is described in anaerobic catabolism of aromatic compounds. This transcriptional regulator interacts with three operating regions present in the promoter catabólico PN inhibiting its activity, having featured for the first time in anaerobic catabolism of aromatic compounds, the nature of the molecule inducing, benzoil-CoA. The new modular structure of the protein BzdR, consisting of a domain-type HTH-XRE typical protein binding to DNA and a domain that provides high identity with siquimato kinases makes BzdR the first member of a new subfamily of transcripcionales regulators.

Author: ÁLVARO PÉREZ CRISTINA DE.
Year: 2004.
University: COMPLUTENSE DE MADRID.
Place of defense: FACULTAD DE FRAMACIA.
Place of preparation: FACULTAD DE FARMACIA.

Summary: The skeletal muscle tissue is an important target for insulin and insulin growth factor type I IGF-I, both from a metabolic, primarily in glucídica homeostasis, as in the processes of differentiation and regeneration muscular.La type 2 diabetes is a progressive metabolic disorder, which is characterized by the body's inability to store and consume glucose in the appropriate way, either by a deficiency of B-cells in the pancreas to secrete necessary amounts of the hormone, or by a lack of response from the peripheral tissues to the action of the insulina.De Indeed, the skeletal muscle is responsible for aprocimadamente the 80% of the utilization of glucose after ingestion of carbohydrate or the direct infusion of sugar and is in the fabric we detect the early signs of a state of insulin resistance, whose main result is a decrease in transport of glucose into the muscle fiber in response to the hormone due to multiple alterations in the signaling via the insulin this tejido.En this paper we described how the TNFA produces insulin resistance in muscle esqueético, reflecting a decline in the translocation of GLUT4 and in the transport of glucose and insulin in response to the attenuation of the signal path hormona.Por Furthermore, the muscle regeneration is a fundamental process after certain injuries, accidents or other pathologies, so knowledge of the molecular and cellular mechanisms that maintain the plasticity muscle permitá define the potential for adaptation to different situations muscle and also, identify new genes and signaling pathways involved in certain patologías.En Here, in this work we studied the molecular mechanisms that modulate muscle differentiation in different situations, such as the transformation of Ras oncogenes and stimulation with growth factors, for example, the FGF.

Author: ROLLÍN TOLEDO RAQUEL.
Year: 2004.
University: COMPLUTENSE DE MADRID.
Place of defense: HOSPITAL CLÍNICO SAN CARLOS.
Place of preparation: HOSPITAL CLÍNICO SAN CARLOS.

Summary: In this work we find the expression of tránscritos of the recipients of the natriuretic peptides (NPRA, NPRB AND NPRC), together with the expression of mRNA of ANP, BNP NPC and in the retina of human subjects controls, providing evidence of there is a system natriurético local.Los levels of gene expression as measured by quantitative real-time RT-PCR showed that there were no significant deferencias between different receptor subtypes of natriuretic peptides in the retina of human subjects controles.La expression of natriuretic peptides were detected by immunohistochemistry in vascular structures, glial (astrocytes) and neural retina of adult normal, suggesting a role of natriuretic peptides in preserving both the vascular integrity of the neural retina matures. In diabetes mellitus, the expression of ANP retinal varies depending on the metabolic status and the degree of retinal ischemia in parallel with changes in the expression of GFAP in the inner layers of retina.Los highlight of this study has been finding that in the proliferative diabetic retinopathy signal inmuorreactiva the PNA disappears (in parallel with a decrease of astrocytes) while the laser treatment increases the signal from the PNA together with the reactive glía (astrocytes and cells MÃ ¼ ller). These data suggest that the PNA could exert a role antiangiogénica in proliferative diabetic retinopathy possibly by inhibiting the production of VEGF or activation latter factor molecules involved in angiogenesis.

Author: TORRES TORRES RAÚL.
Year: 2004.
University: COMPLUTENSE DE MADRID.
Place of defense: FACULTAD DE BIOLOGÍA.
Place of preparation: CENTRO DE INVESTIGACIONES BIOLOGÍCAS (CSIC).

Summary: For Friedreich Ataxia is a neurodegenerative disease caused by the expansion of repetitions GAA.TCC in intrón 1 gene FRDA.En this thesis has studied the effect of this sequence repeated on the progress of the replication forks and the process in recombinant plasmids Escherichia coli.Hemos found that there is a narrowing of the forks when the stream (TTC) n acts as a mold of the thread delayed during replicación.Esta break takes place in the area prior to tripletes.Efectos could be explained as a consequence of the formation of a DNA tríplex within repetition as a result of the homopurina.homopirimidina of secuencia.La volatility of repetition, not only appears to be due to the presence of the pause replication, but it appears to have several other causes. Moreover, we found that regardless of orientation, repetition GAA.TCC induce the accumulation of X-shaped molecules consisting of two molecules plasmídicas united by a union-type Holliday (HJ) located in the repeticiones.Esto could be related to the ability of DNA tríplex to block the migration of unions Holliday. The resolution of these asymmetrical unions Holliday within the repetitions could be a source of instability common to the two orientaciones.No however, we do not rule out other possible causes of blocking unions Holliday as the asymmetric formation of the same within the repetitions or heterogeneity in the number of repetitions between plasmids recombine

Author: HERNÁNDEZ ESCAREÑO JESÚS JAIME.
Year: 2004.
University: AUTÓNOMA DE BARCELONA.
Place of defense: FACULTAD DE VETERINARIA.
Place of preparation: ESCUELA DE POSTGRADO.

Summary: The genus includes Malassezia yeast lipófilas whose main habitat skin of a wide variety of mammals and birds. Classically trained was seen by two species, the species lipodependiente M.furfur, characteristics of the skin and the kind of man not lipodependiente M.pachydermatis, associated with the animals. In the latest revision of the genus taxonomic done, the number of species lipodependientes of written has increased, including a total of six: M.furfur, M.globosa, M.obtusa, M.restricta, M.slooffiae and M.sympodialis. Malassezia pachydermatis is classically considered as zoófila. However, in recent years have been described systemic infections in humans. M.pachydermatis is often associated with otitis externa and different types of dermatitis in pets, especially dogs. It has been accepted that this organism plays a significant role in the production of animal diseases when there is an imbalance in the microclimate of the skin. There are few published studies on the presence and distribution of the different species lipodependientes gender Malassezia using the latest revision of the genus. Recently there have proposed four new species, of which M.dermatits, M.nana and tentatively called "equi Malassezia" features morphological, physiological and genetic similar to M.simpodialis. The routine identification of these species lipodependientes is based primarily on the study of the use and various esters depolioexietilensorbitano. However, these tests are tedious to make and in some cases the results are difficult to interpret, there difficulty in obtaining proper identification. The purpose of this dissertation is the study of the genetic diversity of strains within Malassezia spp.aisladas of pets using DNA techniques, including: 1 - The molecular characterization of strains M.pachydermatis isolated from different pets healthy and with skin disorders through RAPD technique (random amplified DNA polymorphism). 2 - The molecular characterization of strains lipodependientes isolated from different pets near the kind type of M.sympodialis through DNA techniques based on the comparative analysis of the sequences of the fragments D1/D2 26S and ITS-2.8S DNA ribosomal. For the molecular characterization of M.pachydermatis analyzed a total of 55 strains and strain neotipo CBS 1879. With the primer OPT-20 we differentiate 4 genotypes. The predominant genotype (genotype I) was observed in isolates of all animal species and in different anatomical locations. The only genotype was observed in expenses, horse, goat and pig. The other three genotypes were detected only in strains recovered from external ear dog. The genotypes II and IV were observed in dogs with oititis while genotype III of healthy dogs. For the molecular characterization of the strains lipodependientes close to the kind type of M.sympodialis have been analyzed a total of 20 strains. The phylogenetic analysis of sequences from both regions (D1/D2 and ITS-5.8S) allowed us to bring together the strains into four distinct groups. Group I included strains different species of domestic animals (horses, sheep and pigs) and the strain rate M.sympodialis. Group II included strains c 8 aballo, 4d9 agrupándose with the sequence AJ305330 "M.equi." Group III comprised isolates mainly goats. The group was formed by IV isolates of cats, agrupándose with the sequence of M.nana AB075224. It is unclear whether these genetic differences are sufficient to identify new species or only show a genetic variation between strains of different sources within M.sympodialis, which are in the process of differentiation and probably adapt to specific host animal.

Author: GOMEZ MALDONADO JOSEFA.
Year: 2004.
University: MÁLAGA.
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: FACULTAD DE CIENCIAS.

Summary: It has analyzed the pattern of expression of genes coding for glutamine synthetase, GS1 and Gs1b pine during the primary development, determining which presents a tissue and cellular localization not solapante, supporting the hypothesis that both ioenzimas perform a different metabolic function . On the one hand it has been determined that GS1b is the enzyme that is involved in the (re) absorption of nitrogen content in proteins reserve seed and stadiums planturales, its location vascular indicates a role for this enzyme in the biosynthesis of glutamine for transportation to other parts of the seedling. The expression of GS1a associated parenchymal chlorophyll, suggesting their involvement in the provision of nitrogen compounds for photosynthetic function. We have analyzed the structures of the promoters of the two genes GS1 pine, his study dealt with the use of different approaches, such as transient expression system homologous and heterologous in, as well as testing delayed gel. REGARDING GS1A: It has been determined that their region distal promoter presents a sequence AT1 and AT2 binding factors for nuclear AT These boxes are essential for promoting activity and could operate in coordination with the positive element poli-CT present in the region leader . It has been suggested the functionality of these elements in the ECI of GS1a mediated transcriptional activation by light. It has also revealed the existence of negative elements affecting the expression of the gene located in the central area of its structure promoter. REGARDING GS1B: Using the procedure for the analysis of transient expression in protoplasts hormonally treated pine, it has been able to define a region involved in regulating GS1b by gibberellin, which can be seen in the context of the role of this hormone in the development of embryo coordinated with proteolytic enzymes responsible for d degrade proteins reserve seed. The structure promoter GS1b presents a sequence AT functional, which could be involved so accessory in the functionality of the box primigenias, increasing the accessibility of the fund to its specific transcription factors. It has been determined that there is a negative regulatory region in the most distal promoter analyzed the structure, which affects gene expression specifically in cotyledons. Finally, the results obtained using different molecular approaches have led to propose that both Myb1 as Myb4 can regulate gene expression GS1b in tandem with the regulation of the metabolism of fenilpropanoides leading to an efficient (re) absorption of ammonium released in the synthesis of lignin in the vascular tissue. This coordinated regulation appears to be carried out by the interaction of factors Myb with AC specific elements present in the promoter of GS1b.