MOLECULAR BIOLOGY (2)

Author: ZAPATA GONZÁLEZ FERNANDO.
Year: 2004.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAD DE QUÍMICA Y FÍSICA.
Place of preparation: FACULTAD DE BIOLOGÍA.

Summary: The docosahexaenoic acid, a PUFA omega-3 has been associated with immunosuppressive effects in lymphocytes, macrophages and monoliths, but there were no studies of its effects on dendritic cells (DCs). Therefore differed in the presence of DHA monocytes to DCs. Cells treated with the fatty acid showed divergent phenotypic changes depending on the maturity of the same. Thus, in immature stage, there were increases in expression of CD83, HLA-DR and CD36, and decreased CD1a and CD80. However, in mature stage, CD83, CD86 and HLA-DR suffered declines in its expression with respect to the controls. CD80 and CD1a again showed a decreased expression and of CD36 was increased. By measuring the ability of the activation of lymphocyte proliferation of these cells was found that this was diminished. The secretion of IL-10 and IL-12 had also fallen. This set of effects was dependent on the concentration of DHA. DHA was able to develop some sort of immunosuppressive effect on the DCs. Similar experiments were conducted with other fatty acids: EPA, linoleic acid (LA) and oleic acid. With the EPA and the results were similar to those shown by the DHA qualitatively but not quantitatively. The fatty acid DHA was the most powerful and affecting both the fnotipo as to the ability secreting cytokines and activation of lymphocyte proliferation in DCs. These results were similar to those obtained by other authors in the treatment of DCs with activators PPARy which suggested DHA for a possible action through this nuclear receptor. However, the fact that the DHA is an activator PPARy poor compared with EPA, or Rosiglitazone suggested the presence of other avenues activation. Specifically, RXR, which has been shown that DHA is able to interact. DCs were treated with concentrations in the range nanomolar of 9cRA, an activator of CXR obtained that this retinoid was able to interfere with the normal maturation of the DC so that was obtained phenotypic results remarkably similar to those produced by the DHA. Also the ability to stimulate lymphocyte proliferation was reduced. Instead, the DCS treated 9cRA showed a decreased secretion of IL-12 and normal IL-10. The activation of PPARy: RXR simultaneously through both monomers has been linked with additive effects, synergistic or complementary in many cell types. It is believed that this is because the union of molecules coactivadoras up to heterodímero. The simultaneous addition of Rosiglitazone and 9cRA to DCs showed phenotypic effects additives indicating that probably 9cRa was acting through hetreodímero. Similar experiments were carried out combining with 9cRA and DHA DHA with Rosiglitazone. The combination with Rosiglitzona showed changes much more important suggesting that the DHA serving primarily joining RXR in heterodímero. Finally, we added a specific inhibitor of PPARy to the dendritic cells: GW9662, which was able to block the effects produced by the DHA, Rosiglitazone and 9cRA. Furthermore, the inhibitor itself completely alter the process of maturation of dendritic cells implying that PPARy must develop an indispensable role in the process of maturation of the DC and not just act as an activator of immunosuppression and also that the DHA, probably, acting through the monomer RXR of heterodímero PPARy: RXR in Dcs.

Author: HORCAJADA GARRO CRISTINA.
Year: 2004.
University: BARCELONA [www.ub.es].
Place of preparation: UNIVERSIDAD DE BARCELONA.

Summary: The glycogen and starch sitasas are glicosiltransferasas that catalyze the transfer with retention of configuration waste glucosilo the end of a string reducer not growing glucan alpha-1, 4. This process is central to the energy metabolism of most living organisms. In this thesis we have described the crystallographic structure of glycogen synthase from Pyrococcus abyssi, an enzyme which can be used both UDP- as ADP-glucosa as donors glucosilo. Although the technology subunit of this enzyme are similar to those described recently for bacterial synthase from Agrobacterium tumefaciens, its molecular organization presents very significant differences. The synthase of arqueon is homotrimérica on the glass and in solution, as shown by experiments analytical ultracentrifugation and electron microscopy. The provision of subnidades gives the molecule a triangular shape obvious. Comparing protein arqueon with other glicosiltransferasas acting with retention settings have enabled us to identify wastes that determine the specificity of the donor glucosilo, and propose a structural model for explaining the basis for the regulation of glycogen sintasas eukaryotes.

Author: BRAVO ARRANZ MARIA.
Year: 2004.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FARMACIA.
Place of preparation: CENTRO NACIONAL DE ALIMENTACIÓN (AESA).

Summary: The amines biógenas in food are caused by decarboxylase activity of a large number of microorganisms. His concentration and nature is a function of many factors, having detected significant amounts of these compounds in virtually all food groups, especially in fish. The ingestion of high concentrations of amines biógenas may lead to a poisoning that course with symptoms similar to allergies and other types of food poisoning (headache, diarrhea, hives, etc.9. Is unknown with certainty the extent of this toxemia although it is suspected that its incidence is much higher than officially acknowledged. amines biógenas most commonly found in fish are histamna, cadaverina, putrescina, espermina and espermidina. Although it is common to find several of them in the same foods, and that is not known toxicity nor the mechanism by which they act is considered to histamine as the main causative agent of such poisoning. however, there is evidence that the joint presence of several amine enhances the toxic effect of histamine . One of the scenarios raised the matter involves the release of endogenous histamine from cells of the immune system. In the present work we studied the histamine cadaverina, putrescina espermina and espermidina from two points of view: 1 - The sequence that cytotoxic presenting these amines associated with fish and its leverage effect in binary mixtures with histamine using two cell lines: GTR-2 and Chang-Liver. 2, - cell activation basófilas mediated by these amine and their binary mixtures using flow cytometry. From our studies, we have concluded that, among all selected amines, espermina and espermidina have shown the greatest ability to cytotoxicity and cell activation basófilas. mixtures by histamine and the rest of the miana have also expressed clear leverage effect on that activation with respect to their individual concentrations.

Author: ANTÚNEZ RODRIGUEZ CRISTINA.
Year: 2004.
University: MÁLAGA [www.uma.es].
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: FACULTAD DE CIENCIAS.

Summary: Some patologras skin as atopic dermatitis (AD) or psoriasis worsen with stress and this phenomenon has been associated with the local release of neuropeptides (NPs). The NPs, as substance P (SP), gene-related peptide of calcitonin (CGRP), somatostatin and neuropéptido And (NY) moduran or initiate inflammatory processes in the DA. We know that there are proinflammatory cytokines that initiate and regulate these inflammatory processes, and therefore one would expect that NPs influence the production of the same. In the work done in this thesis concludes that ros in vitro production of peripheral blood mononuclear cells from patients with atopic dermatitis, neuropeptides are able to induce changes in the cytokine production of T lymphocytes These changes are different in those with acute injuries with regard to presenting chronic injuries, acting in the way preferential subpopulation of T lymphocytes CLA +. Furthermore, lymphocytes expressed receptors translate this interaction ligando-receptor in a signaling cascade that produces a response. The results of this study indicate that in atopic dermatitis there is a regulation CGRP receptor, since we found a decrease in the component CRLR not found differences between patients with chronic or acute injuries. The study of the expression of the receptor SSTR3 does not reflect significant changes in this condition. Following the separation of memory CD45RO + lymphocytes and the virgin CD45RA + T lymphocytes of patients with chronic DA, it is shown that the effect of the neuropeptides occurs primarily in the memory T lymphocytes and within these cells essentially CLA +. The effect produced by the neuropeptides depends on the pattern of cytokine prior cells. In the case of cells with skipper Th2 the neuropeptides produced an increase in IFN-gy in the case of a pattern Th1 produce an increase in IL-13. All this indicates an immunomodulating effect of the neuropeptides in atopic dermatitis. This influence of NPs in the disease and opens up new areas of research aimed at the research and development of drugs to control this patologra.

Author: CÁMARA NAVARRO YOLANDA.
Year: 2004.
University: BARCELONA [www.ub.es].
Place of preparation: FACULTAD DE BIOLOGÍA - UNIVERSIDAD DE BARCELONA.

Summary: The protein desacopladora mitochondrial UCP3 belongs to the superfamily of conveyors anion mitochondrial and preferentially expressed in skeletal muscle. Their physiological function is unknown, although it is known that in vitro is able to develop an activity desacoplante. We note that the presence of high levels of UCP3 in non-muscle cells, mitochondria gives biochemical behavior characteristic of the presence of a protein desacopladora functional (decoupling induced fatty acids and inhibible by GDP). Under these circumstances, UCP3 does not induce apoptosis but sensitizes cells to stimulation apoptótico involvement with mitochondrial (staurosporina). The muscle cells in culture acquire resistance to the activation of apoptosis to differentiate into myo-tubes, a transient decrease in the content ROS along differentiation, and a decrease in the expression of genes coding for ezimas antioxidant and in the levels of glutathione reduced. The activation and apoptotic pathways (caspase-3, caspase-9) by staurosporina occurs in parallel with an increase in ROS, but the latter phenomenon is not required for the activation of the apoptotic process. The induction of high levels of expression of UCP3 in differentiated muscle cells via gene transfer with a vector adenovírico, in a moderate way diminishes the potential of membrane but does not affect d ROS levels in either oxidative stress in general. The presence of UCP3 no effect on ROS generation and in response to staurosporina and does not protect but even moderately aware of caspase activation in response to staurosporina. Fatty acids, especially palmitate, increasing ROS levels, the cells differentiated muscle (myo-tubes). The presence of UCP3 produces a modest increase ROS sensors and cellular oxidative stress in the context muscle cell. Their presence does not protect cells from the stimulus apoptótico but on the contrary, the sensitized. This must necessarily occur by mechanisms other than potential effects on ROS and may involve interaction indirect (via consequences bioenergéticas) or direct UCP3 with machinery that regulates mitochondrial level to the process of apoptosis.

Author: ROMERO GARCIA M. INMACULADA CONCEPCIÓN.
Year: 2004.
University: CÓRDOBA [www.uco.es].
Place of defense: FACULTAD DE CIENCIAS . UNIVERSIDAD DE CÓRDOBA.
Place of preparation: UNIVERSIDAD DE CÓRDOBA.

Summary: The increasing pollution which is being subjected natural environment is a problem that requires urgent solution. Among the chemical contaminants are oxianiones toxic as arsenide and arsenito used as pesticides, nitrate, used as fertilizer and other oxianiones from industrial processes, such as telurito, seleniato, chlorate, among others. These compounds accumulate in the soil, from where it becomes groundwater and inland waters, preventing its use for consumption. It has been purified and characterized the nitrate reductase periplásmica of Rhodobacter sphaeroides DSM158. Furthermore, it has been characterized metabolism and resistance telurito, arsenate, arsenito, seleniato, selenita, chlorate and perchlorate in Gram negative bacteria photosynthetic Rhodobacter sphaeroides DSM158 which has a nitrate reductase periplásmica (Nap +), Rhodobacter capsulatus E1F1 as nitrate reductase is assimilation (Nas +) and Rhodobacter capsulatus B10 lacking nitrate reductasas (Nap-, Nas-), and in the Gram positive non-photosynthetic bacteria Rhodococcus sp. RB1 (Nas +). It also discussed the possible involvement of nitrate reductasas of organisms involved in reducing these oxianiones because it has been described that these enzymes can catalyze the reduction of oxianiones. Finally, we have to point colorimetric methods for measuring concentrations of some of these oxianiones. The Nap of R. Sphaeroides can reduce chlorate, telurito, seleniato and selenita. This bacterium has the reductasas specific for each of the oxianiones employees except for perchlorate, and as the bacteria Rhodococcus sp. RB1 has telurito and selenita reductasas respiradoras. The bacteria Rhodococcus sp. RB1 also breathes chlorate (Per). This strain in aerobic conditions tolerates arsenate, seleniato and chlorate (Per), but not reduced. It grows with selenite but has a telurito reductase. R. Capsulatus E1F1 and B10 have telurito, seleniato, selenita and reductasas arsenide. They are very sensitive to arsenito and breathe arsenate, seleniato and selenite. The strain B10 also breathes perchlorate.

Author: IVORRA IVORRA CARMEN.
Year: 2004.
University: VALENCIA [www.uv.es].
Place of defense: FACULTAD DE MEDICINA - UNIVERSITAT DE VALÈNCIA.
Place of preparation: INSTITUTO DE BIOMEDICINA DE VALENCIA-CSIC.

Summary: The protooncogenes the family AP-l c-fos and c. Jun desempefian an important role in controlling the prQliferaci6n cell eukaryotic organisms. Its regulation is governed by a multitude of mechanisms, which includes interactions with other proteins. The main objective of the work undertaken in this thesis has been the identificaci6n new targets of interaction of the protein c-Fos. Our experimental approach has allowed us to identify the interacci6n between c-Fos and nuclear matrix proteins of the lamina A / Ce INMP (intranuclear matrix protein), and characterize the interaction c-Fos/lamina A / C. By in vitro tests herrtos identified domains responsible for the formation of heterodímeros c-Fos/lamina A / C. Studies of expression and subcellular localization in cells by immunofluorescence elementary miocito vascular smooth and human HeLa cells have enabled us to demonstrate its colocalización. We have also analyzed Ios effects of this interaction on the activity of c-Fos and its impact on the proliferative capacity of mammalian cells.

Author: GIL MORRIÓ MARÍA JOSÉ.
Year: 2004.
University: POLITÉCNICA DE VALENCIA [www.upv.es].
Place of defense: Dep. Biotecnologia.
Place of preparation: Universidad Politécnica de Valencia.

Summary: An understanding of the molecular mechanisms that control the resistance of the plant compared with pathogens biotrofos is a complex area of research and expanding where it imposes to identify new regulators. Previously it had been described in our laboratory gene P69C that encodes a protease with homology to subtilisinas and whose expression is induced during the interaction planta-patógeno. In order to explore new parts of the plant involved in signaling the defensive response, we proceeded to scrutiny mutant of Arabidopsis thaliana that constitutively and without the presence of any external stimulus was found activated gene expression directed by GUS promoter P69C. In memory of this thesis are extensively described the identification and characterization of mutant csb3 (constitutive subtilisin3). The plants csb3 possess high levels of salicylic acid (SA) and also express genes Depending on the route of SA such as PR-1, PR-2 and GST6. Moreover, the mutant csb3 exhibit a high resistance to oomiceto pathogen Hyaloperonospora parasitica nature biotrofa and pathogenic bacteria also biotrofa Pseudomonas syringae pv.tomato DC3000 (Pst) DC3000. However, resistance to pathogens necrotrofos such as Botrytis cinerea and Plectosphaerella cucumerina unchanged in plants csb3. To analyze the participation of the various components of the signaling path-dependent SA in the event of resistance phenotype of csb3, were analyzed epistático between csb3 and pad4, sid2, eds5, nahG, npr1, dth9 and cpr1. These studies indicate that the high resistance to pathogens biotrofos of plants csb3 requires each and every one of the components of the signaling path dependent HS studied. The gene CSB3 identified by positional cloning consolidates 1-hidroxi-2-metil-2-butenil 4-difosfato (HDS) synthase. This enzyme controls one of the final steps in the biosynthesis of isopentenil diphosphate (IPP) in the path of 2-C-metil-D-eritritol-4-fosfato (MEP), which takes place in the chloroplast. The expression of CSB3 plant wt Punishing partly in response to infection by Pst DC3000. In addition, through the isolation and characterization of suppressor extragénicos of mutant csb3-1. Along with complementing pharmacology fosmidomicina (an inhibitor of rura MEP) phenotype resistance of plants csb3-1 propose that CSB3 could act as a negative regulator of the path-dependent signaling SA leading the resistance to pathogens biotrofos . Moreover, plants csb3-1 presents levels of expression of genes that encode proteases with homology to subtilisinas higher than recorded for plants wt. These two genes csb3-1 presents levels of expression of two genes that encode proteases with homology to subtilisinas higher than recorded for plants wt. These two genes called AtSBT3.3 and AtSBT3.5 are characterized by an expression that is induced so early in response to Pseudomonas syringae and Plectospharella cucumerina and after treatment with H2O2. On the contrary, its expression is completely independent of SA. The loss of function of the gene AtSBT3.3 causes a phenotype hypersusceptibility to pathogens biotrofos. These evidences point to the involvement of AtSBT3.3 and AtSBT3.5 in the mechanism of resistance against pathogens biotrofos.

Author: BARRERO SANCHEZ JOSE MARIA.
Year: 2004.
University: MIGUEL HERNÁNDEZ DE ELCHE [www.umh.es].
Place of defense: CAMPUS DE ELCHE.
Place of preparation: INSTITUTO DE BIOINGENIERIA.

Summary: The salinization of the soil is cultivated one of the biggest obstacles facing agriculture. Isolation and characterization of mutants that express their responses to changes in salinity, with the aim of identifying the corresponding genes and eventually manipulate, it could provide solutions to this problem. With a view to contributing to the resolution of this problem can be isolated in the laboratory of JL Micol mutant strains of Arabidopsis thaliana can germinate in the presence of high salt concentrations. Effective scrutiny of 675,500 seeds were isolated 17 lines expressing mutant germination halotolerante, and were assigned to 5 groups complementation to be named SALOBREÑO1 to 5 (SAÑ1 to 5). We have characterized levels morphological, molecular and physiological 15 mutant of Arabidopsis thaliana, á them isolated earlier in the laboratory JL Micol based on their germination halotolerante. These mutants belonging to three groups of complementarity (SAÑ1, SAÑ3 and SAÑ4). We have demonstrated that these three genes SAÑ encoding enzymes involved in the synthesis of acid abscísico (ABA), which confirms the importance of this fitohormona in the perception of stress and saline suggests that the manipulation of its route biosintética could allow modulation of the halotolerancia in plants. We have cloned posicionalmente mutations sañ1, demonstrating that the gene SAÑ1 is ABA1, whose product is zeaxantina epoxidasa, the enzyme that catalyzes the initial stages of the route síntesjs ABA. We have characterized structurally nine alleles aba1, and correlated with the molecular nature phenotypic effects. The analysis of their phenotype morphological, physiological and cellular suggests that the endogenous ABA promotes vegetative development in the absence of stress. The mutants aba1 manifest desetiolación when they are grown in darkness, a feature not seen in other mutants deficient in the ABA. This suggests that the zeaxantina, the intermediary of the ABA route that accumulates in the mutant aba1, affects escotomorfogénesis. We have cloned posicionalmente genes SAÑ3 and SAÑ4, which proved ABA2 and ABA3. The dehydrogenase gene ABA2 encodes a short-chain alcohols involved in the synthesis of ABA, and ABA3 the sulfurasa a molybdenum cofactor necessary for one of the enzymes in the path of synthesis of this hormone. We structurally characterized two alleles presumably null mutant gene ABA2 and four mutant alleles of the gene ABA3, two of them partially. The study of morphological and physiological phenotype confirmed that the ABA is a promoter of endogenous growth. We have quantitatively analyzed the expression of genes ABA 1, ABA2, ABA3, AAO3 and NCED3, all of whom are involved in ABA biosynthesis, in the mutants aba1-101, aba2-14, aba3-101 and aa03-2 as well as his ancestor wild Col-O. We have also analyzed the response of these genes to NaCI and ABA, which has enabled us to confirm the existence of a mechanism of self-regulation positively on the road biosintética ABA, which is primarily carried out by the genes ABA 1 AND NCED3. We conclude that the key element in the perception of stress is the saline gene NCED3. We have found that the sensitivity of NaCI several Arabidopsis thaliana ecotypes during germination correlates with the inducibility of gene expression NCED3 in response to that salt.

Author: GARCIA SACRISTAN ANA.
Year: 2004.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE CC. BIOLÓGICAS.
Place of preparation: FACULTAD DE CC. BIOLÓGICAS.

Summary: One of the goals of the laboratory where he has conducted the work of this thesis is to identify genes involved in differentiation Erythropoietic using as a model system to study the differentiation of cells induced MEL. Through a cDNA library differential cell MEL pre-diferenciadas has identified the gene cIklSTY as an active gene expression in the early stages of differentiation. ClklSTY is a protein kinase capable of fosforilar residues serine, threonine and tyrosine. Through alternative splicing of exon 4, c1k1STYes capable of generating two transcripts. The inclusion of exon 4 results in a protein catalíticamente active, while the exclusion of exon generates a truncated isoform. In this Doctoral Thesis has shown that clk / STY as well as other family members c1k (clk2, cIk3 YcIk4), increase its expression in differentiated cells MEL. By RT-PCR testing has been observed that increased expression is caused by both the full transcribed by the truncation. In the case of CklSTY, has detected a change in the relationship between the products:-plicing.Así, undifferentiated cells in the early stages of differentiation is the complete transcribed majority, however, in the final stages there is a greater expression of truncated transcribed. It has also been shown that higher exclusion of exon 4 is independent of the action of inducing agent and is directly related to the degree of differentiation of the cells. Through studies have been located in silico consensus binding sites of proteins regulatory If) / Â ¡cing alternative SR proteins and PTB, as introns flanking exon 4 and in the exon 4 By testing transfectantes stable sobreexpresan clk / STY it has been shown that the exclusion of exon 4 of clk / STY favors along the MEL cell differentiation, even when you turn the exogenous expression. These results suggest that CklSTY could contribute to controlling the differentiation Erythropoietic through a mechanism that would involve a balance between the two isoforms. It has been corroborated in MEL cells, the location of clk / STY, as well as its substrate protein SC - 35, is directly related to the degree of phosphorylation. In cells MEL prediferenciadas the localization of both proteins remains a diffuse pattern c'bincidiendo with a greater presence in the manner catalíticamente active. In MEL cells differentiated locating clklSTY and SC - 35 is mostly in speckles concurring in this case with the increase in the truncated protein. On the other hand, we have identified and isolated a new isoform of cIk4 containing a. Additional 59 bp sequence. By their very nature, the stream has been recognized as an exon that can suffer 8plicing alternative generating two new isoforms clk4. Studies in si / ico have helped locate a possible reading frame in the new exon that if used in vivo give rise to a protein kinase domain which would retain part of clk4 modifying the N-terminal domain.

Author: RODRIGUEZ SÁNCHEZ JOSANA.
Year: 2004.
University: AUTÓNOMA DE MADRID [www.uam.es].
Place of defense: DEPARTAMENTO DE BIOLOGÍA MOLECULAR . FACULTAD DE CLENCIAS.
Place of preparation: FACULTAD DE CIENCIAS.

Author: LLEDÓ BOSCH M. BELÉN.
Year: 2004.
University: ALICANTE [www.ua.es].
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: FACULTAD DE CIENCIAS.

Summary: Haloferax mediterranei is a microorganism halófilo extreme category within the domain Archaea. This mircroorganismo is able to grow in minimal medium with glucose as the sole source of carbon and nitrate or nitrite as a sole source of nitrogen. Hfx. Mediterranei manifest a high versarilidad fisológica. When the oxygen availability in the environment is low or zero, this archaeon is able to grow using NO3-como acceptor terminalde the electron transport chain. Despite the recent number of genomes sequenced so far there is no identified gene involved in the uptake of NO3 in archaea. In this work, for the first time for a body of Archaea, has been obtained, sequenced and analyzed the genes for Nas (nasA), NarK (nasB), MobA (nasC) and Nir (nasD). These genes are organized in an operon containing genes nasABC and another monocistrónico. NasD, which are controlled by different promoters. The regulation of the expression level is controlled by transcriptional general systems, which correspond to the availability of a source of nitrogen and preferential specifically controlled, which responds to the availability of NO3-/NO2-. In order to test the usefulness of the heterologous expression of enzymes halophilic to get high amounts of protein were used Nir - Hfx. Mediterranei. The purification and characterization of the enzyme recombianate revealed that their properties are virtually identical to the native enzyme. To complete molecular studies of metabolism NO3-en Hfx. Mediterranei have been sequenced and analyzed the genes involved in the breath of NO3 whose characteristics are those of agencies desnitrificantes. It has been found that transcriptional regulation occurs at being the absence of O2 and the presence of NO3 activators of expression. We have purified and characterized the Nar from cells cultured in conditions microaerófilas and presence of NO3. These results suggest that the enzyme that we have characterized a nitrate recuctasa respiratory membrane.

Author: SALVÀ VILA LLUÍS.
Year: 2004.
University: GIRONA [www.udg.es].
Place of defense: Universidad de Girona.
Place of preparation: UNIVERSIDAD DE GIRONA.

Summary: SUMMARY This thesis focuses on the functional characterization of a heat shock protein from low molecular weight (Small Heat Shock Protein-sHSP) class I to cork corchero when it comes to the ability to protect cells from stress and stabilizing the membranes. The sHsps are proteins that are expressed under conditions of cellular stress. Although some functional aspects of sHsps are known, our work provides new information about the role of the various regions of the protein, in particular the N-terminal region. The specific objective of this work is to determine the role termoprotectora of QsHsp17.4-CI a bacterial model and analyze the importance of the different regions of the protein in that role. This has been partially designed two proteins derived from QsHsp17.4-CI: one that lacks the N-terminal region (C105) and another with virtually the entire domain -cristalino deleccionado (N61) and a third resulting from QsHsp10-CI, which lack the half domain -cristalino (Hsp10). It also examines the potential capacity of stabilizing the membrane and the ability to modify the expression of other Hsps when expressed in a heterologous. Our results demonstrate that the expression of QsHsp17.4-CI protects cells E.coli of heat stress and that the N-terminal region and the consensus region II domain -cristalino are essential for the protection function. Referring to a possible role in the membranes, subcellular localization studies show that QsHsp17.4-CI colocaliza with the membranous portion and that the N-terminal region is necessary for such colocalización. However, it has not been able to prove that the location on the membrane is associated with a protective effect of this: in any case sobreexpressión of proteins alters the composition of fatty acids and only N61, which has no action termoprotectora, alters the state physico the membrane. In studies of de novo expression in E. coli is noted that, unlike other heterologous proteins, N61 activates the expression of the majority of Hsps of E.coli suggesting a possible link between the physical state of the membrane i activation the response to stress. In summary, this study we tested the protective capacity of QsHsp17.4-CI contributing new results on the importance of the N-terminal region i consensus region II domain -cristalino in this function. On the other hand, it is suggested that QsHsp17.4-CI could interact with the membrane of E. coli and that the N-terminal region would be essential for such interaction. Finally, we find that the proteins that cause variations in the state of fluidity of the membrane can trigger the heat shock response by the bacterial cell.

Author: BADIA MARTINEZ DANIEL.
Year: 2004.
University: POLITÉCNICA DE CATALUÑA [www.upc.edu].
Place of defense: Aula B2 de l'ETSEIB.
Place of preparation: ETSEIB, EDIFICI H PLANTA 4 Campus SUD.

Summary: SUMMARY: This work describes the structural determination by X-ray crystallography of the phi29 bacteriophage protein p4, both bound and unbound to DNA. Protein p4 is essential for the bateriophage infective step, as it regulates its genes transcription, thus establishing two clearly differentiated transcriptional phases. This allows temporal separation of the phage protein synthesis: the proteins involved in phage DNA replication are synthesized first and the structural proteins that form the bacteriophage particle later. There are three p4 functional binding sites in phi29 genome, which are located in a 219bp long intergenic region where the three most important promoters (A2b, A2c and A3) are located. In p4 absence, A2b and A2c promoters are active, while A3 promoter, due to not having a -35 box in its sequence, is unable of binding RNA polymerase and remains inactive. When p4 protein is synthesized, it binds to its binding sites, thus producing different effects depending on the promoter. In A2b promoter, protein p4 binding site encloses the promoter -35 box, thus preventing RNA polymerase binding and transcription consequently. In A2c promoter, although protein p4 enhances RNA polymerase binding, the enzyme is unable to perform its function because the complex is overstabilized and so RNA polymerase cannot escape from the promoter. In A3 promotor, protein p4 recruits RNA polymerase, thus allowing transcription initiation. Protein p4 was crystallized in two different space groups, C2221 in presence of a platinum compost and P212121 in its absence. Phases were obtained by MAD, collecting 3 data sets at different wavelengths to have differences in the symmetric reflections intensities due to the effect as an anomal dispersor of the platinium. The protein model was refined up to 3 à in the C2221 space group, and this model was used to perform a molecular replacement in the P212121 crystal. In this former space group, the model could be refined up to 2 à . The complex formed by protein p4 and DNA was crystallized in P2 space group, with one complex per asymmetric unit. Molecular replacement was used for phase determination, taking a protein p4 dimer and an ideal DNA fragment in B conformation as a searching model. P4 is a 125 aminoacid long protein that binds DNA as a dimer. P4 binding to the DNA induces a 70º curvature on it. This binding is specific at the n-terminal region and unspecific at the dimerization area. The contacts with the phosphate backbone of the DNA fix the minor groove of the DNA molecule in a narrowed conformation.

Author: MARTIN ENCABO SUSANA.
Year: 2004.
University: SALAMANCA [www.usal.es].
Place of defense: FACULTAD DE BIOLOGIA.
Place of preparation: INSTITUTO DE BIOLOGIA MOLECULAR Y CELULAR DEL CANCER.

Summary: My work has been based on characterizing the suppressive effect of C3G on the transformation induced by the oncogene Hras. The protein C3G is an interchange of guanine nucleotide (GEF) of Rap1 and R-Ras possessing functional domains counterparts in other GEFs the Ras family, as are the domains CDC25 (or catalytic region), REM and mastery rich prolinas SH3-b (or binding to Crk) (Tanaka et al., 1994). Despite that Rap1 is an antagonist of the transformation induced by K - Ras (Sakoda et al., 1992), C3G is able to reduce the number of foci induced Hras, c-Sis, v-raf cells NIH 3T3 regardless the activation of Rap1 (Guerrero et al., 1998). We have identified that the role suppressor C3G is associated with the domain SH3-by suppresses the transformation induced by several oncogenes as Dbl and R-ras belonging to a number of signaling pathways that determines its overall effect. In particular, the mechanism of suppression exercised by C3G on the Ras-induced transformation is based on reducing the levels of phosphorylated ERK, possibly via activation of phosphatases associated, as it does not alter the activation state of the other members who are over ERK-path Ras-ERK. Also overexpression of C3G reduces the levels of expression of ciclina A, possibly resulting in a reduction in the levels of phosphorylated Rb and less ability to grow independent anchorage to the substrate. Furthermore, the overexpression of C3G also inhibits route PI3K-Akt, but this effect is not related to the suppressor activity on the transformation of Ras. C3G is located in the Golgi and the subcortical region of the actin cytoskeleton, being in the latter region where subcellular possibly C3G exercises its suppressive effect on the transformation oncogénica, perhaps through its interaction with the actin. Levels of C3G present in the cell determines patterns of expression of groups of genes related to the synthesis of extracellular matrix (Adamts5, Lama4, Msln, Ltpb1) and the regulation of the cell cycle (Rbp1, Nmyc, Trib1).

Author: SANZ VICENTE MARIA.
Year: 2004.
University: SALAMANCA [www.usal.es].
Place of defense: FACULTAD DE FARMACIA.
Place of preparation: DEPARTAMENTO DE MICROBIOLOGIA Y GENTICA.

Summary: The fungal cell wall is exoskeletons of fungi and yeast, determining their morphology and participating in numerous cellular processes. Being exclusive fungi and essential for their survival, represents an excellent target for the design of antifungals selective toxicity. The chitin is a polymer minority of the wall but is a structural element essential to the survival fungal. Their synthesis is carried out by the activities Quitín Sintasas (QS). This work focuses on the study of the molecular mechanisms involved in regulating the activity QSIII in the yeast Saccharomyces cerevisiae and in the pathogenic fungus Candida albicans. We have demonstrated that in S. Cerevisiae protein Bni4p is required for the localization of early complex QSIII in the neck by ensuring the proper assembly of the ring chitin, but also develops additional functions related to the assembly of cell septum. The protein Shc1p has been identified as an activator of QSIII specific sporulation, remains essential for the proper maturation of ascosporas through its participation in the synthesis of the layer of chitosan. It is therefore functionally redundant with Chs4p, the trigger / locator the QSIII during vegetative growth. Both are differentially regulated by a combination of mechanisms and transcripcionales postraduccionales that allow not match temporarily in the cell. C. Albicans has a counterpart functional Chs7p, key regulator of the activity QSIII. This implies a functional conservation in the regulation of QSIII. The study has shown that this gene in C. Albicans chitin QSIII-dependiente is not required for the growth or miceliación, but plays an essential role in maintaining the integrity of the cell wall. This integrity is necessary for the proper growth and solid substrates for virulence in murine models.

Author: PRIETO SANCHEZ ROSARIO MARIA.
Year: 2004.
University: SALAMANCA [www.usal.es].
Place of defense: CENTRO DE INVESTIGACION DEL CANCER.
Place of preparation: CENTRO DE INVESTIGACION DEL CANCER.

Summary: The family of GTPasas Rho / Rac is involved in the generation of intracellular responses coordinated after extracellular stimuli. This family is mainly involved in the regulation of actin polymerization of the cytoskeleton, but also in other cellular processes such as gene expression, cell survival and routes endocytosis. This work has focused on the structural and functional characterization of a new member of this family, GTPasa RhoG in cellular signaling processes. First, we have demonstrated that, despite the high structural similarity of RhoG with GTPasa Rac1, signaling mediated by each of these proteins are transmitted via routes functionally independent, however, have common elements. We have also shown that the behavior of both differential GTPasas studied in cellular functions (activation of PAK1, transforming ability and subcellular localization) is controlled by specific regions of the GTPasas and, what is more, for specific amino acids within these regions. Surprisingly, these wastes are located outside the SwitchI and SwitchII, regions that have been traditionally associated with the interaction of GTPasas Rho / Rac their effector proteins. Secondly, we have shown that RhoG participates in the path endocítica mediated caveolas. Thus, RhoG is translocated to the plasma membrane initially, then to intracellular vesicles and finally to the Golgi apparatus following treatment of cells with activators route caveolar (as CTxB). This translocation is linked to changes in the activation state of GTPasa and depends on the integrity of lipid rafts in the cell membrane as the functionality of the dinamina. The constitutive activation of RhoG (but not Rac1) promotes the internalization of caveolas the membrane and blocked traffic after the Golgi apparatus. Besides, the regulatory role of RhoG on endocytosis caveolar is specific to this GTPasa. On the other hand, the generation of mutations in the effector domain of RhoG produce radical changes in the structure of vesicles endocíticas, indicating that their integrity depends on the activation of specific effectors. In any case, the expression of an interfering RNA to RhoG shows that this GTPasa is not essential to the integrity of the route mediated endocytosis by caveolas.

Author: VEGA MORENO FRANCISCO MANUEL.
Year: 2004.
University: SALAMANCA [www.usal.es].
Place of defense: CENTRO DE INVESTIGACION DEL CANCER.
Place of preparation: CENTRO DE INVESTIGACION DEL CANCER.

Summary: VRK1 is the prototype member of the family of kinases of serina-treonina human VRK (Vaccinia-related kinases), kinase with homology to the virus vaccinia B1R, essential for the replication of viral DNA. VRK1 is a kinase widely expressed in various tumor cell lines both as normal and throughout the cell cycle. Through experiments cotransfección show that VRK1 induce the nuclear accumulation and transcriptional activity of p53. This stabilization is due to an effect postraduccional on p53 and dependent on the kinase activity of VRK1 although independent of the action of the kinase "checkpoint" Chk2. VRK1 phosphorylated in Vitro to p53 in the threonine - 18 in the region of binding to Mdm2 and the only phosphorylation of p53 by the kinase disrupts the interaction between these two proteins in Vitro. VRK1 us phosphorylated to Mdm2. In vivo effect of VRJ1 on p53 is at least partly independent of Mdm2. The overexpression of VRK1 promotes phosphorylation at threonine - 18 p53 and its acetylation by coactivador p300, which could explain the increase in transcriptional activity. The suppression of the expression of VRK1 line phenotype of human causes a decrease in proliferation. The protein VRK1 is found at different levels in tumors, and high levels of the kinase correlated with high levels of p53 gene and response to p53 in some tumors, as well as some typical markers of proliferation. The family VRK is also regulated in the murine hematopoietic development.

Author: SABIO BUZO GUADALUPE.
Year: 2004.
University: EXTREMADURA [www.unex.es].
Place of defense: CACERES.
Place of preparation: FACULTAD DE VETERINARIO.

Author: POVEDA GABALDÓN ANA MARÍA.
Year: 2005.
University: VALENCIA [www.uv.es].
Place of defense: BIBLIOTECA DEL CAMPUS DE BURJASSOT.
Place of preparation: FACULTAD DE CIENCIAS BIOLÓGICAS.

Summary: In eukaryotic cells DNA is associated with different types of proteins, empaquetándose in an organized structure, called chromatin. The basic unit of chromatin is the nucleosoma, which consists of 147 bp DNA assembled around a octámero of histones, consisting of two copies of each of the histone, H2A, H2B, H3 and H4, Each histone contains two functional reasons The reason histone fold involved in interactions histona-histona and also interactions histona-DNA, and the tails of the extremes NH2-terminal, which is susceptible to postraduccionales modifications, such as acetylation, methylation, phosphorylation, ubiquitinación and sumoilación. These queues are also involved in the interaction with nucleosomas adjacent. In addition to the internal histones, of eukaryotic chromatin also contains the histone H1, which is available on the outside of nucleosoma, and other non-histone proteins as HMGs (High Mobility Group). The acetylation of histone modifications is one of the most postraduccionales that has been studied. This modification is done by complex hlstona acetyltransferase (HA Ts), and was reversed by complex histone desacetilasa (HDs). Based on the subcellular localization, the preference of histone and the ability to modify histone nucleosomales, the complex HAS T natives have been divided into two groups. The type A HAT enzymes are enzymes nuclear capable of acetilar hlstonas assembled into chromatin, and that frequently are associated with processes of regulation of transcription. The enzymes HAS T type B are primarily cytoplasmic localization and only acetilan histone free, presumably before his deposition in the DNA to form nucleosomas. In yeast described the presence of a HAT type B [Ruiz-García et al., 1998], which was isolated in the fraction citoplásmlca and had a molecular mass of 200 kDa. This enzyme acetila specifically the histone H4 free, but is unable to acetilar nucleosomas, and comprises at least by the catalytic Hat1p and subunit by Hat2p [Lopez-Rodas et al. , 1991, Kleff et al., 1995 AND Parthun et al, 1996]. This work has identified biochemically protein Hif1 (43.3 kDa, 385 aminoácldos), as a subunit of the enzyme B is not identified so far. Further studies were conducted subcellular localization, showing that the protein complex HAT-B are mainly in the nucleus. Similarly, we find a dependence on the subcellular localization of Hat1p with the presence of Hat2p. In this paper we show that the complex HAS TB acetila a soluble fraction of H4 not assembled into chromatin, the lisinas 12 and 5. When this soluble fraction of H4 accumulates for some reason, it is degraded by a path-dependent protein Rad53, a protein kinase of checkpoint. The acetylation of the histone H4 is dependent protein Hat1 and Hat2, but not subunit Hif1p, which does not appear to be involved in the function of acetylation. In addition, both the protein Hat1 as Hat2 are required for the binding of substrate, H4, but Hif1p not involved in this union.