PROTEIN

Author: DAVE COLL NATÁLIA.
Year: 2002.
University: AUTÓNOMA DE BARCELONA [www.uab.es].
Place of defense: FACULTAT DE MEDICINA.
Place of preparation: ESCOLA DE DOCTORAT I FORMACIÓ CONTINUADA.

Summary: The transporter melibiosa (MelB) is a membrane protein from Escherichia coli accumulates alpha or beta-galactósidos inside the cell, thanks to the energy provided by cations Na +, H + or Li +, contrasportados so simporte. We studied the protein solubilizada in detergent docecilmaltósido and reconstituted into lipid d'E. Coli in four different conditions of substrates: H +, Na +, H + and melibiosa, and Na + and melibiosa. The study of MelB in H2O done through infrared transmission spectroscopy revealed that the protein form contains about 50% of propellers, a 20% beta-sheet, a 17% turns reverse, and 12% allocated to other types structures. These data were reinforced by the results of secondary structure obtained in D2O. The quantification of secondary structure is consistent with the model of secondary structure consisting of 12 propellers transmembrane. The results also revealed changes in the structures induced by secondary substrates. Accessibility overall secondary structures of the MelB the aqueous solvent was determined by kinetic exchange D2O. The results revealed a accessibility of 54% when the MelB incubated with Na + and H +. By contrast, the addition of sugar melibiosa resulted in a decline in access to the aqueous solvent in a 5-10%. A more detailed analysis of accessibility revealed that the addition of sugar mainly protects beta-sheet structures. The analysis of the infrared spectra of the MelB in H2O and D2O, obtained with radiation polarized revealed changes in the orientation of the propellers as the substrate united. It was determined that for MelB combined with the H +, with or without melibiosa propellers forming an angle conl or normal membrane 26Â º. By contrast, the addition of Na + decreases the guidance of the propellers to 36Â º.

Author: BOSCH GRAU MONTSERRAT.
Year: 2003.
University: GIRONA [www.udg.es].
Place of defense: CIENCIAS.
Place of preparation: UNIVERSITAT DE GIRONA.

Summary: In order to study the molecular basis of the citotoxicitad of pancreatic ribonuclease is construyeronn several variants dela HP-Rnasa utizando two strategies distintas.En the first generarón variants of the enzyme resistant to the action of the inhibitor protein ribonucleasas (RhI) through the replacement of waste involved in the interface of contact between the ribonucleasas and (rhI). The second reason was added RGD surface in regions of the protein involved in the formation of the complex with (Rhin). This was intended to promote interaction between the enzyme simultaneously with the cell membrane and reduced its intercción with inhibidor.Se found that only variants carrying multiple substitutions were able to avoid intercción with the inhibitor. The study of the percentage of inhibition of protein synthesis in cells incubated with each of the variants showed that only two of them adqurían properties citotóxicas.Sorprendentemente, variente more cytotoxic among these proved to be sensitive to HRI. This is PE5, and presents a value of IC50 only between 5 and 15 times less than those submitted by the onconasa. This result shows that the sensitivity is not an HRI facto imperative for the toxicity of ribonucleasas.Así, none of the alternatives carriers reason RGD acquired cytotoxic abilities. The analysis of internalization of the alternatives marked radiolabelled showed that only onconasa is effectively internalized during the first 7h incubation. It ruled in this way the possibility that the cytotoxic properties of PD5 are the result of greater efficiency of endocytosis. It was also found that the addition of ground RGD is not able to promote internalization of proteins containing it. By Confocal fluorescence microscopy was empezarón to detect variants within human cells only after 24 h of incubation. All variants generated filed a eficíencia catalytic more than 50% dela activity of its protein source, FP5. This result probably indicates that the structure of the active variants has not been affected drastically by the substitutions made. However, in all cases there was a decrease of thermostability compared to FP5. The study of intracellular traffic lanes of the three variants of the HP-Rnasa (PE5, FP5 and an alternative cytotoxic but not resistant Hri) showed that all of them follow the same transportation route, which leads from the surface toward cell lysosomes. This route was not affected by the addition of drugs that alter lasvías transit retrograde, as monensin and brefeldina A. But by the bafilomicina A1, serving neutralizing gradient Ph endosomal and slowing traffic endosonal did lysosomes. According to these data, the values of citotocicidad of the variants were increased significantly only in the presence of the drug, suggesting that the ribonuclease reach the cytoplasm after crossing membrane system endosomal at some point prior to the lysosomes. Characterization macanismo of cytotoxicity of variant PE5 suggested that the presence of a second source of this protein in three Arg gave him a signal of nuclear transport, which would be responsible for its location in this compartment. Thus, the portion of the enzyme which gets reach the cytosol 8 from the 563 s endosomas is conducted rapidly towards the cell nucleus through the mechanism of active transport classic. For his affinity xon rRNA, the enzyme is concentrated below in the nucleolus, which probably makes its catalytic activity. Likewise, the interaction dela variant PE5 with receptors responsible for transport to core (importinas,) which occurs at the level of NLS, would prevent the formation of complex enzima-Hri, thus avoiding the blockade of the enzyme by its inhibitor. The results obtained in this thesis offer a new strategy for designing ribonucleasas cytotoxic based on the addition of NLS segments with the goal of promoting the nuclear transport of enzymes. This capability could allow overcome factors that had so far been regarded as clearly anti-citotóxicidad of pancreatic ribonuclease as són sensitivity of the enzyme to HRI or low internalization.

Author: PALOMARES GRACIA OSCAR.
Year: 2004.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE CC. QUÍMICAS.
Place of preparation: FAC. CC. QUÍMICAS, DPTO. BIOQUÍMICA Y BIOLOGÍA MOLECULAR I.

Summary: The research presented in this Doctoral Thesis is focused towards the study and characterization of proteins alegénicas mustard yellow and olive pollen. In particular has been carried out structural and immunological characterization of the N-and C-terminal domains of Ole and 9, main pollen allergens olive activity 1,3-beta-glucanasa, which occurred in both domains recombinant form in the yeast P.pastoris and were purified to homogeneity. It has been shown that 1,3-beta-glucanasas are involved in processes of cross-reactivity between pollen, food and fruit and vegetables has been established mouse model of allergic to Ole and 9 in which it has tested a prophylactic treatment with fragments recombinants. In addition, it has started a study mapping epitópico the C-terminal of Ole and 9 and has analyzed the clinical relevance of both domains recombinants. On the other hand, there has been the forerunner of Sin to 1, the main allergen yellow mustard, yeast P.pastoris which has enabled analyze its structural and immunological properties and compare them with those of the wild type. This study has permitted to propose that the recombinant protein can be used as a marker awareness mustard. It has also been isolated and identified a new allergen mustard high molecular weight. This is a globulin 11S, called No-2, which has been marked structural and immunologically. Finally, it has cloned the cDNA coding for Sin-2 and analyzed the properties of that sequence.

Author: OLIVA BLANCO M. ÁNGELA.
Year: 2004.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE CIENCIAS BIOLÓGICAS.
Place of preparation: CENTRO DE INVESTIGACIÓN BIOLÓGICAS-CSIC (MADRID).

Summary: FtsZes one of the proteins that intervienenen the divisionof prokaryotes. Ensamblaen site divisiónformando a anillodenominado ring Z, which is the frame on which the rest will be recruited for proteins involved in cell division. FtsZ is highly conserved between prokaryotes and even body has been found in chloroplasts and mitochondria of some eukaryotic organisms. It is a protein homologous to eukaryotic tubulin, and both are GTPasas with the same tertiary structure. Tubulin requires large numbers of molecular and cofactors chaperonas to acquire its native structure and functions, while FtsZ of M.jannaschii does not seem to have the same requirements and is able to fold in vitro from a state denatured. This paper has analyzed the polymorphic of FtsZ assembly of M. Jannaschii, and using techniques of electron microscopy and image processing has been proposed that the basic structure of this protein assembly is a strand consisting of two that run parallel protofilaments. Different lateral interactions between these filaments give rise to different types of polymers across the study. Finally, we analyzed the change conformacionallnducido by nucleotide hydrolysis of GTP joined FtsZ using crystallography diffraction and X-ray The structure of protofilamento of the protein has shown that it forms the same axial contacts during the assembly of tubulin in mlcrotúbuios. It also has characterized the structure of the active site of protein and it has been determined that in order to observe the change conformacional induced nucleotide hydrolysis is required study of nucleotide binding site altogether (the interface between two molecules) and not the monomer (which is the one that has been studied to date).

Author: LÓPEZ AROLAS JOAN.
Year: 2004.
University: AUTÓNOMA DE BARCELONA [www.uab.es].
Place of defense: FACULTAT DE CIÈNCIES.
Place of preparation: ESCUELA DE POSTGRADO.

Author: O`LOGHLEN VELICIA ANA.
Year: 2004.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD CIENCIAS BIOLOGÍCAS.
Place of preparation: HOSPITAL RAMON Y CAJAL.

Summary: The doctoral thesis explores the oxidative stress generated by U2O2 cells PCI2 differentiated with NGF, H2O2 produced a strong inhidirción delo synthesis of proteins in parallel with the increase in fosfonlación of Eif2x, the decline in levels of fosfolación of eIF4E and 4E-BP1 and inhidición of today's eIF4F and the poteina fosfatosa PP1. In addition to all that U2O2 produces cell death which refers to treatment with the antioxidant N, acetil-cisteina (NAC). NAC review prevents inhateción dela sistesis poteica. Lastly, identify and rucieterizan a new form of non-lu eII4E quinusa MNK1, they call MnK1B is located in heart and that is reflected in much tissues.

Author: MERINO MONTERO SANDRA.
Year: 2004.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAD DE FARMACIA.

Summary: The membrane proteins account for between 25 and 30% of the protein in the cells and may be involved in some cases, mechanisms of resistance to certain drugs. It is therefore of great interest to the study of proteins in these models biomiméticos such as proteoliposomas or crystals 2D. The application of techniques manométricas, such as atomic force microscopy (AFM), as well as certain methods fluorescence (calculation of the increase in surface electrostatic potential and the fluorescence anisotropy), allowing the study of the process of reconstruction of protein. The working hypothesis and main objective of this thesis was the detailed study of the process of reconstitution of two proteins transmembranarias, porina Omp1 of Serratia marcescens and lactose permease of Escherichia coli (LacY), modeled to test the applicability of techniques used to this type of molecule, cytochrome c and the melitina. Finally, it was concluded that both proteoliposomas as foils lípido-proteicas obtained were entirely stable in solution and on a flat substrate. The LacY reconstituted in POPC was crystallized in 2D and displayed by AFM, presenting a ground dimérico. The cell parameters were: a = 13.15 nm, b = 16.74 nm and = 116, consistent with a symmetrical type p2.

Author: RAMOS ARANGUREN ALBERTO.
Year: 2005.
University: POLITÉCNICA DE MADRID [www.upm.es].
Place of defense: E.TÉ.S. DE INGENIEROS DE MONTES.
Place of preparation: ESCUELA TÉCNICA SUPERIOR DE INGENIEROS DE MONTES.

Summary: There were marked in the European chestnut cDNA clones of the genes CsTOC1 and CsLHY, counterparts to the elements of the circadian oscillator Arabidopsis. The circadian rhythm of the messenger RNA of genes CsTOC1 and CsLHY at root, stem and leaves of chestnut seedlings grown in standard conditions of light and temperature (22Â ° C and LD) is similar to that described for the clock Arabidopsis. This behavior is also observed in the trees adults during times of growth. It has been found that during the winter dormancy of chestnut take account of changes in the functioning of the circadian clock in the bud as in the stem: the levels of the messenger of CsTOC1 and CsLHY not vary and are permanently high. A similar interruption of induction in the leaves and stems of the seedlings in response to low temperatures (4Â ° C). In Arabidopsis not induce these changes as a result of cold or winter, which suggests the existence of differences in parts or in the mechanisms of regulation of the clocks in both species. The expression of genes CsTOC1 and CsLHY oscillator chestnut endodurmientes or exposed to 4Â ° C retrieves the circadian rhythms when they are transferred to 22Â ° C, which means the alteration of relojo is not intrinsic to the process of endodormancia but a direct consequence of the exhibition is to the low temperatures. However, this does not rule out that such changes may have a role in the development of the same. It discusses the influence they can have the stop watch in the metabolism of trees sleepers, and raises the different behavior of brown and Arabidopsis in response to low temperature implies that the process of acclimatization to woody plants in cold climates temperate must have specific features significant compared with the annual herbaceous plants.

Author: FERNÁNDEZ SÁIZ VANESA.
Year: 2005.
University: PAÍS VASCO [www.ehu.es].
Place of defense: FACULTAD DE CIENCIA Y TECNOLOGÍA.
Place of preparation: BIOQUÍMICA Y BIOLOGÍA MOLECULAR - FAC. CIENCIAS.

Summary: This Doctoral thesis focuses on the study of the C-terminal helical subdomain of the chaperone DnaK, representing the majority of the proteins Hsp70 in E.coli. This group of molecular chaperones is widely distributed in the cytosol and in prokaryotic and eukaryotic organelles. They participate in a broad spectrum of activities such as the assembly of protein complexes, transport across membranes, the folding of proteins synthesized de novo, as well as in the solubilization of protein aggregates formed in stressful situations cell. All this broad set of activities is done through a mechanism that involves concerted conformational changes in the two functional domains that comprise proteins Hsp70. Have been studied by means of biochemical and biophysical techniques the role of the propellers C-terminales of DnaK in communication alostérica between the two domains of the protein and stability in the center of the substrate binding. Likewise, we have identified the interactions that allow the anchor of the propellers C-terminales above the center of substrate binding, and therefore regulating the accessibility of it. Finally, it has studied the interplay of cochaperonas GrpE and DnaJ with DnaK, and the role of the substrate binding domain in this interaction, and their physiological effects.

Author: PALLARÈS GOITIZ IRANTZU.
Year: 2005.
University: AUTÓNOMA DE BARCELONA [www.uab.es].
Place of defense: FACULTAT DE CIENCIES.
Place of preparation: UNIVERSITAT AUTÒNOMA DE BARCELONA.

Summary: In this thesis studies have developed protease isolated structure and complex with their endogenous inhibitors through X-ray crystallography, which have identified in detail their enzymatic mechanisms and inhibició. Likewise, these proteases and inhibitors have been used as models of protein is not involved in diseases that have the capacity to form structures amiloidogenicas des state total and / or fully deployed.

Author: BUENO FERNÁNDEZ MARTA.
Year: 2005.
University: ZARAGOZA [www.unizar.es].
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: FACULTAD DE CIENCIAS.

Author: VILAR VARELA SANTIAGO.
Year: 2005.
University: SANTIAGO DE COMPOSTELA [www.usc.es].
Place of defense: FACULTAD DE FARMACIA.
Place of preparation: FACULTAD DE FARMACIA.

Summary: In this Doctoral Thesis have developed theoretical models for predicting the vasodilator and anti-HIV activity using methods for calculating molecular descriptors in combination with statistical techniques to establish a relationship between molecular structure and biological activity (QSAR methods ). They have also produced calculations interaction fármaco-receptor by type docking methodologies and has done an organic synthesis of a series of coumarin - like resveratol with good vasodilator activity.

Author: NAGANO CELSO SHINITI.
Year: 2006.
University: VALENCIA [www.uv.es].
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: INSTITUTO DE BIOMEDICINA DE VALENCIA.

Summary: The lectin are (glico) origin is not immune proteins, which possess at least one catalytic domain is not capable of uniting and specifies reversibly monkey or oligosaccharides. The lectin are ubiquitous in organizations throughout the evolutionary scale, being involved in many biological processes, both physiological and pathological. The spatial arrangement of places of union sugar is a key element in the recognition of glicanos on the cell surface The overall aim of the thesis was to deepen structural and evolutionary aspects of plant lectin. On the one hand, have characterized the lectin HCA and HML of red marine algae Hypnea cervicomis and Hypnea musciformis, respectively. These lectin containing 90 amino acids and their sequences are very similar. The estructutras elementary HCA and HML consist of two domains counterparts with each other, arranged in tandem and show no similarity to any protein sequence described. We propose that HCA and HML are founding members of a new family of proteins. Furthermore, the thesis focused on determining the structural basis of balance dimero-tetramero pH dependent lectin that some of the tribe Diocleinae the family Papilionoidae of legumes. To this end, an approach has been used based on molecular biology techniques (recombinant expression lectin native and mutant), Biophysics (balance sedmentación) and Structural Biology (determination of the crystal structures of the lectin recombinates). Because of the complex system of processing post-traduccional associated with the biosynthesis of lectin of pulses, the expression of recombinant Escherichia coli called lectin in the design of a synthetic gene that codify the mature protein (alpha chain). The lectin of Dioclea guianensis (r-aDguia) and Dioclea grandiflora (r-aDGL) produced in a recombinant E. Coli from its synthetic genes are bioquimícamente indistinguishable from their counterparts isolated from seeds. It has been successful in generating the balance dimero-tetrámero pH dependent from the lectina r-aDGL, tetrameric pH in the range of 4-8.5, through three mutations E123A, H131 N, K132Q. The existence of equilibrium dimero-tetrámero correlates with a retracted conformation of the loops 117-122, which can not establish contacts with resíduos of subunits opposing stabilized tetrameric association independent of pH.

Author: FORT I BAIXERAS JOANA.
Year: 2006.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAD DE BIOLOGÍA.
Place of preparation: FACULTAT DE BIOLOGIA DE LA UNIVERSITAT DE BARCELONA.

Summary: The family of transporters heterométicos amino acid (HATs) has the unique feature of being composed of two subunits, a heavy subunit (HSHAT) and a light subunit (LSHAT) linked by a disulfide bridge. Mutations in any of its members are responsible for aminoacidurias hereditary, as lisinuria intolerant protein (LPI) or cistinuria. This thesis shows the results obtained from the expression in E. coli and the subsequent purification of the extracellular domain of 4F2hc human, one of the heavy chains of transporters heteroméricos amino acid. The main result is the decision of the three-dimensional structure of this domain of the protein through crystallization and X-ray diffraction It has resolved two types of crystals, monoclinico to 2.1 A (with a molecular in the asymmetric unit) and ortorómbico to 2.8 A (with two molecules in the asymmetric unit coordinating a zinc atom). The ectodominio of 4F2hc has a very similar structure to alfa-glucosidasas insect and consists of a domain A, which is a barrel (alpha / beta) 8 or TIM barrel and a domain C, which consists of 8 leaves beta antiparalelas. The paper presents the structure of 4F2hc and compares with known structures of glucosidasas bacteria. It also presents a structural model for rBAT, the other subunit heavy transporters heteroméricos amino acid. But the production i purification of this protein has enabled us to realize, also, studies of function and interaction with other molecules. This thesis tries to provide information for the study estructura-función of these carriers, but also for the study of the different functions and processes in which 4F2hc hga associated in the liberatura, as a function of integrins and tumorigenesis.