POLIPEPTIDOS AND PROTEINS

Author: SANCLIMENS PÉREZ DE ROZAS GLÒRIA.
Year: 2005.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAD DE QUÍMICA.
Place of preparation: UNIVERSIDAD DE BARCELONA, FACULTAD DE QUÍMICA.

Summary: The main aim of the thesis focuses on the design and synthesis of a family dendrímeros based on sequence poliprolina for application in the medical field as a system of transport and release of drugs. The dendrímeros are macromolecules with an architecture similar to that of a tree. The dendrímeros are typically monodispersos, with a large number of branches, three-dimensional fractal geometry, topology and size defined. Thus, synthesize dendrímeros of poliprolina lets you create proteins biomiméticas, water-soluble polymers and biodendrímeros in a single compound. After an exhaustive study of the stage of growth for the synthesis of dendrímeros, it has been concluded that the optimal synthesis is carried out through in solid phase synthesis withthe incorporation settled block building rich in proline, adding Gly at position N terminal [(Fmoc-Gly-Pro5) 2-Xxx-Oh where Xxx = Amp or Imd], resins funcionalización low. The addition of glycine in the building blocks is important for obtaining the dendrímeros. This estrataegía also offers a wide versatility for the development of new dendrímeros peptídicos in solid phase. During the synthesis of dendrímeros in solid phase has been observed an effect on the balls of resin called saturation voltage or resin (stress). This phenomenon shows that the synthetic methodology described in this report is perfectly feasible for the synthesis of dendrímeros of poliprolina second and third generation. In contrast, for the synthesis of generations reach its upper limit due to the degradation of polymeric support. The circular dichroism analysis concludes that the building blocks are rich in proline plasticity conformacional, acquiring both structures PPI and PPII. In contrast, dendrímeros second generation always retain the structure of PPII. These data show the feasibility of using dendrímeros proline new protéinas sebido their defined and stable secondary structure. The biological studies of a quimioteca of dendrímeros proline modified give their periphery, which show a difference in their surface structure translates into at different properties of internalization and gene transfection. Of all the dendrímeros of quimioteca, dendrímero rich Arg is introducing greater penetration cellular capacity transfectar genes in eukaryotic cells and toxicity. The work done in the dissertation were derived four publications: 1 - G. Sanclimens, L. Crespo, E. Giralt, M. Royo, F. Albericio, Biopolymers (Peptide Science), 2004, 76283-297. 2 - G. Sanclimens, L. Crespo, E. Giralt, F.albericio, M. Royo, J. Org. Chem. 2005,70,6274-6281. 3 - G. Sanclimens, L. Crespo, M. Pons, E. Giralt, F. Albericio, M. Royo, Tetrahedron letters, 2003,44,1751-1754. 4-G Sanclimens, L. Crespo, H. Shen, E. Giralt, F. Albericio, MWSaltzman, M. Royo, Biopolymers (Peptide Science) ,2005,80000-000,

Author: AMORÍN LÓPEZ MANUEL.
Year: 2005.
University: SANTIAGO DE COMPOSTELA [www.usc.es].
Place of defense: FACULTAD DE QUÍMICA.
Place of preparation: FACULTAD DE QUÍMICA.

Summary: This thesis has been synthesized and studied ciclopéptidos containing gamma-aminoácidos capable of forming nanotubes paptídicos or segments of them. Thus, there are different ciclopéptidos: Tetrámeros, hexámros and octámeros in which alternate natural amino acids with gamma-aminoácidos (in particular), with the 3-aminociclohenanocarboxilico = gamma-Ach). Thanks to the studies conducted both in solution and in solid state have been confirmed and established a clear requirement structural and thermodynamic basis of the formation of these novel nanotoubos. There have also been synthesized ciclopéptidos that are inserted into lipid membranes to form channels and allow the transport of ions, as well as the formation of nanotubes in the solid state, which have been able to obtain evidence through the first studies of electron microscopy.

Author: RODRÍGUEZ MIAS RICARD-ALEIX.
Year: 2006.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAD DE QUÍMICA.
Place of preparation: PARC CIENTÍFIC DE BARCELONA.

Summary: The nuclear magnetic resonance is used today in virtually all stages of discovery and characterization of drugs. In this thesis we set out to explore these facets of the NMR modeled different systems related to cancer. First use proteins involved in apoptosis (XIAP and Bcl-XL) to implement several NMR methodologies for the characterization of intermolecular interaction phenomena. They point to selective marking schemes tryptophan and assessed their applicability in the process of screening compounds. Other experiments massive screening (STD, WaterLOGSY) were used to identify new ligands Bcl-XL thereafter reconstruction strategies fragments enabled us to design dual ligands. The same tools are used for the design and development of molecules antiangiogénicas the interaction between VEGF and its receptors. To this end, there were two different strategies in the identification of inhibitors: first sought to design a high affinity ligand peptídico of starting from dipéptidos lower affinity and using strategies to rebuild fragments. A second strategy was to identify the active compounds present in extracts from plants using selective marking schemes for VEGF, namely marking selective metioninas. In the latter case were obtained to identify several compounds flavonoids able to interact with VEGF and its mode of binding was characterized using information derived from NMR experiments. Lastly took place the structural characterization of Kahalalide F by NMR. This despsipéptido of marine origin are currently in clinical phases of development against various types of cancer, and their structural characterization presents tremendous interest in understanding its mode of action. It explored the conformational preferences of the peptide in several ways including: H2O, DMSO, and means miméticos membrane (SDS micelles). The peptide adopts a preferential structure in the presence of SDS micelles, which allows us to propose a structural model for the insertion of monomer Kahalalide F in environments membrane lipid, and this suggests that part of its cytotoxic activity occurs at the level of membrane which the formation of a spin beta in the section of the N-terminal peptide also seems relevant.

Author: LUKOVIC DUNJA.
Year: 2006.
University: VALENCIA [www.uv.es].
Place of defense: BIBLIOTECA CAMPUS DE BURJASSOT.
Place of preparation: BIOQUÍMICA Y BIOLOGÍA MOLECULAR - UNIVERSITAT DE VALÈNCIA.

Summary: The protein SP-C part of pulmonary surfactant, a mixture of lipids and proteins that coats the alveoli, which is essential for breathing. The SP-C e5 a critical component in the preparations used to treat respiratory distress syndrome in premature fetuses. The main objective of this thesis was the development of a new method of producing protein SP-C through recombinant technology that could serve as the basis for obtaining new synthetic surfactants, as well as an excellent tool in the cararterización of determinants estructura-función with the ultimate goal of trying to improve the properties tensioactivas of the protein. We have described a new method of obtaining protein SP-C recombinant bacterial cultures, which is based on a strategy of overexpression of transmembrane proteins in prokaryotes and extractions employed in the functional isolation of the SP-C native from washings pulmonary. This method proved highly effective in preserving the helical structure of the protein, the main caraderistica of their activity. In addition we have designed variants of the SP-C two triptófanos and two cisteinas at positions 5 and 6 to study the effect of resíduos aromatic in the N-terminal segment of the protein. In this case the objective was to ascertain the extent to which greater activity of the protein could be defined by a greater affinity toward the N-terminal segment of the comparatively interfaces with the sequence used initially presents two resíduos of fenidalarina in the positions. Under this scenario, option two triptófanos could show an even bigger that the form of SP-C possessing fenilalaninas, while the stroll doS cisteínas could show a lower activity. The experimental results of the structural analysis showed that all variants have a helical conformation highly essential for biological activity. We have shown that all the alternatives have in vitro activity tensioactiva equal to or greater than that of the native protein isolated from pigs lungs. Furthermore, it has been shown that this important activity tensioadiva not be únicament to the presence of resíduos aromatic at positions 5 and 6 of the sequence of the protein. Finally, it should be noted that similar recombinant the SP-C produced in bacteria combined with the lipid mixture DPPC: POPG (68:31) restored the lung function in premature rabbit fetuses so similar to what makes a surfactant widely currently in clinical application. The three recombinant proteins showed a similar behavior, regardless of the amino acids present in position 5 and 6 of its sequence in these experiments in vivo. In conclusion, we have created a process for the production of protein SP-C for possible inclusion in preparations synthetic surfactant used for the treatment of lung diseases, as well as its use as a powerful tool to generate alternatives that would advance structural studies depending on the protein

Author: SÁNCHEZ MAGRANER LISSETE.
Year: 2006.
University: PAÍS VASCO [www.ehu.es].
Place of defense: FACULTAD DE CIENCIA Y TECNOLOGÍA.
Place of preparation: FACULTAD DE CIENCIA Y TECNOLOGÍA.

Summary: The alfa-hemolisina is a toxin produced stone for pathogenic strains of the bacterium E. coli. This protein is involved in various diseases including meningitis, speticemia, peritonitis or diseases of the urinary tract. In this paper we have studied the relationship estrcutra-función both the native and mutant protein replacement, and disposal of waste and within key domains of the protein. To address this study has used a battery of techniques biochemical, biophysical and structural that has allowed us to clarify the role of the C-terminal region of the protein interaction with the membrane and calcium. It has been determined by the affinity of cominio C-terminal calcium and have been determined by infrared spectroscopy induced changes in the protein fragment for binding to ligand. It has also been characterized stability C-terminal domain of the protein as a function of the concentration of ion Ca2 +. It shows that the fragment is fully deployed in the absence of ligand. This behavior can be drawn significant functional implications of this group of toxins. Only kind saturated calcium can be folded to a kind monomer and stable. Unlike the complete protein there is a transition from highly cooperative folding demonstrating that it is a folding per unit to be. Besides analyzing other variants of the protein complete, paying special attention to waste His859, which is unusual in studies of homology. The surface-active properties of mutants off or doubles, and the species is not acilada has also explored. In their joint work provides important clues about the role of alfa-hemolisina. Furthermore, the results obtained with the C-terminal fragment opens significant potential for the crystallization of the fragment and the subsequent determination of its structure at high resolution, a task that seems impossible with the whole protein.

Author: FRUTOS DOMINGUEZ SILVIA.
Year: 2006.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAD DE QUÍMICA.
Place of preparation: PARC CIENTÍFIC DE BARCELONA.

Summary: The HIV-1 protease (HIV-1 PR) is a protein essential for the maturation and infection of the HIV virus, revealing inhibition in vitro it produces immature and non-infectious particles. A large number of compounds have been developed and some of them have been approved by the FDA for the treatment of AIDS. All compounds approved by the FDA act by inhibiting the active center of the enzyme. However resistance and side effects that these drugs have led to the present development of new strategies to inhibit HIV-1PR as is the use of inhibitors dimerización. The idea of using inhibitors dimerización is based on the fact that HIV-1 PR is active only in the form of dímero so that peptides are designed to interact with the lamina-beta antiparalela formed by the extreme N-and C - terminal manner that can inhibit the enzyme through prevention or disruption of the formation of dímero. Because they were no structural studies on the inhibitors dimerización, this thesis was proposed to study the interaction between this type of inhibitor and HIV-1 PR, combining different markings isotopic selective protein and corresponding NMR experiments. First developed a methodology to obtain HIV-1 PR levados with yields of protein expression by using techniques of molecular genetics. This helped establish the requirements that the expression of this protein required. As one of the objectives was to study the structural inhibitors dimerización with HIV-1 PR, it was necessary to the preparation of any of these compounds, which had nature peptide, which were prepared using synthetic peptides in the solid phase. Once analyzed using a fluorescence test was to study structural. There was marked protein selectively with 19F in the Trp residues and Phe99. To get the marked protein selectively in the residue Phe99 was necessary for the preparation of this synthetically as it was not possible to obtain a recombinant. Based on the experiments NMR 19F was possible to conclude that the signals dela Phe99 and one of the Trp were affected to add the ihibidro of dimerización, indicating the possible interaction. Furthermore, the protein was obtained selectively marked with 13C in the Trp residues, Val, Leu and lle. The marking of the side chain of Trp possible to study in detail the interaction of HIV-1 PR with an inhibitor of dimerización may conclude that the peptide was able to break the dímero and interacts with the region expected. From marking the Val, Leu and lle only conducted preliminary studies. In this thesis is also developing a methodology for massive scrutiny that allows rapid and reliable identification of new inhibitors both from libraries and synthetic blends of natural products, without interference by other methodologies.

Author: PEÑA GULÍN ÓSCAR.
Year: 2006.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAD DE QUÍMICAS.
Place of preparation: PARC CIENTÍFIC DE BARCELONA.

Summary: The peptides and proteins are beginning to take important as active ingredients in the development of new drugs against multiple diseases. Despite this, the material peptídico presents very low bioavailability, which is a major problem in the design of routes of administration. The pharmaceutical industry has developed alternative forms of management which play an important role biopolymers. The poliaminácidos are biopolymers with high potential for the design of new formulations, but there are few examples of the latter in development. It had planned to open a study on the applicability of certain poliaminoácidos (such as acid poliglutámico and poliprolina) in the administration of active ingredients type peptídico for therapeutic purposes. First, it developed the methodology necessary for the preparation of acid poliglutámico and poliprolina and their precursors (preparing Nalfa-carboxianhídridos). The manipulation of macromolecules of this work also demanded an effort to its characterization, commensurate with the needs. This led to the use of light scattering techniques (light Scattering) as a key element in the characterization, both polymers and proteins. The handling of acid poliglutámico allowed obtaining a derivative polymer funcionalizado (poliGlu (Cys) No), which allows its reticulation in oxidizing conditions, resulting in a polymer matrix type hydrogel. The properties quimico-fisicas study suggest a high potential of this material for the design of a controlled-release formulation for the administration of peptides and / or proteins. Studies of entrapment and release of models péptidicos, hydrogels of poliGlu (Cys) No, confirmed the ability of the hydrogels described to house a protein (such as human growth hormone, hGH) on the inside and releasing post in response to environmental stimuli (as a function of pH). Experiments with different techniques (MALDI-TOF SEC, DC NMR) also suggest that the protein suffers minimal chemical alterations as a result of his entrapment within the polymer matrix. In parallel, the poliprolina was used to obtain conjugated proteína-polímero (hGH-PP), which will be studied to determine various properties of these conjugates introduced by the poliaminoácido. In this paper we present the results obtained in the determination of Rh these conjugates and their comparison with combined peer hGH-PEG described above. The purpose of this last point is the design of conjugated proteína-PP to prevent their removal to maintain its level renal bioactivity.

Author: BARBARROJA PUERTO NURIA.
Year: 2006.
University: CÓRDOBA [www.uco.es].
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: FACULTAD DE CIENCIAS.

Summary: The Acute Myelogenous Leukemia (LAM) is generated by the accumulation in the bone marrow of mieloblastos with a variable degree of myeloid differentiation, which are beyond the control of programmed cell death to enhanced intracellular pathways that inhibit apoptosis. In fact, the LAM cells is common alteration in the expression and activity of different cell receptors, factors proangiogénicos, transcription factors and intracellular routes. These molecules are involved in the processes of coagulation, the anomalous signs of survival, proliferation, differentiation, and angiogenesis in the process leucémico. Thus, knowledge of the intracellular mechanisms involved in the origin and the pathogenesis of LAM is essential to identify new molecules that block so specific routes or intracellular enzymes essential for the development of leukemia, such as specific inhibitors of RTKs, the MAP kinases, Pl3K and other intracellular routes. With this premise raised the following objectives: 1-Analysis of the cellular and molecular mechanisms involved in the response of cells promielocítics treatment with retinoids derived (AM80 and CD437). 2, - The study of the molecular mechanisms involved in the expression of PML / RARa in Acute Leukemia Promielocítica (LAP). 3-Analysis of the status of expression and activation of several signal transduction important in the initiation and development leucemogénico: FLT3, MAP kinases, STAT5, NFkB and Pl3K/Akt in patients with LAM. 4-Study of the altered expression associated molecular pathogenesis wing and development leucémico through proteomic analysis of cells LAM. These results suggest that routes intracellular Pl3K/Akt-MEK/ERK mediate the activation effects of AM80 on anticoagulants APL cells, while the CD437 regulates apoptosis via the route MEK / ERK. These results have revealed the existence of a thin intracellular regulation of the response of cells to LAP derivatives retinoids, which allow valuing alternation or combination of treatments during follow-up of patients APL. Moreover, the expression of oncoporteian PML / RARa appears to be linked to the constitutive activation of the road MEK / ERK, route intracellular responsible for the pathogenesis of LAP. Combination treatment of new drugs alternative to conventional chemotherapy, as ATRA or As2O3, with the inhibitor of this route, the power degradation of oncoproteína, thus promoting the remission of the neoplastic process. Therefore, the use of inhibitors of intracellular route MEK / ERK in combination with these drugs could be an effective therapeutic strategy in the treatment of APL. Following the analysis of the activation of intracellular different routes in 28 samples LAM, it was noted that there is a constitutive activation of the three major signal transduction pathways responsible for the proliferation, differentiation and cell survival. Such signals are altered so heterogeneous among different subtypes of myeloid leukemia, and even between different patients. This heterogeneity suggests the need for individual studies prior to each patient for a better election and specific combination of antineoplastic therapy. Proteomic studies conducted in 13 samples LAM have shown that many pathological alterations observed in the LAM are also reflected in the pattern of expression of proteomic cells blastic. Therefore, identification of this pattern could represent a path of discovery of new tumor markers.