BIOCHEMICAL GENETICS

Author: RIPOLL SAMPER JUAN JOSE.
Year: 2003.
University: MIGUEL HERNÁNDEZ DE ELCHE [www.umh.es].
Place of defense: MEDICINA.
Place of preparation: FACULTAD DE MEDICINA - UNIVERSIDAD MIGUEL HERNÁNDEZ.

Summary: Arabidopsis thaliana presents excellent properties as a model organism to carry out both genetic and molecular studies of the development process. The analysis of the development of the fruit in this plant model is generating considerable progress in understanding this phenomenon. With the objective of contributing to the understanding of this development program, we carried out a scrutiny of mutants affected in the morphology of fruit in a collection of T-DNA insertion lines. Several mutants were isolated with fruits of abnormal appearance. One of them, called pepper1-1 (pep1-1) shows a phenotype pleiotrópico characterized by various traits among these is the emergence of more than two valves in the fruits. The cross sections of fruit pep1-1 to observe three or four repla delimiting three or four valves, respectively. This phenotype was transmitted to the progeny following a recessive pattern characteristic and absolutely linked to resistance to the antibiotic kanamycin, conferred by the presence of T-DNA. By I-PCR and TAIL-PCR were characterized genomic regions adjacent to the edges of the T-DNA, finding sequences belonging alos chromosomes IV and V. In addition, it was found that the locus PEP1 shows absolute linkage to markers SSLP both chromosomes, suggesting a reciprocal translocation. The results of Southern hybridization and semiesterilidad that prsentan the heterozygote pep1-1 is ecuentran consistent with the type of chromosomal aberration. All this suggests that the phenotype of splantas pep1-1 is the result of a complex combination of genetic functions. However, tests alelismo between pep1-1 and various lines of insertion in one of the genes potentially affected by the aberrción chromosome show that these estripes mutants fail to complement, appearing fruits which have extra valves. The new alleles (pep1-2 to pep1-5) presents a single T-DNA insertion in a gene encoding a polypeptide with three domains of interaction with KH-type RNA. This belongs to a large family of 26 members in Arabidopsis and is highly conserved across the plant phylogeny. The introduction of transgenic constructs carrying the wild version of the gene phenotype rescued wild pep1-1, demonstrating that mutations in this gene are the cause of the mutant phenotype observed. PEP1 expressed in the principal organs of the plant (roots, stems, leaves, flowers and fruits), which is consistent with the degree of pleiotropía observed in their mutants. By in situ hybridization has located the transcribed from PEP1 in outlying regions of meristemo floral and in the region for the replum. Moreover, the sinteracciones genetic suggest that PEP1 involved in the morphogenesis of the plant through various development processes as a means of signal transduction CLAVATA or route autonomous control of flowering time. Proceeds from PEP1, an alleged protein binding to RNA, could be playing a role in regulating postranscripcional of gene products important in developing these routes.

Author: MUÑOZ MUÑOZ IVAN.
Year: 2004.
University: AUTÓNOMA DE BARCELONA [www.uab.es].
Place of defense: FACULTAT DE VETERINÁRIA.
Place of preparation: ESCUELA DE POSTGRADO.

Summary: The phosphatases type Z, encoded by genes PPZ1 and PPZ2 develop an important role in the biology of the yeast Saccharomyces cerevisiae. Specifically, exert a negative control on salt tolerance and cell cycle progression and positive in the maintenance of cell integrity. At the time of initiating this work only knew of a regulatory subunit of these phosphatases, known as Hal3 or Sis2. This protein acts as an inhibitor of all functions concoidas of Ppz1. However, their mechanism of action is unknown. The primary aim of the thesis has been trying to deepen the regulation carried out phosphatases Ppz1 and Ppz2 in controlling cell cycle in yeast. This has created a double mutant condiconal sit4 hal3 introducing a stop between phases G1 and S cycle. This strain has been transformed with two gene banks mutlicopia different. As a result it has been possible asilar a series of genes, multicopia are able to suppress the phenotype of this strain. Among them are regulators of the cell cycle already known, some phosphatases, elements involved in homeostasis saline and three genes whose function is unknown at the moment, which have been renamed VHS1, VHS2 and VHS3. Secondly, we have investigated the mechanism of action of Hal3 on phosphatase Ppz1. This has been studied elements found in the sequence of Hal3 possibly relevant to the function of this protein. In addition, there has been a strategy of random mutagenesis of the most preserved Hal3 followed by a search for lost function. This has enabled halalr a series of changes aminoacídicos only that interfere with the function of Hal3. The biochemical characterization post has identified two regions in the sequence of HAl3 important for the union to Ppz1 and important region for a third to inhibit phosphatase. Finally, it has studied the biological role of YFR003c an ORF whose function was unknown for the time being. These studies have shown that encodes a protein with characteristics similar to those phosphatase inhibitor type 1 human. Moreover, it has been able to demonstrate that Yfr003c is able to unite and inhibit in vitro Glc 7 (the catalytic subunit of the phosphatase type 1 of yeast) and, less efficiency Ppz1. Subsequent studies in vivo reinforcing the role of Yfr003c as inhibitor Glc7, why has received the name of Ypi1 (Yeast Phosphatase 1 Inhibitor).

Author: IGLESIAS SERRET DANIEL.
Year: 2004.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: UNIVERSITAT DE BARCELONA.

Summary: In the Doctoral Thesis has studied the transcriptional regulation and traduccional of genes involved in controlling the apoptótico process, as well as routes transducciones signals involved in its regulation. We have studied the regulation of gene Mcl-1, the family of Bcl-2, in line limfocitaria Jurkat during induction of apoptosis by drugs estaurosporina and aspirin. The two drugs induce apoptosis by mechanisms dependent and independent of the activation of caspases. The estaurosporina takes effect transcriptional level and traduccional, while aspirin induces stop the synthesis of proteins in the cell, at least for phosphorylation factor eIF2alfa. Our results indicate that the disappearance of Mcl-1 may be necessary but not sufficient to induce apoptosis in cells Jurkat. Our results indicate that these inserts are a polymorphism in the population and does not appear to be associated with a poor prognosis in the diagnosis. We have studied the regulation of gene BIM, the family of Bcl-2 in primary cultures of LLC-B during apoptosis induced by different drugs and during treatment by various factors of survival. It also has studied the region 5'-UTR gene antiapoptótico XIAP, the family of TSIs.

Author: MAYOR OLEA ÁLVARO.
Year: 2004.
University: MÁLAGA [www.uma.es].
Place of defense: FACUTAD DE MEDICINA.
Place of preparation: DPTO. DIOQUÍMICA , FACULTAD MEDICINA.

Summary: OBJECTIVES: To study the involvement of different genetic polymorphisms of angiotensin Renina Systems (MRS): delección insertion (I / D) of the gene Enzina Conversora of Angiotensin (RCT), the polymorphism A1166C gene receptor AT1 of angiotensima and polimordismo M235T the angiotensinogen gene, in the choice of drug treatment of Hypertension (ETS). METHODS: A retrospective study based on the genetic analysis of 250 patients (58% female) treated as monotherapy (ACE inhibitors or ARAII) with hypertension control over tiempo.La response to antihypertensive therapy was analyzed based on the control of ETS over time and treatment options for each patient based on their medical history and pharmacology. RESULTS: With regard to treatment with ARAII, we found no statistically significant differences in allelic frequencies genotypic not in any of the polymorphisms studied. The pacentes treated with ACE inhibitors showed significant differences (p greater 0.05) in terms of the presence of D allele polymorphism L / R RCT, which this is more common in non-responders (0.73) compared with responders (0, 58). was also observed statistically significant differences in genotype frequencies II turned out to be from 0.21 in responders and 0.05 in the group of patients who were non-responders (p greater 0.05). Regarding the polymorphism A1166C and M235T no significant differences were observed in the response to drugs. CONCLUSIONS: polymorphisms A1166C gene receptor AT1 and M235T of angiotensinogen gene are not predictors of response to treatment of essential hypertension with ARAII or ACE inhibitors in monoterapia.La polymorphism genotyping of the L / R RCT will be a a useful tool in the future for when choosing a treatment for essential hypertension, and treatment with ACE inhibitor beneficiosoo in patients with homozygous genotype II.

Author: HERNÁNDEZ RODRÍGUEZ CARMEN SARA.
Year: 2004.
University: VALENCIA [www.uv.es].
Place of defense: BIBLIOTECA DEL CAMPUS DE BURJASSOT.
Place of preparation: FACULTAD DE CIENCIAS BIOLÓGICAS-UNIVERSITAT DE VALÈNCIA.

Summary: Bacillus thuringiensis is the biopesticida most used in the world and expresi6n their toxins Ien plant transgenic crops to offer protection to front insect pests along dE entire crecimientO the plant. In this thesis is estudiala distribution of various tóxinas B. In thuringiensis isolates of this species was described and the interaction of these binding sites with specific membrane vesicles from different insect pest. The estudlo of distribuciónn ~ b-exotoxina tlpo I found that this metabolite was produced by strains of type serovars thuringiensis, tolworthi and darmstadiensis. There was correlation of production or absence of certain exotoxin serovars of B. Thuringiensis. In a study with isolated from Bolivia, was found B. Thuringiensis in 60% of samples from the potato-producing regions in Bolivia. The most toxic isolated against Spodoptera exigua and Phthorimaea operculella contenian crystals bipiramidales, a band of 130 KDa and genes of families cryl and cry2. These isolates were identified a gene cryl again. Most of the isolates from 4 collections from established habitat and types of samples divers as contenian crystals bipiramidales. The presence of crystals bipiramidales related to the amplification of genes crylA and cry2, while the presence of crystals rounded was asocidada with the absence of these genes. The overall frequency of genes crylA and cry2 was 61.5% and 59.2%, respectively, and there was a tendency for these genes appear conjuntaniente. In a collection of 507 strains of B. Thuringiensis, the frequency of genes vip was de110% for gene viply vip2, and 49% for gene vip3. The strategy of PCR-RFLP used permiti6 identify and classify these genes vip based on the ten different patterns found. Two not expected patterns showed the presence of two genes vip new wings belonging families vipl and vip3. It showed that the lyophilization of intestines lepidopteran larvae is a method otimo of preservaci6n which allows realizaci6n studies union of Cry toxins of B. Thuringiensis and vesículas membrane of the intestine yes conservation method most suitable for introduction this biological material. Studies union of Cry toxins to vesículas membrane concluded that the toxins d ~ Cry1Ac, CrylFa and Cry1Ja share the binding site on the membrane in Helicoverpa armigera, Helicoverpa zea and Spodoptera exigua. In these insects, not ericontraron sites additional uni6n to Cry1Ac and Cry1Fa. These results, together with previous experiments, supporting the idea that toxins Cry1A, Cry1Fa and Cry1Ja have a common binding site on most species of Lepidoptera.

Author: BLANCH MOCHALES ÁLVARO.
Year: 2004.
University: AUTÓNOMA DE MADRID [www.uam.es].
Place of defense: FACILTAD DE MEDICINA.
Place of preparation: FACULTAD DE MEDICINA.

Summary: The hereditary angioedema (HAE) is a rare disease which is characterized by recurrent swelling in the subcutaneous tissue or submucoso gastrointestinal tract and / or upper respiratory vias, and that is potentially lethal because of the risk of suffocation that produce swelling of glottis. The HAE is inherited as an autosomal dominant and is caused by mutations in the gene C1 NH producing a deficiency cUantitativa (HAE type 1) or functional (HAE type 11) of C1 inhibitor (C1-lnh), which is an inhibitor of serin proteases regulating systems complement, calicreína-cininas, coagulation and fibrinolysis. This study has analyzed the C1NH in a series of 131 patients belonging to 93 families throughout Spain, and this analysis has focused on large deletions and insertions, and point mutations in exon eight, the two types of mutations most frequently produce HAE. For the characterization of mutations have been used techniques SSCP, Southern transfer, multiple quantitative PCR and sequencing. In some specific cases has been completed study by RT-PCR analysis, PCR fragments lengthy transfer wesiern and ELlSA. As a result we have identified 20 point mutations in exon eight in 30 families different (32%), 13 of which had not been previously described. Among them is the first ~ description of a patient with a mutation in homocigosis in the coding region of C1 NH, which produces a profile of consumption complement similar to that found in patients with acquired angioedema. It also has identified 13 families (14%) with large deletions or insertions in the C1 NH. Among them is the first description of an overlapping exons 5 and 6, and two cases of deletion of exon 4 in which it has sequenced the cutoff point of the deletion. These data support the hypothesis that large deletions and duplications in the C1 NH are produced by homologous recombination between unequal repetitions of the Alu family. Despite the diversity of mutations, their analysis has revealed and / or confirmed certain hot spots of mutation, some of whom are responsible for the high number of mutations nava occurring in this disease. The genetic study has demonstrated its clinical utility to help differentiate these cases nava of cases of acquired angioedema, and to reach the final diagnosis of HAE in some patients with levels of complement in the limits of normal and / or family histories sometimes mistaken diagnosis.

Author: CABALLERO REYES ANTONIO.
Year: 2004.
University: GRANADA [www.ugr.es].
Place of defense: ESTACIÓN EXPERIMENTAL DEL ZAIDIN CSIC.
Place of preparation: ESTACIÓN EXPERIMENTAL DEL ZAIDÍN CSIC.

Summary: The nitroaromático 2,4,6-trinitrotolueno TNT has been used extensively as an explosive military during much of the last century. Its manufacturing and use, as well as the dismantling of instalaiones treatment of this explosive has caused the contamination of aquifers and large areas of land. His high toxicity, in addition to its mutagenic, in addition to the extremely difficult it is their degradation, are the reasons that have convestido one of the contamiantes priority to the elimination by many governmental environmental agencies. Toxicity and character recalcitante are due mainly to the presence of three nitro groups, very electornegativos distributed symmetrically around the aromatic ring that prevents an attack by enzymes oxigenasas. Therefore, the elimination of these groups nitro compounds that can be generated metabolize and degrade to carbon dioxide and water. Pseudomonas putida JLR11 is a bacterium that was isolated in the group Degradation Toxic Organic (DOT) to be able to absorb up to 85% of the nitrogen of TNT, thus favoring its degradation. The objects of this thesis has been focused on the study of the routes assimilation N-TNT in this strain, and has been demonstrated for the first time the existence of two alternative routes able to metabolize the nitro groups of TNT in the same bacterium, so that they can free themselves from the aromatic ring in the form of nitrite or ammonium. The characterization of mutants in key steps for asimiliación of these sources of nitrogen demustra that nitrogen of TNT were incórpora to carbon skeletons of the bacterium through the route GS-GOGAT. In addition, seha marked an enzyme nitroreductrasa, called PnrA, which is a key participant in the metabolism of nitrogen TNT by reducing it to 4-hidroxilamino-2, 6 dinitrotoluno (4-HADNT). In a mutant in the gene pnrA demonstrates that reducing this enzyme conducts claver for the release of nitro groups in the form of ammonia. These results clearly demonstrate the versatility that has metabolic pseudomonas JLR11 to degrade a compound so contamiente as TNT.

Author: ALVAREZ GOMEZ ENRIQUE.
Year: 2004.
University: AUTÓNOMA DE MADRID [www.uam.es].
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: CENTRO DE BIOLOGIA MOLECULAR SEVERO OCHOA.

Summary: The initiation of translation plays an important role in the regulation of gene expression in mammalian cells. The translation initiation factor eIF4G, attached to eIF4E and eIF4A, constitutes the eIF4F complex which is a key component to promote the binding of ribosomes to mRNAs. eIF4G has emerged as a key target for the regulation of translation in mammalian cells infected with different viruses. Thus, cleavage of eIF4G by viral proteases from picornaviruses, as well as from human immunodeficiency virus type 1 (HIV-1), leads to inhibition of protein synthesis directed by capped cellular mRNAs. Notably, this cleavage of eIF4G does not hamper translation directed by mRNAs containing an internal ribosome entry site (IRES) structure. In the present work, cleavage of both eIF4GI and eIF4GII has been examined with the proteases encoded within the genomes of several members of the family Retroviridae, e.g., Moloney murine leukemia virus (MoMLV), mouse mammary tumor virus, human T-cell leukemia virus type 1, HIV-2, and simian immunodeficiency virus. All retroviral proteases examined were capable to cleave the initiation factor eIF4GI both in intact cells and in cell-free systems, albeit with different efficiencies. The pattern obtained from the hydrolysis of eIF4GI with HIV-1 and HIV-2 proteases were very similar to each other, but differed from that obtained with MoMLV protease. Both eIF4GI and eIF4GII were cleaved very efficiently by the MoMLV protease. Notably, eIF4GII was a poor substrate for HIV proteases. Proteolytic cleavage of eIF4G by retroviral proteases led to a profound inhibition of cap-dependent translation, while protein synthesis driven by mRNAs containing IRES elements remained unaffected or was even stimulated in transfected cells and in cell-free systems. Our present findings illustrating those retroviral proteases hydrolyze eIF4G point to the idea that these viruses may follow a mechanism similar to that described for picornaviruses to regulate translation in the infected cells.Rec Recently, the poly (A) binding protein (PABP), which plays an important role in the initiation of translation, has been shown to be a target for proteases from several picornaviruses and caliciviruses. In addition, our findings reveal by the first time that infection of cells with HIV-1 also leads to proteolysis of PABP. Purified HIV-1 PR cleaves PABP both in transfected cells and in the test tube. The PABP hydrolysis by retroviral PR can be extended to other members of the family Retroviridae such as HIV-2 and MMTV. These findings indicate that several retroviruses share with picornaviruses the capacity to proteolyze PABP.

Author: REGUERA VIDAECHEA JUAN JAVIER.
Year: 2004.
University: AUTÓNOMA DE MADRID [www.uam.es].
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: CENTRO DE BIOLOGIA MOLECULAR SEVERO OCHOA.

Summary: ABSTRACT / RESUMEN EN INGLÉS. The assembly and stability of a nonenveloped spherical virion must depend on the establishment of a number of non-covalent interactions between the protein subunits that form its capsid. Interactions between the capsid and the enclosed viral nucleic acid could also contribute to the stability of the virion. This doctoral thesis presents the results of a thorough mutational analysis of the protein-protein interfaces between the subunits of a viral capsid, and of the visible protein-nucleic acid interfaces in the virion. As a model we have used the parvovirus minute virus of mice, one of the structurally simplest spherical virions known. The analysis has allowed to identify amino acid residues at the protein subunit interfaces that are involved in capsid assembly, stability and/or conformation, and those at the capsid-nucleic acid interfaces that are involved in nucleic acid packaging, virion stability and/or infectivity. The specific results obtained can be summarized as follows. 1) Removal of some interactions predicted to be important for holding trimers of the capsid protein together led to the isolation of stable trimeric structures, and confirmed that the MVM capsid assembles through trimeric intermediates. 2) Systematic mutation to alanine of 28 selected residues at the intertrimer interfaces showed that only a few residues per subunit (W203, F247, W543, Y522, T249, Q129, K153) are individually critical for MVM capsid assembly. All but one of these residues correspond to those involved in the highest number of hydrophobic contacts or in hydrogen bonds or salt bridges between trimers. The vast majority of the interfacial residues are not individually needed for capsid assembly or contribute substantially to capsid stability against dissociation. 3) The interfacial residues located around the base of the pores at the 5-fold symmetry axes of the capsid (V40, S43, N149, N170, D263, F526) are also needed for viral infectivity. These residues are involved in a conformational change of the capsid that is associated with the externalization of the N-terminal segment of capsid protein VP2. 4) Other residues at the intertrimeric interfaces that are not critical for capsid assembly or stability are, however, involved in virus viability. Together, the residues that delimit the intersubunit interfaces in a spherical virus capsid are involved in several functions needed for the completion of the viral life cycle. 5) The involvement of capsid residues that interact with the viral nucleic acid on the stability and function of a spherical virus has been established. Several amino acid residues at the capsid-DNA interfaces are involved in the conformational stability of the MVM virion. 6) One residue (D273) in each capsid subunit, negatively charged and located at the internal surface of the capsid, is involved in the packaging of the viral DNA inside the MVM virion.

Author: GUTIERREZ PULGAR JOSE RAMON.
Year: 2004.
University: SALAMANCA [www.usal.es].
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: INSTITUTO DE MICROBIOLOGIA Y GENETICA.

Summary: The riboflavin is an essential vitamin in humans, has been implicated in many redox processes in the cell. To improve the production of this vitamin, has studied the route biosintética of purines in the body superproductor Ashbya gossypii to increase the availability of precursor GTP. In this paper we studied two Metabolism IMP: IMP dehydrogenase and IMP nucleotidasa. The objective of our work with IMP dehydrogenase enzyme encoded by the gene AgGUA1, was the characterization of the center of inhibition by GMP. The purification, crystallization and resolution of the structure of this enzyme in the presence of GMP, allowed us to identify the amino acids involved in the interaction proteína-inhibidor, and establish the mechanism of inhibition. We 22 mutations per directed mutagenesis, for the analysis of this center mutacional inhibition and found to be resistant to inhibition. He also conducted the identification of ORF coding for the enzyme IMP nucleotidasa both in S. Cerevisiae as in A. Gossypii. This identification was carried out through the purification of the protein with FPLC and subsequent analysis by mass spectroscopy. The pattern of expression of this gene shows how there is a strong increase in the expression in terms of oxidative stress. That is why we see the effect of H2O2 on growth and the concentration of intracellular nucleotides in strain isn1. Treatment with H2O2 leads to the accumulation of IMP and slows the growth of the strain isn1 while in the wild strain shows an increase of inosine. In the absence of oxidative agent, the strain isn1 shows low levels of intracellular hipoxanthine and inosine, although there was no significant difference in growth. These results indicate that the gene ISN1 plays an important role in preventing the imbalance in the concentration of nucleotide and the genotoxic effect of this under certain stress conditions.

Author: SANDOVAL DEL AMOR JUAN.
Year: 2005.
University: VALENCIA [www.uv.es].
Place of defense: BIBLIOTECA DEL CAMPUS DE BURJASSOT.
Place of preparation: FACULTAD DE MEDICINA - UNIVERSITAT DE VALÈNCIA.

Summary: The pancreatitis Acute Necrotica (PA) has a relatively high mortality rate due to multiple organ failure in the final stages of the disease. Much work has been invested in this area of research. However, the molecular mechanisms that initiate the disease are not solved so far. In this work Doctoral Thesis has studied the regulation of gene expression of different genes that are involved in the initiation and desarro110 of acute pancreatitis. It has focused research on the characterization of the modifications of histones, and the signal transduction remodelamiento of chromatin in the promoters of genes: early growth response 1 (EGR-1), "inter-cellular adhesion molecule 1" (ICAM-1), interleukin 6 (IL-6) and "tumor necrosis factor a." (TNFa.). For a thorough and comprehensive analysis has been used in a model of PA alive in the rat by administraci6n of taurocolata sodium and a model in vitro cell culture acinar AR42J. During the course of this work we have developed a new aplicaci6n the methodology inmunoprecipitación chromatin (ChiP), we lIamado RNApoI-ChIP. This aplicaci6n enables analysis of the transcript genica in real time and consists of the detection of RNA polymerase II in the coding region of genes. Furthermore, we have studied the action of pentoxifylline, a potential therapeutic agent in front of the PA, in the expression of genes selected. Our results support the use of pentoxifylline as a therapeutic agent in the initial stages of the pathology, as active as a regulator for the expression of the genes involved in the development of the PA.

Author: Garcia Martinez Miguel.
Year: 2005.
University: AUTÓNOMA DE BARCELONA [www.uab.es].
Place of defense: F.Veterinaria.
Place of preparation: Facultad de Veterinaria.

Summary: The reaction catalysed by 6-fosfofructo-1-quinasa (PFK-1) is the limiting step of the glucólisis. There are 3 genes coding for the three isoenzymes described the PFK-1: muscle, liver and platelet. The loss of muscle isoenzyme of PFK-1 is the cause of the glucogenosis type VII or disease Tarui. It is characterized by the absence of activity PFK-1 in skeletal muscle and a deficit in part eritrocito. The most common symptoms are myopathy with exercise intolerance and hemolytic anemia compensated. Due to its low incidence and the lack of a mouse model, now are little known biochemical and physiological adaptations, as well as the interaction between different tissues affected by the shortfall of PFK-1M. This work has generated a mouse model of glucogenosis type VII techniques through homologous recombination in ES cells. This animal reproduced the symptoms characteristic of this disease associated with a high mortality. Thus, the skeletal muscle of these animals showed a blockage glucolítico resulting in the accumulation of hexosas monophosphate and glycogen. This blockade glucolítico was accompanied by a severe intolerance to exercise, which is characterized by a depletion of the ATP muscle. Also, the study of muscles involved in breathing revealed a severe morphological and biochemical impairment associated with a possible functional alteration of this process. Likewise, the cardiac muscle was also affected by the lack of PFK-1M. Similarly, consideration of whether this animal reproduced alterations eritrocitarias described in patients. Thus, the mice deficient in PFK-1M had a chronic hemolysis characterized by a large number of reticulocytes in blood. There was also a splenomegaly associated with an increase in the number of hematopoietic precursors in this body. The biochemical analysis of eritrocito revealed a blockage glucolítico with a marked reduction of 2.3 DPG. This decrease resulted in a response to hypoxia in the skeletal muscles characterized by the expression of HIF-1a. As a result of these muscle and eritrocitarias the skeletal muscle of animals Pfkm- /-could not compensate for the lack glucolítica provoking a situation of chronic stress energy that was reflected in a loss of muscle function.

Author: Grazú Bonavía María Valeria.
Year: 2005.
University: AUTÓNOMA DE MADRID [www.uam.es].
Place of defense: Inst.de Cat. y Petro. CSIC. U.A.M..
Place of preparation: Inst.de Cat. y Petro. CSIC. U.A.M..

Summary: This thesis proposes a new strategy for achieving a high degree of stabilization of industrial enzymes: rigidificación oriented very strong in a particular region of the enzyme surface through optimizing the union covalent multipuntual on activated pre-existing media. The main objective of this dissertation involved the development of the following sub-objetivos: a. The preparation as media disulfuro-epóxido containing: i) - a small proportion of groups tiol-reactivos able to detain them selectively to the enzyme through cysteine residue embedded in the surface protein ii) - a high concentration of epoxide groups capable of conducting an intense union covalent irreversible multipuntual groups nucleotide protein on the surface near the area where he was introduced cysteine residue. In turn, took out the optimization process union covalent multipunutal in mixed media developed. - B. Obtaining different mutant containing a cysteine residue in various regions of the surface with a high protein content native lisinas. This will be assessed three-dimensional structure of penicillin G acilasa (CPA) of E. Coli, and different amino acids were selected to perform the mutation. After evaluating via molecular dynamics simulation that the introduction of different mutations without causing alterations in the conformation of the enzyme, the mutant selected were obtained by directed mutagenesis. C.-rigidificación targeted each of the various mutants, in order to identify areas of the surface protein more susceptible to stabilization, which proved to be a region near the center of the active enzyme (region close to waste B380 the enzyme). D.-enrichment lysine residues of the selected area (were inserted until 8 new waste lisinas in the area close to the surface protein residue aminoacídico B380) Thus, we were able to significantly improve the level of rigidificación of this zone by union covalent multipuntual. E. Evaluation of different media activated in order to get as much rigidificación targeted enzyme mutated and thus the best factors of stabilization. Support glioxil-agarosa allowed to immobilize enzyme selectively through the region enriched in lysine residues, and promoted a union covalent multipuntual very high (around 20 residues lisinas seem to be involved in this union covalent multipuntual). Through the search for the most sensitive areas of the front surface protein at different denaturing agents, and the optimization of rigidificación this area (increasing the number of lisinas present and using a medium, glioxil-agarosa, which provides levels of interaction ), it was possible to maximize the geometric congruence between two rigid structures are as different as the enzyme and support. Thus, the best derived enzyme obtained proved to be 300,000 times more stable to temperature or enzyme-soluble derivative where the enzyme is linked so unipuntual to the stand. This stabilizing factor is not only better than those reported so far for the PGA of E. Coli, but is the highest that we have been able to find in the scientific literature for any stabilization methodology used so far with industrial enzymes. Faced with organic solvents were also seen stabilization similar factors. In turn, the intense rigidificación oriented in the area close to the surface enzyme residue B380 yielded derivatives enz 8 imáticos 2d7 with an improved performance in the synthesis cinéticamente controlled beta-lactámicos antibiotics.

Author: de la Peña Ingelmo Pablo.
Year: 2005.
University: AUTÓNOMA DE MADRID [www.uam.es].
Place of defense: Inst.de Inv. Bioméd."Alberto Sols" CSIC.
Place of preparation: Dpto. Bioquímica Facultad de Medicina.

Summary: The replication of mitochondrial DNA (mtDNA) is one of the fundamental processes that occur during mitochondrial biogenesis. Recently there have characterized several factors machinery mtDNA replication, but the basic mechanism of synthesis of the two chains of DNA is in discussion and unknown elements that regulate the process. The use of D. Melanogaster as a model system to be an animal is very useful experimental tool for the study of the components that make up the machinery baseline replication, transcription and maintenance of mtDNA. In our group we have analyzed how it regulates the expression of various genes encoding essential for the metabolism of mtDNA in Drosophila. Contrary to what happens with the catalytic subunit of mitochondrial DNA polymerase (pol), the expression of the subunit accessory (pol) is subject to strict transcriptional regulation, and the levels of messenger pol increase prior to the activation of synthesis the mtDNA during embryonic development. In this dissertation we have delved into the mechanisms that control the expression of this gene potentially key in the regulation of mitochondrial replication. We have shown that Pol b expressed in Drosophila in a messenger bicistrónico functional possesses characteristics procarióticas. This also mRNA encodes subunit C of Glutamyl tRNAGln amidotransferasa (GatC). We have shown that GatC is located in the mitochondria and is essential for the biosynthesis process of protein, suggesting that mitochondria animals use a route transamidación for the synthesis of tRNAGln. Various experimental evidence suggests that GatC is a protein bifuncional which is also involved in regulating the number of copies of mtDNA, which is the first example of a protein that controls in a manner directly or indirectly mtDNA replication at the cellular level. The study of the mechanisms that regulate the expression of both proteins showed that, unlike what happens in many mRNAs bicistrónicos prokaryotes, translating polb does not depend on the synthesis prior GatC but occurs independently. However, the expression of pol - bse found regulated at traduccional. This control is mediated by a fragment of DNA from 93 nucleotides located at the far 3Â 'of the coding sequence of gatC exercising strong repression on the translation of pol-b

Author: ROJO SANCHIS ANA ISABEL.
Year: 2005.
University: AUTÓNOMA DE MADRID [www.uam.es].
Place of defense: U.A.M. Fcd. DE MEDICINA. Dpto.BIOQ..
Place of preparation: U.A.M. Fcd. DE MEDICINA. Dpto.BIOQ..

Summary: The origin of the disease PArkinson still not well understood, but epidemiological studies suggest that environmental toxins are a major risk factor. But this hypothesis is not formally proven because the models developed in experimental animals based on the entry of toxins through pathways of entry unnatural. In this paper we develop for the first time a new model for him Parkinson's disease in mice based on intranasal inoculation chronicle of MPTP. Previous work showed that the group of Akt kinase plays an important role as a sensor oxidative stress, however the molecular mechanisms have not been fully elucidated. In this thesis show as at least partly Akt exercises its antioxidant role inducing trasncripción enzyme SOD-1 through the regulation of transcription factor NF-kB.

Author: RIOLA MACÍAS JOSÉ.
Year: 2005.
University: EXTREMADURA [www.unex.es].
Place of defense: FACULTAD DE INGENIERÍA INDUSTRIAL.
Place of preparation: ÁREA DE GENÉTICA.

Author: GÓMEZ LÓPEZ GONZALO.
Year: 2005.
University: AUTÓNOMA DE MADRID [www.uam.es].
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: HOSPITAL RAMÓN Y CAJAL.

Summary: The overall survival of cancer patients has not changed significantly in recent years. The main reason for this failure is the expression of an early invasive phenotype in primary tumors. The valuation of multiple markers associated with the tumor is essential for determining the presence of cells microdiseminadas and potentially metastatic. But the shortage demarcadores of microdiseminación tumor early remains a constraint on today. In this paper we applied molecular biology techniques for the study of tumor spread and micrometastases in clinical samples. It conducts a study by RT-PCR marker tirosinasa (TYR) in lymph node tissue, blood and plasma from patients with malignant melanoma. They also apply analysis techniques multiple differential gene expression on a model of human cell lines and metastatic, in order to propose new candidates for gene markers of metastasis. Finally, we study the patterns of gene expression in the human cell line and metastatic PCR3, in the presence of zoledronato with the aim of proposing therapeutic targets.

Author: AYUSO LÓPEZ EDUARDO.
Year: 2005.
University: AUTÓNOMA DE BARCELONA [www.uab.es].
Place of defense: FACULTAD DE VETERINARIA.
Place of preparation: FACULTAD DE VETERINARIA Y CENTRO DE BIOTECNOLOGÍA Y TERAPIA GÉNICA.

Author: Bernal Guzmán Patricia.
Year: 2006.
University: GRANADA [www.ugr.es].
Place of defense: Estación Experimental del Zaidín.
Place of preparation: Estación Experimental del Zaidín.

Summary: The sheath cell bacteria is the barrier at the same time allows the isolation and selective communication with the external environment as needed. It is composed mainly of lipids and proteins. The study of phospholipids to abiotic stress as the lyophilization and resistance to organic solvents has shown the importance of the same and the existence of a regulation that allows you to adjust the components of membrane so that it remains functional. Pseudomonas putida KT2440 and DOT-T1E are of great interest because of their possible application in biotechnological processes. Q. Putida KT2440 is a bacterium saprofita soil that colonizes the roots of plants and can stimulate the growth of the same. The study of the long-term conservation of this strain is important because of its potential use agrobiotecnológico. Q. Putida DOT-T1E is a bacterium resistant to organic solvents isolated in the group of Toxic Organic Degradation in Grenada that can be used in bioremediation processes and systems biotransformations in two phases. In P. Putida KT2440 we have demonstrated the importance of the fatty acids ciclopropanos to increase survival after lyophilization process. In P. Putida DOT-T1E demonstrate the role of cardiolipina in resistance to organic solvents as well as the effects of compensatory mutations with opposite effects on membrane fluidity. The bacteria in general and Pseudomonas putida in particular, tend to have a plastic membrane as possible that can adapt to different environments that surround trying to minimize the damage that different agents cause stressful.

Author: GARCIA OCAÑA MARCOS.
Year: 2006.
University: OVIEDO [www.uniovi.es].
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: DEPARTAMENTO DE BIOQUIMICA Y BIOLOGIA MOLECULAR. FACULTAD DE MEDICINA.

Summary: The osteomyelitis is a difficult treatment of bone infection characterized by a progressive inflammatory destruction of bone. It is influenced by factors related to bone injury and microorganisms inoculated in this, but they can also play a role genetic factors and the immune system. In this thesis studies were conducted to establish the role of neutrophils, cells essential innate immunity in the pathogenesis of this disease. These studies highlight a possible delay in neutrophil apoptosis of peripheral blood of patients with osteomyelitis in relation to apoptosis in neutrophils from healthy controls, associated with high levels of IL-6 in the serum of these patients. In addition, a model developed in vitro osteomyelitis in which neutrophils stimulated with bacterial control is typical of this infection as S. Aureus. In this model has been reproduced in vitro delay apoptótico of neutrophils observed in vivo, and in addition it was found that it was dependent on the synthesis of substances reactive oxygen and proinflammatory cytokines (IL-6, TNF-ae IL-1b) by the neutrophil and was governed by an increase in gene expression Bcl-xL and a decline in the Bax. Studies were also carried out to find genetic susceptibility factors for the development of infection, we found that polymorphisms bax G (-248) A cts-g A125G were significantly more common in patients of osteomyelitis. The advance in the understanding of genetic and immunological factors associated with the development of osteomyelitis, could help the future development of tools prediagnóstico as chips for DNA analysis of different genetic polymorphisms associated with osteomyelitis. The results presented here could also lead to the development of techniques for modifying the apoptosis of neutrophils (inhibitors of the synthesis of cytokines such as IL-6) that could help prevent infection of the bone is cronifique.