MOLECULAR BIOCHEMISTRY

Author: SANTIAGO JOSEFAT MARIA BELEN.
Year: 2002.
University: EXTREMADURA [www.unex.es].
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: FACULTAD DE CIENCIAS.

Summary: Much of the studies carried out on the dioxin receptor (AhR) in recent years has focused on defining their contribution to physiological processes and the maintenance of cellular homeostasis. The so-called endogenous function of AhR mean, per se, the existence of molecular mechanisms of transcriptional activation independent interaction with exogenous ligands. The work presented in this Doctoral Thesis we have provided a possible mechanisms for regulating the activity of AhR in the absence of exogenous ligands. These studies have focused on the regulation carried out by the cellular machinery mediated proteolysis by proteosoma. Thus, we noticed that exite relationship between inhibition of this proteolytic machinery and the level of activation of AhR through a mechanism which could involve an increase in levels of ARNT in through the phosphorylation of Sp1 by PKC. Moreover, we have approached the analysis of genes differentially expressed between cutlivos primary mouse embryonic fibroblasts (MEFs) and wild "knockout" for the AhR (Ahr- /-). Here, we have focused our study on the gene that encodes for a protein binding of latent transforming growth factor beta 1 (TGF-Beta1), called LTBP-1. This gene is sobreexpresa in mice Ahr- / - with respect to wild mice. Moreover, the levels of TGF-beta, both of the active form of the latent form, detected in MEFs from mice Ahr- / - are significantly higher than those found in MEF2 of wild mice. Moreover, in the culture medium of MEFs Ahr- / - we noticed a lower activation of MMP-2, either pro-MMP-2 as how active.

Author: POSADAS MAÑANES SINFORIANO JOSE.
Year: 2003.
University: MÁLAGA [www.uma.es].
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: FACULTAD DE MEDICINA.

Summary: This paper explores key mediators in the immune response such as cytokines in the monitoring of allergic reactions to drugs. This is a descriptive study describing gene expression and protein from a group of these mediators through various immunological and biochemical techniques of molecular biology, such as chain reaction (PCR) polymerase, PCR competitive PCR real time and enzimo-inmunoensayo. The groups of individuals (patients and controls) included in the study were validated in the diagnosis of various clinical understood grouped as immediate reactions to drugs and reactions no-inmediatas to them. The checks were healthy individuals and exposed to the same drugs that caused allergic reactions in patients. We were able to establish partnerships between various cytokines, measured by different techniques. The main conclusion of the thesis is that allergic reactions to drugs remain the paradigm Th1/Th2 corresponding profile of the first cytokines to the reactions no-inmediatas and the second to the immediate reactions to medications. It was also possible to confirm that conclusion when analyzed the transcription factors involved in controlling the synthesis of cytokines tested. Lastly also could be shown that the mediators involved in cell apoptosis as perforina, granzima B yFas-L involved in the mechanisms of effector cells in the immune system reactions no-inmediatas.

Author: TITOS RODRÍGUEZ ESTHER.
Year: 2003.
University: BARCELONA [www.ub.es].
Place of defense: BIOLOGIA.
Place of preparation: FACULTAD DE BIOLOGIA,UNIVERSIDAD DE BARCELONA.

Summary: The liver fibrosis is the result of inflammation and tissue repair in response to a lengthy and liver damage is defined as the accumulation of connective tissue in the liver due to the significant imbalance between production and degradation of matrix extracelular.Mientras the liver fibrosis is a reversible process, its final state, cirrhosis is considered the estdio irreversible end of the emfermedad.Cuando cirrhosis is severe and occurs complete desestrcturación parenchymal hepatic giving rise to liver failure and hemodynamic disturbances graves.Una the first hemodynamic complications is the aparión of hipertención portal that takes place as a result of an increase in resistance to the passage of intrahepatic portal blood flow as well as an increase intríseco of vascular tone of the microvasculature liver. There is an enormous scientific interest not only to shed light on the cellular mechanisms involved in the development of liver fibrosis and cirrhosis, but also by identifying new therapeutic strategies aimed at preventing or reversing the prograsión this liver disease. One possible therapeutic targets by the way biosintética of 5-LO. The 5-LO is the key enzyme responsible for the synthesis of LTS, important mediators lipídos derivatives AA.Este group eicosanoides includes LTB4 involved mainly in the inflammatory response and cisteinil-LTS (LTC4/LTD4/LTE4), which are potent agents vasosontrictores able to increase vascular permeability and exercise hepatic perfusion as extravasation of fluid vascular.Además, clinical studies demonstrate that in patients with hapatopatía there is an increase in the production of these eicosanoides which has been associated with the development of an increase in resistance intrahepatic there is a increase in production of these eicosanoides which has been associated with the development of an increase in the intrahepatic resistance and the emergence of colestasis.Sin enbargo and to the relationship of this draft doctoral thesis, not we knew the exact cellular mechanisms of synthesis of these eicosanoides in the liver as well as pathophysiological implications of these compounds in the development of liver cirrhosis and its complications. Therefore, the general object of this study were: 1. Characterize biosynthesis and functional role of cistenil-LTS in sinusoide liver. 2. To investigate the role of cell Kupffer and the 5-LO in the pathogenesis of experimental hepatic fibrosis. In summary, the results of this study indicated that not only in the liver cell Kupffer contributes to the endogenous production of cisteinil-LTS but hepatocytes contribute significantly to its production through metabolism transcelular. Addition these icosanoides can exert actions paracrinas on liver cells estralladas and thus contribute to increasing the resistance intrahepáticas since the onset of hypertension portal.Además, the results provide the first evidence that the active presence of the metabolic pathway of 5-LO is critical to the survival of Kupffer cells and showed that the inhibition of the path of 5-LO in Kupffer cells may reduce the progression of fibrosis in chronic liver damage pilot.

Author: DUFLOT SYLVIE.
Year: 2003.
University: BARCELONA [www.ub.es].
Place of defense: BIOLOGÍA.
Place of preparation: UB.

Summary: The functions of the liver are imports due to its strategic location between the digestive system and the rest of the body (the systemic circulation). Generation antibodies against transporters nucleoside sodio-dependientes Rcnt1 (pyrimidine Preferred) and rCNT2 (purine Preferred ) helped identify the presence of these two trasportadores in hígado.El first objective of this thesis is to establish and ditribución subcellular localization of Rcnt1-y Rcnt2 in rat hepatocytes. The hepatocytes represent a 60-80% of the cell mass totaldel liver: cells are highly polarized and characterized po have a distinct plasma membrane in three different functional domains: the apical membrane and lateral.Por these features and be very po assets metabolitamente, liver cells are a good model to study the routes endocíticas.Examinamos location sbcelular in three ways distintas.Primero, detectmos by technical transportation and Western blot expression CNT1 and CNT2 in fraciones purified from endosomas of rat liver, by using antibodies against disease. Third by electron microscopy techniques. These studies indicate that the transporter nucleoside CNT1 comes at the canalicular membrane, in which the conveyor is biologically active, by an indirect route via the basolateral membrane. His insertion is performed near microdominios specialized enriched caveolas. CNT2 detected has been almost exclusively on preparations enriched membranes plasmáricas basolaterales in which the conveyor is activo.Estos studies tell us that the two transport CNT1 and CNT2 have a different location in liver cells, so that these two carriers appear to be diferenicalmente regulados.En the second part of this thesis we examine the interactions between conveyor concentrative adenosine CNT2 and recipients purinérgicos.Las liver cells previously treated, in the short term, with the agonist-receptor A-1, (-) - N ( 6) - (2-phenylisopropy) adenosine (RPIA) produces expanding transportation of CNT2.Este effect is inhibited in the presence of inhibitors channels KATP and mimics in the presence of activators such canales.Estos studies show that liver cells, the activity of transporting CNT2 is regulated by activation of receptor A-1, via canles KATP.Se has also shown that the induction of transport activity of CNT2 by opening delos channels KATP is dependent on the activation of the PKC.Por Finally, we note that the glycemia and matabolismo of glucose could affect regulation purinérgica of CNT2 since the induction of activity CNT2 through the receiver A-1 can be modulated by the concentration of glucose in the medio.Estudiamos in the third part of this thesis the role of nitric oxide and in the PKC activation post-traduccional of CNT2 cells hepáticas.Medimos transport activity of CNT2 in Fao cells and primary culture of hapatocitos after incubating cells with different activators and inhibitors of nitric oxide synthase (NOS). The result was that the NOS inhibition causes an increase in activity was transporting CNT2 a 60%. Through inhibitors of the activity of PKC and channel blockers KATP, determine quela decrease in the concentration of intracellular nitric oxide contributes to the activation, in the short term, transporter CNT2 through activation of PKC regardless of the channels katp.

Author: FIGUEROA MORALES SALVADOR.
Year: 2003.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE FARMACIA.
Place of preparation: INSTITUTO DE BIOQUҍMICA-FACULTAD DE FARMACIA.

Summary: NO is a molÃÂ © cula gas with many implications fisiolÃÂ ³ gicas and patolÃÂ ³ gicas. In this paper we have studied both strands of molÃÂ © cula on primary cultures of cortical neurons of rat brain, using as a giver of NO S-nitrosotiol S-nitroso-N-acetilpenicilamina (SNAP). The system under consideration, NO expresses a dual effect neurotÃÂ ³ neuroprotective and Mexico, which is dose-dependent and time exposiciÃÂ ³ n the giver NO. AsÃÂ, low dose of NO were able to counteract apoptosis -mediada by caspase 3-induced by the withdrawal of support trÃÂ ³ fico the culture medium. This neuroprotective effect of NO in front of the privaciÃÂ ³ n serum course with an increase of intracellular content of proteÃÂnas antiapoptÃÂ ³ acteristics of the family of Bcl-2, especially Bcl-XL, compared with proteÃÂnas proapoptÃÂ ³ policies, as Bax, ademÃÂ ¡s a disminuciÃÂ ³ n the activity of caspase 3. Doses above those that induce neuroprotecciÃÂ charges produce a disminuciÃÂ charges of cell viability, through a process apoptÃÂ ³ tico involved in the mitochondria: despolarizaciÃÂ ³ n of the mitochondrial membrane internal pore opening of mitochondrial permeability transition under certain cirunstancias, liberaciÃÂ charges of cytochrome c from space intermembranal the cytoplasm and activaciÃÂ charges of caspases. At high doses mÃÂ ¡s NO employed, there is a coexistence of processes death carÃÂ ¡cter necrÃÂ ³ tico and apoptÃÂ ³ tico. Along with the evidence of apoptosis was detected liberaciÃÂ ³ n of the ATP enzyme LDH and the culture medium, which is indicative of cell lysis. The estrÃÂ © s oxidative desempeÃÂ ± an important role in cell death induced by high concentrations of NO, as evidenced by the increase in the intracellular content of reactive species of oxÃÂgeno (ROS) and detecciÃÂ charges of peroxidaciÃÂ ³ n lipÃÂdica. Along with the NO-mediated effects on cell viability, this paper presents data from the acciÃÂ ³ n NO as a modulator of neurotransmisiÃÂ ³ n. NO is capable of increasing liberaciÃÂ ³ n basal neurotransmitter (glutamate, aspartate, GABA and glycine), asÃÂ as to regulate the secrecciÃÂ charges induced by people despolarizante the membrane plasmÃÂ ¡tica (KCI) and receptor agonists glutamatÃÂ © rgicos.

Author: TORRES GUZMÁN RAQUEL.
Year: 2003.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE CIENCIAS BIOLÓGICAS.
Place of preparation: FACULTAD DE BIOLOGÍA (UCM).

Summary: ABSTRACT penicillins, GoV, industrially produced through processes fermentativos in quetambién occur, so collateral, penicillins other side chain of linear (penicillin F dihidro FyK). The presence of these penicillins hinders subsequent extraction and purification of 6-APA, the starting material for obtaining penicillins, semi. Penicillin acilasa from Streptomyces lavendulae has been described as a penicillin V acilasa able to hydrolyse, besides those penicillins aliphatic, has not been described another penicillin acilasa with this substrate specificity. This is one of the reasons why it was interesting study of this enzyme, with a view to their possible use in industrial bioreactors for the procurement of 6;. APA. The penicilma acilasas have also been included within a superfamily of proteins described recently, Ntn-hidrolasas. In this group are included primarily enzymes hydrolyzed penicillin Go glutaril 7-ACA, but little is known about the specific enzyme penicillin V, that is another reason why it is interesting to study the enzyme from Streptomyces lavefuJulae as an example penicillin V acilasa 1. A method has been optimized production. Penicillin acilasa from Streptomyces lavendulae using milk, skimmed as culture medium, in terms of agitation orbital 20Â ° C. This situation has got to produce enough enzyme to. His after purification, characterization studies address. The production of the enzyme repression suffered by catabolito in the presence of sugars (glucose and lactose). 2. It got a simple method of purification, in four steps. That yielded a preparation of penicillin acilasa pure from, fermentation broths Streptomyces lavendulae 3. Penicillin acilasa from Streptomyces lavendulae, is composed of two subunits of different sizes, a subnidad to the 21.4 KDa and submridad beta 59 IDa, forming a heterodímero alpha beta 80 KDa. 4. The activity Ía penicillin acilrisa: Stréptomyces lavendulae is enhanced in the presence of cosolvente organic aqueous. This activation depends on the nature of organic solvent and its concentration in the reaction medium. Alcohols and polyols. May participate in the catalysis acting as nucleófi1o alternative to water. Solvents polar apróticos, disrupt, half physicochemical properties thus affecting lasolubilidad of substrate and product of the reaction and the flexibility conformacional of the enzyme. 5. Penicillin acilasa from Streptomyces lavendulae acts on a mechanism cmético sorted Uní-Di, where the acid 6-arninopenicilánico is the first product released. The reaction trascurre through the formation of an intermediate acil-enzirna, who suffered the attack nucleófilo of a water molecule, with the consequent release of acid fenoxiacético. 6. A residue of cationic nature with a pK of 6.45 has been identified as essential for the activity. This waste would be involved in the catalysis and in the union of the substrate. Taking into account the results of variation Vmax_Vmax / KM and KÂ ¡with pH, the tests of disturbance with solvent and the results of the modification of the protein with specific reagents, the residue proposed as responsible for the catalytic activity of the penicillin acilasa from Streptomyces lavendulae is serine terminal located in the beta subunit (being beta 1). 7. Penicillin acilasa from Streptomyces lavendulae hydrolyzed preferentially substrates with hydrophobic side chain and presents a good catalytic efficiency (KcalKu) to hydrolyse penicillins of side chain alifática. As the penicillins F dihidroF and K. 8. The binding site .. portion acilo of the substrate has a marking characters 8 er hidró 396 fóbico, in the form of tunnel. Capable of hosting chains hidrocarbonadasde at least eight carbon atoms. 9. Both results lead us to propose that the enzyme as a Ntn-hidrolasa That is the serine beta 1 would nucleófilo attacking carbon carbonílico of substrate to form the interim acil-enzima.

Author: CUESTA DE LA PLAZA ISABEL.
Year: 2003.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE CIENCIAS BIOLÓGICAS.
Place of preparation: F.CC. BIOLÍGICAS (UNIVERSIDAD COMPLUTENSE DE MADRID).

Summary: The virus respiratory sincital human (VRSH) is a pneumovirus the family paramyxoviridae, which cause serious infections in the lower respiratory tract in niñós small, immunocompromised individuals or cardiovascular disease and the elderly. Facts such as antigenic variability of the virus, affecting populations as diverse and the inability of primary infection to develop a protective immune response and lasting suggest that the most appropriate strategies to follow for him sanitary control of the infection is to develop antiviral compounds and generate through reverse genetics live attenuated virus vaccine. The ribonucleoproteínas are internal components of the pratículas viral and unidads functional processes for viral replication and transcription. They are composed of viral RNA, negative polarity, non-segmented strongly linked to the N protein constituting the nucleocápsida viral, it binds to this the viral RNA polymerase, L protein, and its cofactors the fosfoproteínas Py M2-1. Proteins of RNPa are multifuincionales and are involved in viral specific functions, which makes them suitable targets for the action of antiviral compounds and carriers of mutations that confeirían attenuation to live virus. In order to understand how these proteins perform their duties during the growth cycle of the virus at the molecular level, etudiamos some of the interactions that establish with different viral components. We demonstrate that the protein M2-1 binds RNA, with a kinetic cooperative and recognizes the sequence of Black leader in the context of an RNA 86nt. The region of binding to RNA was located between residues 33-37,43-48 and 59-62 of the protein. It was determined that the T59 and S58 are the residues that are fosforilan and that such an amendment decreases binding to RNA. The interaction of the protein M2-1 with RNA through residues 33-37 and 43-48 is essential for the activity antiterminadora and / or elongadora that the protein plays during the viral transcription. It purified protein N-free RNA and demonstrated that it has any ability to unite RNA, no sequence specificity. We have located debris from the N protein that contact with the RNA, are any or all of those in positions I242, F243, W246, F247 ,258-271, E355, Q356 and E359, all of them located inside the body dela globular protein . The C-terminal tail (residues 365-391) protects a region of the interaction of RNA located at the extreme N-terminal (residues 87-92). We have also identified waste through which interaciciona with the P protein (A244, G245, M248MQ356, E359 and G361) and find which are the same or very close to those interaccinan with RNA, indicating that both interactions can be exclusive. These findings help delineate regions of the two proteins VRSH (Ny M2-1) targets for interaction with specific antiviral compounds for VRSH.

Author: AGUILAR IZQUIERDO SUSANA.
Year: 2003.
University: POMPEU FABRA [www.upf.edu].
Place of defense: DEPARTAMENTO DE CIENCIAS EXPERIMENTALES Y DE LA SALUD.
Place of preparation: DEPARTAMENTO DE CIENCIAS EXPERIMENTALES Y DE LA SALUD.

Summary: Pancreatic cancer is the fifth leading cause of death in the Western world and one of the most aggressive tumors. The plasminogen system is important in the intravascular thrombolysis and biological processes that require cell migration and angiogenesis and tumor progression. The plasminogen activation mediated by tissue plasminogen activator (tPA) and plasminogen activator type uroquinasa (uPA), resulting in the degradation of extracellular matrix proteins and the activation of MMPs and latent growth factors. The objective of this thesis was to study the role of tPA in tumor progression in vivo using murine models of pancreatic cancer and analyze its mechanism of action. As a model of pancreatic tumorigenesis were used animale stransgéncios Ela1-Tag and Ela1-myc. It was also used transgenic animals Ela1-CCK2 and MT-TGF-a, as a model of metaplasia acinar-ductal. The results show that tPA not detected in any epithelial cell in the normal pancreas or in ducts metaplásicos or acinar cells in tumor found sobreexpresado in duxtos neoplastic pancreas. On the other hand, anexina A2 is detected in the apical domain of duct epithelium of normal and complex ductales and is sobreexpresada and despolarizada in neoplastic ducts. In order to determine the effect of the inactivation of tPA to the development of tumors of mice Ela1-myc crossed these animals transgenic mice to knock out tPA (tPA- /-). Mice hybrid Ela1-myc; tPA- / - also develop pancreatic tumors and tumors with the proportion of ductal component is unchanged. However, there was an increase in survival in animals that do not express tPA, indicating that this protease promotes tumor progression. An analysis of factors related to tumor progression showed that the tumors of mice Ela1-myc: tPA- /-vascularization and show lower rate of proliferation, while the invasion is not affected. To study the effects of direct expression of tPA in the pancreas, acinar cells or ductales, transgenic mice were generated that sobreexpresan tPA under the control of the promoter of elastasa1 (Ela1-tPA) or citoqueratina 19 (CK19-tPA) respectively. The results of the analysis changes in the pancreas. But we need a more comprehensive study of phenotype.

Author: PEY RODRIGUEZ ANGEL LUIS.
Year: 2003.
University: AUTÓNOMA DE MADRID [www.uam.es].
Place of defense: FACULTAD DE CIENCIAS,AUTONOMA DE MADRID.
Place of preparation: FACULTAD DE CIENCIAS DE LA UNIVERSIDAD ATONOMA DE MADRID.

Summary: In this study have been performed expression analysis of mutations that cause phenylketonuria (PKU) in order to characterize its effects on the folding, stability and function of the protein PAHs. The results have deepened in the correlation genotipo - fenotipo and in the mechanisms responsible for the response to treatment with the cofactor (BH4) in PKU. It has also been given the rate of degradation of PAHs proteins expressed in a cell-free system through experiments pulso-caza. Mutations analyzed can be classified into two groups: 1) structural change, affecting the folding and stability of the protein, and are characterized by a decrease in the levels of soluble protein PAHs, in ways oligoméricas functional (tetrámeros and dimers) and lower thermal stability. For these mutations also have seen an accelerated rate of proteolytic degradation, as experiments pulso-caza. The coexpresión of chaperoninas bacterial GroEL and GroES (permissive conditions) increases the activity for residual PAH most of these mutations, confirming the effect on the folding of PAHs. The PAH residual activity for most of these mutations correlated well with the metabolic phenotype in patients, and when we look at the correlation genotipo - fenotipo inconsistencies in patients for some of them, the potential modulation observed in conditions permissive to suggest that differences individual quality control cell protein (chaperonas and systems proteolíticos) could explain these inconsistencies observed in patients. 2) mutations catalytic, which negate the role likely to affect waste enzyme essential for catalysis in the center of the enzyme active and having a high performance forms oligoméricas functional lack of activity. For these mutations, modulation conditions permissive does not alter the PAH residual activity, and these changes are clearly associated with severe phenotypes. Fourteen PKU mutations associated with response in patients have been expressed in E. coli and in vitro in a cell free system. It has implemented a combination of kinetic analysis (steady state), titration calorimetry of isotherm (union under equilibrium conditions), CD spectroscopy (effect of BH4 on the thermal stability) and expression in a cell-free system eucariota (effect on the activity) in order to assess possible defects in the affinity for BH4 and characterize the effect of cofactor on the activity and stability of the PAHs. Five mutations introduced an affinity diminished by the cofactor in steady-state conditions, while three of them also showed defects in a position to balance these effects contributing to the response to BH4 in patients. All mutations studied show alterations in the regulatory and catalytic properties, affecting the cooperatividad and activation L-Phe, and in some cases reducing the apparent affinity for the substrate. These results suggest that the abnormal behavior observed kinetic could also contribute to the pathogenesis of PKU. In addition, there has been a possible rel 8 ation in 52f tre activation state, the affinity for BH4 diminished and a different sensitivity to thermal stabilization by the cofactor for some of the mutations studied. The expression in the cell-free system has revealed a moderate increase in activity PAHs when proteins are synthesized in the presence of BH4, in addition to protecting the face of oxidative inactivation once synthesized. In conclusion, our experimental approaches suggest that the answer to BH4 is a multifactorial process, in which there are contributions from defects in the affinity for BH4 and the protective effect of cofactor versus oxidative inactivation.

Author: CAPILLA RUBIO SILVIA.
Year: 2003.
University: ZARAGOZA [www.unizar.es].
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: FACULTAD DE MEDICINA.

Summary: Y. EnterocoLiticcr 0:3 is a bacteria that can cause gastroenteritis is transmitted through contaminated water or food and in some cases it may be necessary antibiotic treatment. The objective of this study is to analyze the mechanisms of resistance to quinolones and other antibiotics in a range of clinical isolates of Y. Enterocolítica 0:3 resistant to nalidixic acid. To this end we have studied the presence of mutations in the target of quinolones and possible alterations in the permeability as mechanisms of resistance. The results show that a mutation in GyrA is responsible for the resistance to quinolones although the presence of bombs expulsion can modulate the minimum inhibitory concentration (MIC) of this antimicrobial. We conducted an analysis of the resistance to aminoglycosides, and the results have demonstrated the presence of an enzyme-modifying of aminogIicósidos (ANT (3 ") (9)) as the principal involved. Parallel, in the study of resistance to chloramphenicol have found the the presence of the enzyme, chloramphenicol acetyl transferase responsible for the resistance but the presence of bombs expulsion may also modulate the CIM. We have studied the presence of plasmids and integrones as mechanisms of horizontal transmission and the results show that the plasmids containing genes involved the virulence and in the majority of integrones we have found the presence of the gene aadA coding for the enzyme-modifying of amiglicósidos ANT (3 ") (9). Finally, the results of the epidemiological study of all strains using the technique of pulsed field using different restriction enzymes suggests that Y. Enterocolitica 0:3 is a bacterium with little genetic diversity.

Author: RODRIGUEZ SANTIAGO BENJAMÍN.
Year: 2004.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAD DE BIOLOGÍA.
Place of preparation: FACULTAT DE BIOLOGÍA , UNIV DE BCN.

Summary: Study on the involvement of the mitocondría in patients with disease alzheimer type esporádico.Se have analyzed autopsy samples from three regions of our brain and blood. The molecular analysis included studies of nutDNA to detect point mutations, rearrangements, changes in the amount of nutDNA and changes in the gene expression of various RNAS mitocondriales.También were performed biochemical studies to evaluate the effect on the function of the respiratory chain mitocondríal responsible to cover the 90% of cellular energy needs. Comparison between patients and controls showed no significant different in studying nutDNA and function of the respiratory chain mitocondríal.

Author: CARRASCAL PEREZ MONTSERRAT.
Year: 2004.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAD DE BIOLOGIA.
Place of preparation: FACULTAD DE BIOLOGIA.

Summary: In pathological situations, as in the case of autoimmunity, the epithelial cells of tissues express ectopicamente meléculas MHC class II.El study of the mechanisms leading to the formation of the complex MHC-péptido, his presentation on the surface of APC and the recognition of same by T cells is essential to understand not only the immune response to infections, but also as relevant diseases such as cancer, autoimmunity or rejection of and / or maintenance of the disease . This thesis has dealt with the study of the presentation of peptides by molecules of class II MHC using various strategies proteómicas.En First, the work focused on the characterization of codes peptídicos associated with a CPA and epithelial cells expressing molecules MHC class II secuenciándose more than 300 ligand peptídicos, including various peptides presented in ex vivo thyroid affected by a disease autoinmune.Mediante This study showed that there are differences in codes associated with the epithelial cells compared to those submitted by the CPA , both in the characteristics of peptides as those of proteins which derivan.Asimismo, shown as the expression of chaperonas HLA-DM and Li affects the characteristics of repertorio.Finalmente, identified peptides Class II presented in human thyroid affected by the disease Graves, including a peptide derived from the thyroglobulin, a protein described as autoantígeno this enfermedad.En a second part of the work has studied the effect of trasfección of HLA-DR molecules , HLA-DM and Li on the proteome of the epithelial cells and have identified several proteins involved in this process, showing that despite the absence of drastic changes in the proteome overall, some proteins involved in cellular metabolism varies his speech. Also, we have identified 30 proteins that are associated directly or through other molecules with molecules HLA-DR4 in the cell, including several chaperonas and enzymes oxidoreducción and proteins involved in the transportation associated with expression of membranas.La these proteins also depended on the expression of chaperonas li and DM it is suggested that some could be involved in the generation, processing and presentation of peptides by the molecules of class II MHC.

Author: MOORE CARRASCO RODRIGO ERNESTO.
Year: 2004.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAD DE BIOLOGÍA.
Place of preparation: UNIVERSIDAD DE BARCELONA.

Summary: The wasting syndrome is a frequently associated with tumor growth, as well as other pathological conditions such as sepsis, AIDS (acquired immunodeficiency syndrome), diabetes and so on. The cachexia is characterized by a significant and progressive loss of body weight due mainly to the disappearance of fat reserves and reduced muscle mass. It is also accompanied by anorexia, nausea, fatigue, weakness, altered hormonal homeostasis and immunosuppression. About transcription factors that could be involved in the process of muscular observed in the cachexia (in vivo), very little is known, but some factors appear as potential candidates both in the outbreak in the regulation of this process. NF-KS and AP-1 are two transcription factors that are activated in different tissues in inflammatory processes. It has been described that NFKSy AP-1 have a differential regulation in rat skeletal muscle during the process of sepsis. The same group has determined that C / ESP, another transcription factor involved in the inflammation process, enhances its binding to DNA activity in muscle septic rats, and that this process is dependent on glucocorticoids. The objectives of this study are: 1) analyzing the regulation of certain factors ranscripción in skeletal muscle during cachexia induced tumor growth. 2) reversing the muscular associated with tumor growth through strategies based on transcription factors from a drug. 3) approaches to therapy against cachexia by an adenovirus for gene transfer of a dominant negative AP-1. The results allow us to conclude that the transcription factor AP-1 presents a differential regulation in skeletal muscle during tumor growth, suggesting that this factor is involved in the development of cachexia associated with cancer. By contrast, other transcription factors studied (NFKS, C / ESP And Sp-1) do not appear to be directly involved in the development process caquéctico at least the muscle. The factor miogénico MyoD introduced significant changes in their levels in skeletal muscle of different experimental models of tumors caquécticos, as well as the muscle of patients with pancreatic cancer. The acute administration of LPS to rats also markedly affects muscle levels of MyoD. These data suggest an implication of this factor in the development of cachexia associated with tumor growth and other pathological processes. The administration of SP100030, inhibitor NF-KSy AP-1, succeeds partially reverse the effects associated with the growth of hepatoma ascítico Yoshida AH-130. This effect is associated with an inhibition of the activity of union AP-1 to the DNA, an increase in the expression of MyoD, and a decrease in muscle proteólisis. Treatment with GW1929, agonist PPARy, induces a recovery in the weight of the extensor Digitorum longus muscles of mice bearing Lewis lung carcinoma, associated with a restoration of the levels of MyoD. This compound also induced a decrease in proteólisis these muscles in vitro. The overexpression of Tam67, dominant negative AP1, completely reverses the effects caused by the addition of TNF to the growing medium myoblast C2C12 in differentiation. This effect is produced by the restoration program dei muscle differentiation, mediated by a decrease of ciclin_élDJ a recovery levels MyoDy unaumentode the proteínaMHCen these cells. The in vivo gene transfer of dominant negative AP-1 Tam67 by adenovirus produces a recovery in the weight of the muscles and a atenuaciónen the falling standards of MyoD in muscle gastrocnemius of animates 8 lesporta 1a1 'tumor.

Author: MARTRAS DELGADO SÍLVIA.
Year: 2004.
University: AUTÓNOMA DE BARCELONA [www.uab.es].
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: ESCUELA DE POSTGRADO.

Summary: The retinoic acid, in its forms todo-trans- and 9-cis, serving as a ligand of specific nuclear receptors, and it is essential processes of growth, development and maintenance epithelial. The training route of retinoic acid oxidation includes two steps, retinol and retinal, and retinoic acid to retinal. It has been described several enzymes that can participate in the first step, and how the ADH RDH, but was unaware of the contribution of each enzyme in the process. It has been cloned, expressed and purified ADH1B1, ADH2B2 and ADH4 human, and also ADH1 and ADH4 mouse, for the first time, have been characterized as recombinant. We have determined the kinetic constants of all these ADHs with todo-trans- and 7-cis-, 9-cis-, 11-cis- and 13-cis-retinol and corresponding retinales. In general, ADH4 human and mouse shows kinetic constants similar, and are more efficient than the ADH1. All ADHs used as sustratros both 11-cis-retinol as 11-cis-retinal compounds relevant in the visual cycle. It has been observed that ADH4 shows specificity for the oxidation of 11-cis-retinol on reducing 11-cis-retinal, a property unique among all ADHs for any pair of substrates alcohol / aldehyde. By methods of molecular simulation, and colonación, expression and characterization of mutant of the ADH4 human M141L, we have shown that the residue 141, located in the middle of the tunnel hydrophobic region of the active site of ADH, it is essential to define the specific nature of the ADH4 on ADH1. The inmunolocalización of ADH4 in the pigmented epithelium and muhcas layers of the retina supports the participation of the ADH4 in different reactions with retinoids. The cytosolic activity of the ADH4 in the pigmented epithelium can be complementary to the activity 11-cis-retinol deshidrogenasas of RDH5 needed to complete the visual cycle, and may also be involved in the generation of retinoic acid in the layers of the neural retina. Another family consists of retinoids derived oxidized in ring cilohexeno, such as 4-oxo- ,4-hidroxi- and 3,4-dideshidroretinol and corresponding retinales. Despite compounds that are little studied, it is known that some derivatives, such as acid 4-oxo- and 3,4-dideshidroretinoico may interact with nuclear receptors. The kinetics of enzymes ADH1B1, ADH2B2 and ADH4 human, and also ADH1 and ADH4 mouse, indicate that 4-oxo-retinal and 4-hidroxi-retinol are substrates with a higher catalytic efficiency among all retinoids, especially with respect the AHD4, while the 3,4-dideshidroretinoides presented an activity similar to those of the todo-trans-retinoides. The data obtained in vitro support the existence of a metabolic pathway for the formation of acid retinoicos oxidized in the ring from correpsondientes retinoles, with the participation of ADH. Finally, we found that the presence of Tween 80 leads to a decrease in activity resulting in an apparent competitive inhibition in the kinetics of ADH for todo-trans-retinol, with an increase in Km and decreased the catalytic efficiency increasing the concentration of detergent. This implies that the real values of Km are much lower than those published so far, traditionally obtained in the presence of 0.02% Tween 80. Thus, ADHs presented Km values close to those of the RDH and it tatno, the contribution in the metabolism of retinoids may be similar for both enzyme systems. We have seen espectrofotométricamente and HPLC, the Tween 80 maintains the stability of the aqueous solution of retinoids, and perm 8 ite obte 310 nera reproducible and comparable results between different ADHs.

Author: GIMFERRER MIGUEL IDOIA.
Year: 2004.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: FACULTAD DE MEDICINA . UNIVERSIDAD DE BARCELONA.

Summary: The T cells are activated when recognized through your receiver or the TCR peptide antigen presented by the Main System Histocompatibility (MHC, Major Histocompatibility complex). This is the first signal and has to be amplified for proper activation of T lymphocytes (LT) And this amplification is performed by couples ligando-receptor function co-estimuladora as B7-CD28, which give the so-called second signal. Then the contact zone between the LT and the cell presentadota (APC) called immune synapse (IS), and accessory molecules and signaling of both cells are reorganizing Buddha rmando a structure called Supramolecular Complex Association or SMAC. CD6 belongs to the superfamily of receptors with rich domains in cisteínas type Scavenger (SRCR, Scavenger Receptors Cistein Rich). LT is expressed in mature and immature cells of Chronic Lymphocytic Leukemia, a subtype B cell called Bla and central nervous system. Traditionally has been described as a receiver function co-estimuladora but the mechanism that performs this function is not known. This doctoral thesis aims to expand the limited knowledge about this protein. Our objectives were: 1. Study of possible associations CD6 with other receivers linfocitarios. 2. Implication of CD6 and CD5 (member of the same SF-SRCR with which is very homology) in the processes of lymphocyte activation. By studying its presence in structures related to the activation and immune synapse (IS) and rafts, and the involvement of their respective ligands in these processes. 3. Possible partnerships undertaken by the cytoplasmic region of CD6. 1. Study of possible associations CD6 with other receivers linfocitarios: Using biochemical analysis (co-inmunoprecipitación) and cell biology (FRET and modulation), we detected the association physics CD6 to CD5 and complex CD3/TCR and suggest a possible association with others two molecules function accession as CD44 and ICAM-3. The association CD6 with CD5 occurs in lymphocytes mature in thymocytes. This physical proximity allows the hypothesis of a possible coordination of their roles during the process of activation and differentiation of LT, giving similar signals and / or complementary. The association physics CD6 the complex CD3/TCR is independent of the presence of CD5 and makes CD6 in a co-receptor this complex enabling to modulate their signals for lymphocyte activation processes. 2. Implication of CD6 and CD5 in the process of lymphocyte activation: studies conducted training IF between LT and antigen presenting cells (APC) and note that both CD5 as CD6 were rec1utados to the contact zone between the two types of cells form antigen dependent. This showed that both receivers were rec1utados to SI during activation of the LT. More detailed studies indicated that both receivers were accumulated in the central area of the IS, as well as the ligand of CD6 (ALCAM). Furthermore we studied the presence of CD6 in rafts, lipid rafts rich in cholesterol and esfingolípidos that have the ability to secrete proteins involved in signaling and have proved to be important for proper activation of LT. Within this second goal also wanted to explore the involvement of ligands CD5 and CD6 in the process of activating the LT. To do this we used soluble proteins formed by the three extracellular domains of CD5 and CD6 with layer 8 cidad of 1386 blocking the interaction of these receptors to their respective ligands. Then we study as affecting the presence of these proteins to the maturation of the IS and note that the presence of recombinant soluble protein (rs) CD6 (rsCD6) resulted in inhibition of between 37% Yun 46% in the proportion of SI mature. The presence of rsCD5 or albumin did not affect the maturation of the IS. We then proliferation studies with different stimuli and this protein also showed inhibitory capacity on a dose-dependent stimulation of proliferative given by AcMo anti-CD3 (OKT-3) and PHA. The protein rsCD5 surprisingly also was able to form dose dependent inhibition of proliferation induced OKT-3. These inhibitory effects were not as toxic maximum doses of these proteins were unable to inhibit crop stimulated with Pokewit mitogen (PWM), cells alogénicas (cropping) and superantigeno (SEA). Therefore, the interaction of CD5 and CD6 their respective ligands is important for the proper activation of the LT Yla proliferative response to certain stimuli, proving to be capacity immunomodulator. It should be stressed that there are natural soluble forms of CD5 and CD6 serum of healthy people and higher levels in sera of patients with autoimmune processes that might have this feature immunomodulator. 3. Possible partnerships undertaken by the cytoplasmic region of CD6: By double-hybrid tests detect the association of the C-terminal region of CD6 with subunit A phosphatase PP2A (PP2A-A) Yla protein adapter syntenin-l. The interaction CD6-PP2A-A we have not yet been able to demonstrate a system in mammalian cells, but we detected by tests dual hybrid direct interaction with the regulatory subunit this area CD6 between aa A613 and P647. The protein syntenin-l contains an N-terminal region followed by two ADS domains in tandem. It was first described by its interaction with syndecan. Syntenin-l interacts through its ADS domains with the four aa more C-terminales of target proteins. For this interaction is necessary integrity of its two domains ADS, but each domain individually have a preferred one type of protein. It has been described that syntenin-l could make a bridge between two proteins joining a domain for each ADS. In addition it must be stressed that syntenin-l also interacts by its N-terminal area with transcription factors such as Sox-4. Our test results dual hybrid direct precipitation and demonstrated that the interaction CD6-syntenin-l involved in the two domains ADS of syntenin-l well as aa more C-terminales of CD6. Also the interaction in PBLs human demonstrated as well as the accumulation of syntenin-l in central IS co-localizando with CD3. This ínteracción between CD6 and syntenin-lsugiere that syntenin-l could function as an adapter protein joining CD6 the actin cytoskeleton of other signaling proteins and could be involved in the recruitment of CD6 to the central area of the IS. CONCLUSIONS 1-all results of this study suggest that the recipient lymphocytic CD6 is responsible for interactions relevant to the processes of lymphocyte activation and differentiation. 2-CD6 is physically associated with CD5, suggesting that these two receptors may form a functional unit signals sharing similar or complementary. 3-CD6 is physically associated with the complex CD3/TCR independently CD5. This partnership makes CD6 in a co-receptor this complex CD3/TCR and 10 trains to modulate signals mediated by this complex. 4-CD6 ys ligand accumulate in the central part of the IS co-localizando with CD5 and complex CD3/TCR. This location is in line with its potential to modulate lymphocyte activation. 5 - The region intracellular CD6 ínteracciona with syntenin-l, a protein domain ADS. The demonstration of the presence of syntenin-l in central IS suggests that this protein functions as an adapter protein allowing the union of CD6 to cytoskeletal proteins or signaling during training and / or maturity of the IS. 6 - The way recombinant soluble CD6 (rsCD6) has been shown to be capable immunomodulator. This demonstrates the relevance of this receptor-mediated interactions in the process of lymphocyte activation and raises the potential therapeutic value.

Author: GONZÁLEZ ROCA EVA.
Year: 2004.
University: AUTÓNOMA DE BARCELONA [www.uab.es].
Place of defense: FACULTAT DE CIÈNCIES.
Place of preparation: ESCUELA DE POSTGRADO.

Author: LLORENS TORRES FRANCESC JOSEPH.
Year: 2004.
University: AUTÓNOMA DE BARCELONA [www.uab.es].
Place of defense: FACULTAT DE CIÈNCIES.
Place of preparation: ESCUELA DE POSTGRADO.

Summary: This work is included in a comprehensive project initiated in the group of Dr. Itarte that seeks to explore some of the cellular processes in which the protein kinase CK2 exerts its functions. The project began with the observation that various chromatographic steps for the purification of CK2 in rat liver, CK2 co-purificada protein separation of which was very difficult, later identified as chaperone grp94 (endoplasmina) and the factor the initiation of protein synthesis elF2. The latter interaccionaba with CK2 through the subunitats beta gamma i, and test activity CK2 in the presence of elF2beta, there was an increase in Km of CK2 on beta-caseína. These works well as data provided in the literature where describing elF2beta as substrate in vitro CK2 led the development of this thesis, which has been characterized and have explained the functional interrelationships between CK2 i elF2beta, both of interaction between the two proteins, and at the level of phosphorylation of elF2beta, trying to deepen the role that fosforilaciónpuede have on the functionality of the process of protein synthesis where elF2 has a key role in its regulatory mechanism. The results indicate that elF2beta interacts in vitro and in vivo as CK2, both with their subunits isolated as with the way holoenzimática of the kinase. This interaction should be sought through the C-terminal domain of elF2beta and the cluster of basic residues and the segment activation deCK2. The interaction elF2beta-CK2 affects the activity CK2, which resulted in an inhibition of the kinase activity on other substrates CK2 as beta-caseína or calmodulina. In turn CK2 phosphorylated in vitro and in vivo enlF2beta in serinas 2 and 67. The mutation of these centers causes a partial inhibition of protein synthesis in cells Hela re-estimuladas with serum. The serinas phosphorylated found in the N-terminal domain of elF2b, which to be removed by mutagenesis causes severe defects in protein synthesis and in cell viability. It is postulated that these effects can be produced by the elimination of the center anchor for elF5 (GAP protein of elF2).

Author: ARESTÉ CALERO M. CRISTINA.
Year: 2004.
University: AUTÓNOMA DE BARCELONA [www.uab.es].
Place of defense: HOSPITAL UNIVERSITARI VALL D¡HEBRON.
Place of preparation: ESCUELA DE POSTGRADO.

Author: JURADO SÁNCHEZ SANDRA.
Year: 2004.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE VETERINARIA.
Place of preparation: FACULTAD DE VETERINARIA.

Summary: The hypothesis of this research is that the enzymes involved in the path of NO / cGMP granule neurons in the rat cerebellum may be subject to a mechanism for regulating their activity levels and amount of protein, which ran a differential expression in the course of development. The close relationship between NMDA receptor isoform NOS and I assumed the possibility of a way of modulating the activity and expression of these proteins by pharmacological compounds that act through these receptors (agonists and antagonists). We characterized the expression of proteins involved in the path of NO / cGMP in the rat cerebellum and the neurons in culture checking not only the existence of all proteins of the track under consideration, but a pattern of differential expression of this protein in the transcurdo development. In addition prolonged treatment with NMDA produces a clear increase in the activity of GCs stimulated with exogenous NO donor, the DEA / NO, and an increase in the messenger of the subunits that make up this protein. We have delved into the study of the molecular mechanism responsible for the regulation of subunit a2 of the GCs, demonstrating that the NMDa increased the stability of messenger of subunit a2, which until then had not been cloned, which has many streams rich uracilo related binding proteins involved in the stabilization / destabilization of messengers as AUF1 and HuR. Finally, we demonstrated that treatment with NMDA decreases the expression of AUF1 significant way through a mechanism dependent PKG.

Author: SANTAMARÍA MARGALEF ANNA.
Year: 2004.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAD DE BIOLOGÍA.
Place of preparation: HOSPITAL VALL D'HEBRÓN - CONSEJO SUPERIOR DE INVESTIGACIÓN CIENTÍFICAS.

Summary: This thesis is divided into 3 parts: Part I and Central analyzes the functional characterization of the protein PTOV1. This protein was found in an experiment conducted with differential display samples of prostate adenocarcinoma and normal samples. PTOV1 is sobreexpresada in prostate cancer (PC) and PIN lesions. PTOV1 is required for cell proliferation. When sobreexpresa, as in CP, cells entering proliferative state. Furthermore, the overexpression of PTOV1 in tumors also correlates with increased their proliferative state. PTOV1 interacts with Flotillin-1, protein majority of genetic lipid rafts not caveolares. PTOV1 seems to be required for entry to kernel Flotillin-1. The overexpression of Flotillin-1 also induced cell proliferation and both appear to depend on the mutual its mitogenic activity. Flotillin-1 also appears to intervene in regulatory mechanisms of mitosis. On the other hand, PTOV1 induces an increase in the invasive ability of the cells in vitro. The overexpression and PTOV1 induces an increase in the levels of proteass system plasminogen uPA and uPAR. PTOV1 interacts with the protein RACK1. Both are translocated to the membrane under stimulation with IGF-1 or LDCs. Both could participate in processes polarization and / or migration of cells. It examines in detail the impact that promotes a form of intracellular protein transfectada both transient and stable in this cell line. The overexpression of how nuclear Clusterin1 induces arrest in G2/My apoptosis of cells. Finally, the third part of the thesis deals is studying the antiproliferative effects of the exact lines of Pygeum africanum on prostate tumor as well as explants from benign prostatic hyperplasia.