La conservación del patrimonio agroindustrial. Propuestas para la reutilización de los antiguos mataderos municipales y análisis de costes

La creciente demanda de alimentos en las áreas urbanas, junto con los avances en los transportes y la aparición de nuevas fuentes de energía propiciaron a finales del siglo XIX la construcción de diversas industrias agroalimentarias en el medio rural español. Un ejemplo de este patrimonio agroindustrial son los antiguos mataderos municipales, edificados en un gran número de localidades de la geografía española con el objetivo de evitar sacrificios clandestinos de ganado y de mejorar las condiciones higiénicas de la carne. Las cada vez mayores exigencias técnicas y sanitarias para este tipo de construcciones a partir de la década de los 70 y la progresiva sustitución de las instalaciones municipales por mataderos privados más modernos y de mayor capacidad provocaron el cierre y posterior abandono de muchos de estos edificios en las décadas siguientes. En el presen te trabajo se muestran cuatro propuestas concretas para la reutilización de este tipo de construcciones. A partir de la información recopilada de varios proyectos fin de carrera desarrollados por alumnos de la Escuela Técnica Superior de Ingenieros Agrónom os de Madrid, se describen las actuaciones y trabajos de rehabilitación necesarios en cada caso y se ofrece un estudio comparativo de costes entre proyectos de reutilización y obra nueva

Idoneidad del grado de rigidez de las uniones en pórticos metálicos de naves a dos aguas en edificación agroindustrial

Para el análisis de la respuesta estructural, tradicionalmente se ha partido de la suposición de que los nudos son totalmente rígidos o articulados. Este criterio facilita en gran medida los cálculos, pero no deja de ser una idealización del comportamiento real de las uniones. Como es lógico, entre los nudos totalmente rígidos y los nudos articulados existe una gama infinita de valores de rigidez que podrían ser adoptados. A las uniones que presentan un valor distinto de los canónicos se les denomina en sentido general uniones semirrígidas. La consideración de esta rigidez intermedia complica considerablemente los cálculos estructurales, no obstante provoca cambios en el reparto de esfuerzos dentro de la estructura, así como en la configuración de las propias uniones, que en ciertas circunstancias pueden suponer una ventaja económica. Del planteamiento expuesto en el párrafo anterior, surgen dos cuestiones que serán el germen de la tesis. Estas son: ¿Qué ocurre si se aplica el concepto de uniones semirrígidas a las naves industriales? ¿Existen unos valores determinados de rigidez en sus nudos con los que se logra optimizar la estructura? Así, surge el objetivo principal de la tesis, que no es otro que conocer la influencia de la rigidez de los nudos en los costes de los pórticos a dos aguas de estructura metálica utilizados típicamente en edificios de uso agroindustrial. Para alcanzar el objetivo propuesto, se plantea una metodología de trabajo que básicamente consiste en el estudio de una muestra representativa de pórticos sometidos a tres estados de carga: bajo, medio y alto. Su rango de luces abarca desde los 8 a los 20 m y el de la altura de pilares desde los 3,5 a los 10 m. Además, se considera que sus uniones pueden adoptar valores intermedios de rigidez. De la combinatoria de las diferentes configuraciones posibles se obtienen 46.656 casos que serán objeto de estudio. Debido al fin economicista del trabajo, se ha prestado especial atención a la obtención de los costes de ejecución de las diferentes partidas que componen la estructura, incluidas las correspondientes a las uniones. Para acometer los cálculos estructurales ha sido imprescindible contar con un soporte informático, tanto existente como de creación ex profeso, que permitiese su automatización en un contexto de optimización. Los resultados del estudio consisten básicamente en una cantidad importante de datos que para su interpretación se hace imprescindible tratarlos previamente. Este tratamiento se fundamenta en su ordenación sistemática, en la aplicación de técnicas estadísticas y en la representación gráfica. Con esto se obtiene un catálogo de gráficos en los que se representa el coste total de la estructura según los diferentes valores de rigidez de sus nudos, unas matrices resumen de resultados y unos modelos matemáticos que representan la función coste total - rigideces de nudos. Como conclusiones se puede destacar: por un lado que los costes totales de los pórticos estudiados son mínimos cuando los valores de rigidez de sus uniones son bajos, concretamente de 5•10³ a 10•10³ kN•m/rad; y por otro que la utilización en estas estructuras de uniones semirrígidas con una combinación idónea de sus rigideces, supone una ventaja económica media del 18% con respecto a las dos tipologías que normalmente se usan en este tipo de edificaciones como son los pórticos biempotrados y los biarticulados. ABSTRACT Analyzing for structural response, traditionally it started from the assumption that joints are fully rigid or pinned. This criterion makes the design significantly easier, but it is also an idealization of real joint behaviour. As is to be expected, there is an almost endless range of stiffnes value between fully rigid and pinned joints, wich could be adopted. Joints with a value other than traditional are referred to generally as semi-rigid joints. If middle stiffness is considered, the structural design becomes much more complicated, however, it causes changes in the distribution of frame stresses, as well as on joints configuration, that under certain circumstances they may suppose an economic advantage. Two questions arise from the approach outlined in the preceding subparagraph, wich are the seeds of the doctoral thesis. These are: what happens when the concept of semirigid joints is applied to industrial buildings? There are certain stiffness values in their joints with which optimization of frame is achieved? This way, the main objective of the thesis arise, which is to know the influence of stiffness of joints in the cost of the steel frames with gabled roof, that they are typically used in industrial buildings. In order to achieve the proposed goal, a work methodology is proposed, which consists in essence in a study of a representative sample of frames under three load conditions: low, middle and high. Their range of spans comprises from 8 to 20 m and range of the height of columns cover from 3,5 to 10 m. Furthermore, it is considered that their joints can adopt intermediate values of stiffness. The result of the combination of different configurations options is 46.656 cases, which will be subject of study. Due to the economic aim of this work, a particular focus has been devoted for obtaining the execution cost of the different budget items that make up the structure, including those relating to joints. In order to do the structural calculations, count with a computing support has been indispensable, existing and created expressly for this purpose, which would allows its automation in a optimization context. The results of the study basically consist in a important amount of data, whose previous processing is necesary. This process is based on his systematic arrangement, in implementation of statistical techniques and in graphical representation. This give a catalogue of graphics, which depicts the whole cost of structure according to the different stiffness of its joints, a matrixes with the summary of results and a mathematical models which represent the function whole cost - stiffness of the joints. The most remarkable conclusions are: whole costs of the frames studied are minimum when the stiffness values of their joints are low, specifically 5•10³ a 10•10³ kN•m/rad; and the use of structures with semi-rigid joints and a suitable combination of their stiffness implyes an average economic advantage of 18% over other two typologies which are used typically in this kind of buildings; these are the rigid and bi-articulated frames.

Replacement of the proximal heme thiolate ligand in chloroperoxidase with a histidine residue

Chloroperoxidase is a versatile heme enzyme which can cross over the catalytic boundaries of other oxidative hemoproteins and perform multiple functions. Chloroperoxidase, in addition to catalyzing classical peroxidative reactions, also acts as a P450 cytochrome and a potent catalase. The multiple functions of chloroperoxidase must be derived from its unique active site structure. Chloroperoxidase possesses a proximal cysteine thiolate heme iron ligand analogous to the P450 cytochromes; however, unlike the P450 enzymes, chloroperoxidase possesses a very polar environment distal to its heme prosthetic group and contains a glutamic acid residue in close proximity to the heme iron. The presence of a thiolate ligand in chloroperoxidase has long been thought to play an essential role in its chlorination and epoxidation activities; however, the research reported in this paper proves that hypothesis to be invalid. To explore the role of Cys-29, the amino acid residue supplying the thiolate ligand in chloroperoxidase, Cys-29 has been replaced with a histidine residue. Mutant clones of the chloroperoxidase genome have been expressed in a Caldariomyces fumago expression system by using gene replacement rather than gene insertion technology. C. fumago produces wild-type chloroperoxidase, thus requiring gene replacement of the wild type by the mutant gene. To the best of our knowledge, this is the first time that gene replacement has been reported for this type of fungus. The recombinant histidine mutants retain most of their chlorination, peroxidation, epoxidation, and catalase activities. These results downplay the importance of a thiolate ligand in chloroperoxidase and suggest that the distal environment of the heme active site plays the major role in maintaining the diverse activities of this enzyme.

Measures of residue density in protein structures

A hierarchy of residue density assessments and packing properties in protein structures are contrasted, including a regular density, a variety of charge densities, a hydrophobic density, a polar density, and an aromatic density. These densities are investigated by alternative distance measures and also at the interface of multiunit structures. Amino acids are divided into nine structural categories according to three secondary structure states and three solvent accessibility levels. To take account of amino acid abundance differences across protein structures, we normalize the observed density by the expected density defining a density index. Solvent accessibility levels exert the predominant influence in determinations of the regular residue density. Explicitly, the regular density values vary approximately linearly with respect to solvent accessibility levels, the linearity parameters depending on the amino acid. The charge index reveals pronounced inequalities between lysine and arginine in their interactions with acidic residues. The aromatic density calculations in all structural categories parallel the regular density calculations, indicating that the aromatic residues are distributed as a random sample of all residues. Moreover, aromatic residues are found to be over-represented in the neighborhood of all amino acids. This result might be attributed to nucleation sites and protein stability being substantially associated with aromatic residues.

A glutamate residue in the catalytic center of the yeast chorismate mutase restricts enzyme activity to acidic conditions

Chorismate mutase acts at the first branchpoint of aromatic amino acid biosynthesis and catalyzes the conversion of chorismate to prephenate. Comparison of the x-ray structures of allosteric chorismate mutase from the yeast Saccharomyces cerevisiae with Escherichia coli chorismate mutase/prephenate dehydratase suggested conserved active sites between both enzymes. We have replaced all critical amino acid residues, Arg-16, Arg-157, Lys-168, Glu-198, Thr-242, and Glu-246, of yeast chorismate mutase by aliphatic amino acid residues. The resulting enzymes exhibit the necessity of these residues for catalytic function and provide evidence of their localization at the active site. Unlike some bacterial enzymes, yeast chorismate mutase has highest activity at acidic pH values. Replacement of Glu-246 in the yeast chorismate mutase by glutamine changes the pH optimum for activity of the enzyme from a narrow to a broad pH range. These data suggest that Glu-246 in the catalytic center must be protonated for maximum catalysis and restricts optimal activity of the enzyme to low pH.

Identification by mass spectrometry of the phosphorylated residue responsible for activation of the catalytic domain of myosin I heavy chain kinase, a member of the PAK/STE20 family

Myosin I heavy chain kinase from Acanthamoeba castellanii is activated in vitro by autophosphorylation (8–10 mol of P per mol). The catalytically active C-terminal domain produced by trypsin cleavage of the phosphorylated kinase contains 2–3 mol of P per mol. However, the catalytic domain expressed in a baculovirus–insect cell system is fully active as isolated without autophosphorylation in vitro. We now show that the expressed catalytic domain is inactivated by incubation with acid phosphatase and regains activity upon autophosphorylation. The state of phosphorylation of all of the hydroxyamino acids in the catalytic domain were determined by mass spectrometry of unfractionated protease digests. Ser-627 was phosphorylated in the active, expressed catalytic domain, lost its phosphate when the protein was incubated with phosphatase, and was rephosphorylated when the dephosphorylated protein was incubated with ATP. No other residue was significantly phosphorylated in any of the three samples. Thus, phosphorylation of Ser-627, which is in the same position as the Ser and Thr residues that are phosphorylated in many other kinases, is necessary and sufficient for full activity of the catalytic domain. Ser-627 is also phosphorylated when full-length, native kinase is activated by autophosphorylation.

A conserved residue in the tip proteins of CS1 and CFA/I pili of enterotoxigenic Escherichia coli that is essential for adherence

Enterotoxigenic Escherichia coli associated with human diarrheal disease utilize any of a limited group of serologically distinguishable pili for attachment to intestinal cells. These include CS1 and CFA/I pili. We show here that chemical modification of arginyl residues in CS1 pili abolishes CS1-mediated agglutination of bovine erythrocytes, which serves as a model system for attachment. Alanine substitution of the single arginyl residue in CooA, the major pilin, had no effect on the assembly of pili or on hemagglutination. In contrast, substitution of alanine for R181 in CooD, the minor pilin associated with the pilus tip, abolished hemagglutination, and substitution of R20 reduced hemagglutination. Neither of these substitutions affected CS1 pilus assembly. This shows that CooD is essential for CS1-mediated attachment and identifies specific residues that are involved in receptor binding but not in pilus assembly. In addition to mediating agglutination of bovine erythrocytes, CFA/I also mediates agglutination of human erythrocytes. Substitution of R181 by alanine in the CooD homolog, CfaE, abolished both of these reactions. We conclude that the same region of the pilus tip protein is involved in adherence of CS1 and CFA/I pili, although their receptor specificities differ. This suggests that the region of the pilus tip adhesin protein that includes R181 might be an appropriate target for therapeutic intervention or for a vaccine to protect against human diarrhea caused by enterotoxigenic E. coli strains that have serologically different pili.

Solution structure of a 142-residue recombinant prion protein corresponding to the infectious fragment of the scrapie isoform

The scrapie prion protein (PrPSc) is the major, and possibly the only, component of the infectious prion; it is generated from the cellular isoform (PrPC) by a conformational change. N-terminal truncation of PrPSc by limited proteolysis produces a protein of ≈142 residues designated PrP 27–30, which retains infectivity. A recombinant protein (rPrP) corresponding to Syrian hamster PrP 27–30 was expressed in Escherichia coli and purified. After refolding rPrP into an α-helical form resembling PrPC, the structure was solved by multidimensional heteronuclear NMR, revealing many structural features of rPrP that were not found in two shorter PrP fragments studied previously. Extensive side-chain interactions for residues 113–125 characterize a hydrophobic cluster, which packs against an irregular β-sheet, whereas residues 90–112 exhibit little defined structure. Although identifiable secondary structure is largely lacking in the N terminus of rPrP, paradoxically this N terminus increases the amount of secondary structure in the remainder of rPrP. The surface of a long helix (residues 200–227) and a structured loop (residues 165–171) form a discontinuous epitope for binding of a protein that facilitates PrPSc formation. Polymorphic residues within this epitope seem to modulate susceptibility of sheep and humans to prion disease. Conformational heterogeneity of rPrP at the N terminus may be key to the transformation of PrPC into PrPSc, whereas the discontinuous epitope near the C terminus controls this transition.

Detection of residue contacts in a protein folding intermediate

Protein folding can be described in terms of the development of specific contacts between residues as a highly disordered polypeptide chain converts into the native state. Here we describe an NMR based strategy designed to detect such contacts by observation of nuclear Overhauser effects (NOEs). Experiments with α-lactalbumin reveal the existence of extensive NOEs between aromatic and aliphatic protons in the archetypal molten globule formed by this protein at low pH. Analysis of their time development provides direct evidence for near-native compactness of this state. Through a rapid refolding procedure the NOE intensity can be transferred efficiently into the resolved and assigned spectrum of the native state. This demonstrates the viability of using this approach to map out time-averaged interactions between residues in a partially folded protein.

A single serine residue controls the cation dependence of substrate transport by the rat serotonin transporter

The serotonin transporter (SERT) is a member of the Na+/Cl−-dependent neurotransmitter transporter family and constitutes the target of several clinically important antidepressants. Here, replacement of serine-545 in the recombinant rat SERT by alanine was found to alter the cation dependence of serotonin uptake. Substrate transport was now driven as efficiently by LiCl as by NaCl without significant changes in serotonin affinity. Binding of the antidepressant [3H]imipramine occurred with 1/5th the affinity, whereas [3H]citalopram binding was unchanged. These results indicate that serine-545 is a crucial determinant of both the cation dependence of serotonin transport by SERT and the imipramine binding properties of SERT.