Nuevo concepto de elementos estructurales horizontales de vidrio : placa autoportante y pretensada en forma de pi

En los últimos años, podemos darnos cuenta de la importancia que tienen las nuevas aplicaciones de vidrio especialmente en edificios turísticos donde el vértigo juega un papel importante en la visita. Sin embargo los sistemas constructivos no tienen un especial interés porque el vidrio laminado está siempre soportado por otro elemento de acero o incluso vidrio en forma de retícula. En la presente tesis voy a desarrollar una nueva solución de elemento autoportante de vidrio de gran tamaño haciendo seguro el uso del elemento para andar en el aire. El sueño de muchos arquitectos ha sido diseñar un edificio completamente transparente y a mí me gustaría contribuir a este sueño empezando a estudiar un forjado de vidrio como elemento estructural horizontal y para ello debemos cumplir requerimientos de seguridad. Uno de los objetivos es lograr un elemento lo más transparente y esbelto posible para el uso de pasarelas en vestíbulos de edificios. Las referencias construidas son bien conocidas, pero por otro lado Universidades europeas estudian continua estudiando el comportamiento del vidrio con diferentes láminas, adhesivos, apilados, insertos, sistemas de laminado, pretensado, pandeo lateral, seguridad post-rotura y muchos más aspectos necesarios. La metodología llevada a cabo en esta tesis ha sido primeramente diseñar un elemento industrial prefabricado horizontal de vidrio teniendo en cuenta todos los conceptos aprendidos en el estado del arte y la investigación para poder predimensionar el elemento. El siguiente paso será verificar el modelo por medio de cálculo analítico, simulación de elementos finitos y ensayos físicos. Para realizar los ensayos hay un paso intermedio teniendo que cambiar la hipótesis de carga uniforme a carga puntal para realizar el ensayo de flexión a 4 puntos normalizado y además cambiar a escala 1:2 para adaptarse al espacio de ensayo y ser viable económicamente. Finalmente compararé los resultados de tensión y deformación obtenidos por los tres métodos para extraer conclusiones. Sin embargo el problema de la seguridad no ha concluido, tendré que demostrar que el sistema es seguro después de que se produzca la rotura y para ello sólo dispongo de los ensayos como medio de demostración. El diseño es el resultado de la evolución de una viga tipo “I”; cuando es pretensada para obtener más resistencia, aparece el problema de pandeo lateral y éste es solucionado con una viga con sección en “T” cuya unión es resuelta con un cajeado longitudinal en la parte inferior del elemento horizontal. Las alas de éste crecen para recoger las cargas superficiales creando a su vez un punto débil en la unión que a su vez se soluciona duplicando la sección “TT” y haciendo trabajar dicho tablero de forma tan óptima como una viga continua. Dicha sección en vidrio como un único elemento pretensado es algo inédito. Además he diseñado unas escuadras metálicas en los extremos de los nervios como apoyo y placa de pretensión, así como una hendidura curva en el centro de los nervios para alojar los tirantes de acero de modo que al pretensar el tirante la placa corrija al menos la deformación por peso propio. Realizados los cambios geométricos de escala y las simplificaciones en el laminado y el adhesivo se programan la extracción de resultados desde 3 estadios diferentes: Sin pretensión y con pretensión de 750 Kg y de 1000Kg en cada nervio. Por cada estadio y por cada uno de los métodos, cálculo, simulación y ensayos, se extraen los datos de deformación y tensión en el punto medio de un nervio con el objetivo de hacer una comparación de resultados para obtener unas conclusiones, siempre en el campo de la elasticidad. Posteriormente incrementaré la carga hasta el momento de la rotura de la placa y después hasta el colapso teniendo en cuenta el tiempo y demostrando una rotura segura. El vidrio no tendrá un comportamiento plástico pero habrá sido controlado su comportamiento frágil manteniendo una carga y una deformación aceptable. ABSTRACT Over the past few years we have realized the importance of the new technologies regarding the application of glass in new buildings, especially those touristic places were the views and the heights are the reason of the visit. However, the construction systems of these glass platforms are not usually as interesting, because the laminated glass is always held by another steel substructure or even a grid-formed glass element. Throughout this thesis I am going to develop a new solution of a self-bearing element with big dimensions made out of glass, ensuring a safe solution to use as an element to walk on the air. The dream of many architects has been to create a building completely transparent, and I would like to contribute to this idea by making a glass slab as a horizontal structural element, for which we have to meet the security requirements. One of the goals is to achieve an element as transparent and slim as possible for the use in walkways of building lobbies. The glass buildings references are well known, but on the other hand the European Universities study the behaviour of the glass with different interlayers, adhesives, laminating systems, stacking, prestressed, buckling, safety, breakage and post-breakage capacity; and many other necessary aspects. The methodology followed in this thesis has been to first create a horizontal industrial prefabricated horizontal element of glass, taking into account all the concepts learned in the state of art and the investigation to be able to predimension this element. The next step will be to verify this model with an analytic calculus, a finite element modelling simulation and physical tests. To fulfil these tests there is an intermediate step, having to change the load hypothesis from a punctual one to make the test with a four points normalized deflexion, and also the scale of the sample was changed to 1:2 to adapt to the space of the test and make it economically possible. Finally, the results of tension and deformation obtained from the three methods have been compared to make the conclusions. However, the problem with safety has not concluded yet, for I will have to demonstrate that this system is safe even after its breakage, for which I can only use physical tests as a way of demonstration. The design is the result of the evolution of a typical “I” beam, which when it is prestressed to achieve more resistance, the effect of buckling overcomes, and this is solved with a “T” shaped beam, where the union is solved with a longitudinal groove on the inferior part of the horizontal element. The boards of this beam grow to cover the superficial loads, creating at the same time a weak point, which is solved by duplicating the section “TT” and therefore making this board work as optimal as a continuous beam. This glass section as a single prestressed element is unique. After the final design of the “π” glass plate was obtained and the composition of the laminated glass and interlayers has been predimensioned, the last connection elements must be contemplated. I have also designed a square steel shoe at the end of the beams, which will be the base and the prestressed board, as well as a curved slot in the centre of the nerves to accommodate the steel braces so that when this brace prestresses the board, at least the deformation due to its self-weight will be amended. Once I made the geometric changes of the scale and the simplifications on the laminating and the adhesive, the extraction on results overcomes from three different stages: without any pretension, with a pretension of 750 kg and with a pretension of 1000 kg on each rib. For each stage and for each one of the methods, calculus, simulation and tests, the deformation datum were extracted to obtain the conclusions, always in the field of the elasticity. Afterwards, I will increase the load until the moment of breakage of this board, and then until the collapse of the element, taking into account the time spent and demonstrating a safe breakage. The glass will not have a plastic behaviour, but its brittle behaviour has been controlled, keeping an acceptable load and deflection.

Deficient DNA-ligase activity in the metabolic disease tyrosinemia type I

Hereditary tyrosinemia type I (HT1) is an autosomal recessive inborn error of metabolism caused by the deficiency of fumarylacetoacetate hydrolase, the last enzyme in the tyrosine catabolism pathway. This defect results in accumulation of succinylacetone (SA) that reacts with amino acids and proteins to form stable adducts via Schiff base formation, lysine being the most reactive amino acid. HT1 patients surviving beyond infancy are at considerable risk for the development of hepatocellular carcinoma, and a high level of chromosomal breakage is observed in HT1 cells, suggesting a defect in the processing of DNA. In this paper we show that the overall DNA-ligase activity is low in HT1 cells (about 20% of the normal value) and that Okazaki fragments are rejoined at a reduced rate compared with normal fibroblasts. No mutation was found by sequencing the ligase I cDNA from HT1 cells, and the level of expression of the ligase I mRNA was similar in normal and HT1 fibroblasts, suggesting the presence of a ligase inhibitor. SA was shown to inhibit in vitro the overall DNA-ligase activity present in normal cell extracts. The activity of purified T4 DNA-ligase, whose active site is also a lysine residue, was inhibited by SA in a dose-dependent manner. These results suggest that accumulation of SA reduces the overall ligase activity in HT1 cells and indicate that metabolism errors may play a role in regulating enzymatic activities involved in DNA replication and repair.

Nrf2 is essential for protection against acute pulmonary injury in mice

Nrf2 is a member of the “cap ‘n’ collar” family of transcription factors. These transcription factors bind to the NF-E2 binding sites (GCTGAGTCA) that are essential for the regulation of erythroid-specific genes. Nrf2 is expressed in a wide range of tissues, many of which are sites of expression for phase 2 detoxification genes. Nrf2−/− mice are viable and have a normal phenotype under normal laboratory conditions. The NF-E2 binding site is a subset of the antioxidant response elements that have the sequence GCNNNGTCA. The antioxidant response elements are regulatory sequences found on promoters of several phase 2 detoxification genes that are inducible by xenobiotics and antioxidants. We report here that Nrf2−/− mice are extremely susceptible to the administration of the antioxidant butylated hydroxytoluene. With doses of butylated hydroxytoluene that are tolerated by wild-type mice, the Nrf2−/− mice succumb from acute respiratory distress syndrome. Gene expression studies show that the expression of several detoxification enzymes is altered in the Nrf2−/− mice. The Nrf2−/− mice may prove to be a good in vivo model for toxicological studies. As oxidative damage causes DNA breakage, these mice may also be useful for testing carcinogenic agents.

Cisplatin increases meiotic crossing-over in mice

Genetic mapping of traits and mutations in mammals is dependent upon linkage analysis. The resolution achieved by this method is related to the number of offspring that can be scored and position of crossovers near a gene. Higher precision mapping is obtained by expanding the collection of progeny from an appropriate cross, which in turn increases the number of potentially informative recombinants. A more efficient approach would be to increase the frequency of recombination, rather than the number of progeny. The anticancer drug cisplatin, which causes DNA strand breakage and is highly recombinogenic in some model organisms, was tested for its ability to induce germ-line recombination in mice. Males were exposed to cisplatin and mated at various times thereafter to monitor the number of crossovers inherited by offspring. We observed a striking increase on all three chromosomes examined and established a regimen that nearly doubled crossover frequency. The timing of the response indicated that the crossovers were induced at the early pachytene stage of meiosis I. The ability to increase recombination should facilitate genetic mapping and positional cloning in mice.

On the reaction mechanism of l-lactate oxidase: Quantitative structure-activity analysis of the reaction with para-substituted l-mandelates

The rate constants for reduction of the flavoenzyme, l-lactate oxidase, and a mutant (in which alanine 95 is replaced by glycine), by a series of para-substituted mandelates, in both the 2-1H- and 2-2H- forms, have been measured by rapid reaction spectrophotometry. In all cases, significant isotope effects (1H/2H = 3–7) on the rate constants of flavin reduction were found, indicating that flavin reduction is a direct measure of α-C-H bond breakage. The rate constants show only a small influence of the electronic characteristics of the substituents, but show a good correlation when combined with some substituent volume parameters. A surprisingly good correlation is found with the molecular mass of the substrate. The results are compatible with any mechanism in which there is little development of charge in the transition state. This could be a transfer of hydride to the flavin N(5) position or a synchronous mechanism in which the α-C-H is formally abstracted as a H+ while the resulting charge is simultaneously neutralized by another event.

Superoxide-mediated clastogenesis and anticlastogenic effects of exogenous superoxide dismutase

Superoxide-mediated clastogenesis is characteristic for various chronic inflammatory diseases with autoimmune reactions and probably plays a role in radiation-induced clastogenesis and in the congenital breakage syndromes. It is consistently prevented by exogenous superoxide dismutase (SOD), but not by heat-inactivated SOD, indicating that the anticlastogenic effect is related to the catalytic function of the enzyme. Increased superoxide production by activated monocytes/macrophages is followed by release of more long-lived metabolites, so-called clastogenic factors, which contain lipid peroxidation products, unusual nucleotides of inosine, and cytokines such as tumor necrosis factor α. Since these components are not only clastogenic, but can stimulate further superoxide production by monocytes and neutrophils, the genotoxic effects are self-sustaining. It is shown here that anticlastogenic effects of exogenous SOD are preserved despite extensive washing of the cells and removal of all extracellular SOD. Using flow cytometry and confocal laser microscopy, rapid adherence of the fluorescently labeled enzyme to the cell surface could be observed with slow uptake into the cell during the following hours. The degree of labeling was concentration and time dependent. It was most important for monocytes, compared with lymphocytes, neutrophils, and fibroblasts. The cytochrome c assay showed significantly diminished O2− production by monocytes, pretreated with SOD and washed thereafter. The preferential and rapid binding of SOD to monocytes may be of importance not only for the superoxide-mediated genotoxic effects, described above, but also from a therapeutic standpoint. It can explain the observation that beneficial effects of injected SOD lasted for weeks and months despite rapid clearance of the enzyme from the blood stream according to pharmacodynamic studies.

Tyrosine replacement in P-selectin glycoprotein ligand-1 affects distinct kinetic and mechanical properties of bonds with P- and L-selectin

Selectins are adhesion molecules that initiate tethering and rolling of leukocytes on the vessel wall. Rolling requires rapid formation and breakage of selectin–ligand bonds that must have mechanical strength to resist premature dissociation by the forces applied in shear flow. P- and L-selectin bind to the N-terminal region of P-selectin glycoprotein ligand-1 (PSGL-1), a mucin on leukocytes. To define determinants on PSGL-1 that contribute to the kinetic and mechanical properties of bonds with selectins, we compared rolling of transfected preB cells expressing P- or L-selectin on transfected cell monolayers expressing wild-type PSGL-1 or PSGL-1 constructs with substitutions in targeted N-terminal residues. Rolling through P- or L-selectin required a Thr or Ser at a specific position on PSGL-1, the attachment site for an essential O-glycan, but required only one of three nearby Tyr residues, which are sites for Tyr-SO3 formation. The adhesive strengths and numbers of cells rolling through P- or L-selectin were similar on wild-type PSGL-1 and on each of the three PSGL-1 constructs containing only a single Tyr. However, the cells rolled more irregularly on the single-Tyr forms of PSGL-1. Analysis of the lifetimes of transient tethers on limiting densities of PSGL-1 revealed that L-selectin dissociated faster from single-Tyr than wild-type PSGL-1 at all shears examined. In sharp contrast, P-selectin dissociated faster from single-Tyr than wild-type PSGL-1 at higher shear but not at lower shear. Thus, tyrosine replacements in PSGL-1 affect distinct kinetic and mechanical properties of bonds with P- and L-selectin.

Topoisomerase II-mediated site-directed alkylation of DNA by psorospermin and its use in mapping other topoisomerase II poison binding sites

Psorospermin is a plant natural product that shows significant in vivo activity against P388 mouse leukemia. The molecular basis for this selectivity is unknown, although psorospermin has been demonstrated to intercalate into DNA and alkylate N7 of guanine. Significantly, the alkylation reactivity of psorospermin at specific sites on DNA increased 25-fold in the presence of topoisomerase II. In addition, psorospermin trapped the topoisomerase II-cleaved complex formation at the same site. These results imply that the efficacy of psorospermin is related to its interaction with the topoisomerase II–DNA complex. Because thermal treatment of (N7 guanine)–DNA adducts leads to DNA strand breakage, we were able to determine the site of alkylation of psorospermin within the topoisomerase II gate site and infer that intercalation takes place at the gate site between base pairs at the +1 and +2 positions. These results provide not only additional mechanistic information on the mode of action of the anticancer agent psorospermin but also structural insights into the design of an additional class of topoisomerase II poisons. Because the alkylation site for psorospermin in the presence of topoisomerase II can be assigned unambiguously and the intercalation site inferred, this drug is a useful probe for other topoisomerase poisons where the sites for interaction are less well defined.

Nuclear Ferritin Protects DNA From UV Damage in Corneal Epithelial Cells

Previously, we identified the heavy chain of ferritin as a developmentally regulated nuclear protein of embryonic chicken corneal epithelial cells. The nuclear ferritin is assembled into a supramolecular form indistinguishable from the cytoplasmic form of ferritin found in other cell types and thus most likely has iron-sequestering capabilities. Free iron, via the Fenton reaction, is known to exacerbate UV-induced and other oxidative damage to cellular components, including DNA. Since corneal epithelial cells are constantly exposed to UV light, we hypothesized that the nuclear ferritin might protect the DNA of these cells from free radical damage. To test this possibility, primary cultures of cells from corneal epithelium and stroma, and from skin epithelium and stroma, were UV irradiated, and DNA strand breaks were detected by an in situ 3′-end labeling method. Corneal epithelial cells without nuclear ferritin were also examined. We observed that the corneal epithelial cells with nuclear ferritin had significantly less DNA breakage than other cell types examined. Furthermore, increasing the iron concentration of the culture medium exacerbated the generation of UV-induced DNA strand breaks in corneal and skin fibroblasts, but not in the corneal epithelial cells. Most convincingly, corneal epithelial cells in which the expression of nuclear ferritin was inhibited became much more susceptible to UV-induced DNA damage. Therefore, it seems that corneal epithelial cells have evolved a novel, nuclear ferritin-based mechanism for protecting their DNA against UV damage.

Femtosecond observation of benzyne intermediates in a molecular beam: Bergman rearrangement in the isolated molecule

In this communication, we report our femtosecond real-time observation of the dynamics for the three didehydrobenzene molecules (p-, m-, and o-benzyne) generated from 1,4-, 1,3-, and 1,2-dibromobenzene, respectively, in a molecular beam, by using femtosecond time-resolved mass spectrometry. The time required for the first and the second C-Br bond breakage is less than 100 fs; the benzyne molecules are produced within 100 fs and then decay with a lifetime of 400 ps or more. Density functional theory and high-level ab initio calculations are also reported herein to elucidate the energetics along the reaction path. We discuss the dynamics and possible reaction mechanisms for the disappearance of benzyne intermediates. Our effort focuses on the isolated molecule dynamics of the three isomers on the femtosecond time scale.

Rescue of arrested replication forks by homologous recombination

DNA synthesis is an accurate and very processive phenomenon; nevertheless, replication fork progression on chromosomes can be impeded by DNA lesions, DNA secondary structures, or DNA-bound proteins. Elements interfering with the progression of replication forks have been reported to induce rearrangements and/or render homologous recombination essential for viability, in all organisms from bacteria to human. Arrested replication forks may be the target of nucleases, thereby providing a substrate for double-strand break repair enzyme. For example in bacteria, direct fork breakage was proposed to occur at replication forks blocked by a bona fide replication terminator sequence, a specific site that arrests bacterial chromosome replication. Alternatively, an arrested replication fork may be transformed into a recombination substrate by reversal of the forked structures. In reversed forks, the last duplicated portions of the template strands reanneal, allowing the newly synthesized strands to pair. In bacteria, this reaction was proposed to occur in replication mutants, in which fork arrest is caused by a defect in a replication protein, and in UV irradiated cells. Recent studies suggest that it may also occur in eukaryote organisms. We will review here observations that link replication hindrance with DNA rearrangements and the possible underlying molecular processes.

Single-strand interruptions in replicating chromosomes cause double-strand breaks

Replication-dependent chromosomal breakage suggests that replication forks occasionally run into nicks in template DNA and collapse, generating double-strand ends. To model replication fork collapse in vivo, I constructed phage λ chromosomes carrying the nicking site of M13 bacteriophage and infected with these substrates Escherichia coli cells, producing M13 nicking enzyme. I detected double-strand breaks at the nicking sites in λ DNA purified from these cells. The double-strand breakage depends on (i) the presence of the nicking site; (ii) the production of the nicking enzyme; and (iii) replication of the nick-containing chromosome. Replication fork collapse at nicks in template DNA explains diverse phenomena, including eukaryotic cell killing by DNA topoisomerase inhibitors and inviability of recombination-deficient vertebrate cell lines.

DNA replication meets genetic exchange: Chromosomal damage and its repair by homologous recombination

Proceedings of the National Academy of Sciences Colloquium on the roles of homologous recombination in DNA replication are summarized. Current findings in experimental systems ranging from bacteriophages to mammalian cell lines substantiate the idea that homologous recombination is a system supporting DNA replication when either the template DNA is damaged or the replication machinery malfunctions. There are several lines of supporting evidence: (i) DNA replication aggravates preexisting DNA damage, which then blocks subsequent replication; (ii) replication forks abandoned by malfunctioning replisomes become prone to breakage; (iii) mutants with malfunctioning replisomes or with elevated levels of DNA damage depend on homologous recombination; and (iv) homologous recombination primes DNA replication in vivo and can restore replication fork structures in vitro. The mechanisms of recombinational repair in bacteriophage T4, Escherichia coli, and Saccharomyces cerevisiae are compared. In vitro properties of the eukaryotic recombinases suggest a bigger role for single-strand annealing in the eukaryotic recombinational repair.

A eukaryotic enzyme that can disjoin dead-end covalent complexes between DNA and type I topoisomerases.

The covalent joining of topoisomerases to DNA is normally a transient step in the reaction cycle of these important enzymes. However, under a variety of circumstances, the covalent complex is converted to a long-lived or dead-end product that can result in chromosome breakage and cell death. We have discovered and partially purified an enzyme that specifically cleaves the chemical bond that joins the active site tyrosine of topoisomerases to the 3' end of DNA. The reaction products made by the purified enzyme on a variety of model substrates indicate that the enzyme cleanly hydrolyzes the tyrosine-DNA phosphodiester linkage, thereby liberating a DNA terminated with a 3' phosphate. The wide distribution of this phosphodiesterase in eukaryotes and its specificity for tyrosine linked to the 3' end but not the 5' end of DNA suggest that it plays a role in the repair of DNA trapped in complexes involving eukaryotic topoisomerase I.

Diverse transposable elements are mobilized in hybrid dysgenesis in Drosophila virilis.

We describe a system of hybrid dysgenesis in Drosophila virilis in which at least four unrelated transposable elements are all mobilized following a dysgenic cross. The data are largely consistent with the superposition of at least three different systems of hybrid dysgenesis, each repressing a different transposable element, which break down following the hybrid cross, possibly because they share a common pathway in the host. The data are also consistent with a mechanism in which mobilization of a single element triggers that of others, perhaps through chromosome breakage. The mobilization of multiple, unrelated elements in hybrid dysgenesis is reminiscent of McClintock's evidence [McClintock, B. (1955) Brookhaven Symp. Biol. 8, 58-74] for simultaneous mobilization of different transposable elements in maize.