El proyecto de vivienda como laboratorio de estrategias para Sejima y Nishizawa de 1987 a 2010

Como indica el título, esta tesis plantea el estudio de la arquitectura doméstica elaborada por los arquitectos japoneses Kazuyo Sejima y Ryue Nishizawa. Más concretamente, la investigación se ciñe a un conjunto integrado por veinticinco casas que los arquitectos proyectaron entre 1987, momento en el que Sejima establece su propia oficina, y 2010, fecha en la que el reconocimiento del trabajo que ambos venían desarrollando queda certificado a nivel internacional, ya que ese año son galardonados con el premio Pritzker y comisarían la Bienal deVenecia. Del estudio conjunto y sistemático de estos proyectos, y de sus distintas versiones se espera poder obtener una serie de criterios exegéticos que permitan comprenderlos y explicarlos mejor, relacionándolos entre sí de manera coherente. A estos criterios es a lo que hemos denominado estrategias. En el momento en el que se inicia esta investigación se detectó un vacío editorial respecto al tema tratado, apenas había textos, propios o ajenos que abordaran esta faceta de la obra de Sejima y Nishizawa en profundidad. No en vano, algunos críticos han acuñado el apelativo de "arquitectos sin palabras" para referirse, tanto a ellos como a la generación de arquitectos que la pareja encabeza, dando a entender que se trata de una arquitectura carente de explicación.1 Sin embargo, esta investigación sostiene que el hecho de que los arquitectos no hablen en exceso de su obra y mantengan una actitud centrada en desarrollar de forma práctica su trabajo no quiere decir que prescindan de realizar operaciones compositivas altamente idealizadas y abstractas, refrendadas por un marco cultural y teórico que se pueda describir. Tras recopilar, ordenar y analizar un número lo suficientemente amplio como para ser significativo de las manifestaciones verbales que los arquitectos han realizado a lo largo del periodo estudiado, se ha observado que de entre todos los términos que los dos socios que integran SANAA emplean para describir sus proyectos, uno de los más relevantes y quizás también el más general resulta la palabra “sistema”.2 El modo en el que ambos describen los principios de su arquitectura, diferenciando entre los componentes del programa, y las relaciones que se establecen entre éstos, y entre ellos y el exterior permiten sostener que no se trata de un uso accidental término. Paralelamente, al mismo tiempo que se indaga sobre el corpus intelectual de esta teoría se intenta detallar las circunstancias que favorecieron tal trasvase de ideas entre Occidente y Japón y como acabaron llegando al ámbito de estos arquitectos. Al amparo de este marco teórico y tras redibujar las viviendas documentadas, se intentará describir los rasgos de la estructura material de las viviendas analizadas, así como cartografiar los patrones organizativos que las caracterizan. Para ello se empleará el rigor instrumental que aporta la teoría de grafos como método habitual para la representación, estudio y caracterización de sistemas. Los resultados de la investigación evidencian que hay una serie de estrategias — tanto materiales como organizativas— que enunciadas en sus primeros proyectos se van desarrollando en obras posteriores, conformado sistemas que están paulatinamente más organizados. Y que llegado un punto en la trayectoria de estos arquitectos, se observa que tales estrategias, se van superponiendo de distinta forma en diversos proyectos, por lo que es posible agruparlos y hablar de ellos atendiendo a características comunes. Finalmente, el estudio concluye que tanto a nivel material como reladonal, bien podría decirse que en el periodo estudiado, las estrategias empleadas por Sejima y Nishizawa para elaborar su arquitectura doméstica persiguen un objetivo común que se fundamenta en la elaboración de planteamientos sintéticos que les permiten explorar y responder creativamente ante las disyuntivas previamente establecidas, precisamente explotando el potencial de las paradojas que las originan. ABSTRACT As the title suggest itself, this thesis deals about the study of domestic architecture developed by Japanese architects Kazuyo Sejima and Ryue Nishizawa. More specifically, research focus its attention on a group of twenty five houses that both architects projected from 1987, when Sejima establishes her own practice, and 2010, as the moment in which their work obtains international acknowledgment, since this year they are awarded the Pritzker prize and cúrate the Venice Biennale. From the combined and systematic study of all these projects, and their different versions are expected to obtain a series of exegetical criteria to relate to each other, understand and explain better. These criteria are what we cali strategies. By the time when this research began, an editorial emptiness about the treaty issue was detected; there were barely texts that addressed this aspect of the work of Sejima and Nishizawa in depth, neither the ones written by the architects themselves ñor by other authors. Some critics have coined the ñame "wordless generation" to refer to both them as to the generation of architects that the couple leads, implying that it is an architecture devoid of explanation.3 However, this study argües that the fact that architects do not speak too much about his work and keep themselves focused on developing practical work attitude does not mean that dispense perform highly idealized and abstract compositional operations, fueled by a frame cultural and theoretical that can be described. After collect, sort and analyze a large enough number of verbal statements done by the architects about their work as to be meaningful, it was observed that of all the terms that the two partners that intégrate SANAA used to describe their projects, one of the most important and, perhaps one of the most general, is the word "system".4 The way in which both describe the principies of their architecture distinguishing between program components and the relationships established between them, and between them and the outside allow us the view that it is not accidental use of a term. Similarly, while it investigates the intellectual corpus of this theory it attempts to explain some of the circumstances that favored such transfer of ideas between the West and Japan and how eventually reaching the scope of these architects. Under this framework and after redraw the documented houses we attempt to describe the characteristics of the material structure of the projects tested, as well as mapping the organizational patterns that characterize them. For this, we use the instrumental rigor that brings graph theory, as a regular method of representation, study and characterization of systems used. The research results show that there are a number of strategies -both material and organizational level- that once they are set out in its first projects are developed in later works. Bringing up systems that are gradually more and more organized. And at one point in the career of these architects, such strategies are observed, they are superimposed differently on various projects, making it possible to group them and discuss them according to common characteristics. Finally, the study condueles that both materially and organizational it could be said that in the period studied, the strategies employed by Sejima and Nishizawa to develop its domestic architecture pursue a common goal, which is based on the development of synthetic approaches that allow them explore and respond creatively to the previously established dilemmas precisely exploiting the paradoxical potential that originates them

Protein-Protein Interactions within Late Pre-40S Ribosomes

Ribosome assembly in eukaryotic organisms requires more than 200 assembly factors to facilitate and coordinate rRNA transcription, processing, and folding with the binding of the ribosomal proteins. Many of these assembly factors bind and dissociate at defined times giving rise to discrete assembly intermediates, some of which have been partially characterized with regards to their protein and RNA composition. Here, we have analyzed the protein-protein interactions between the seven assembly factors bound to late cytoplasmic pre-40S ribosomes using recombinant proteins in binding assays. Our data show that these factors form two modules: one comprising Enp1 and the export adaptor Ltv1 near the beak structure, and the second comprising the kinase Rio2, the nuclease Nob1, and a regulatory RNA binding protein Dim2/Pno1 on the front of the head. The GTPase-like Tsr1 and the universally conserved methylase Dim1 are also peripherally connected to this second module. Additionally, in an effort to further define the locations for these essential proteins, we have analyzed the interactions between these assembly factors and six ribosomal proteins: Rps0, Rps3, Rps5, Rps14, Rps15 and Rps29. Together, these results and previous RNA-protein crosslinking data allow us to propose a model for the binding sites of these seven assembly factors. Furthermore, our data show that the essential kinase Rio2 is located at the center of the pre-ribosomal particle and interacts, directly or indirectly, with every other assembly factor, as well as three ribosomal proteins required for cytoplasmic 40S maturation. These data suggest that Rio2 could play a central role in regulating cytoplasmic maturation steps

Structure of the pre-60S ribosomal subunit with nuclear export factor Arx1 bound at the exit tunnel

Pre-ribosomal particles evolve in the nucleus through transient interaction with biogenesis factors, before export to the cytoplasm. Here, we report the architecture of the late pre-60S particle purified from Saccharomyces cerevisiae through Arx1, a nuclear export factor with structural homology to methionine aminopeptidases, or its binding partner Alb1. Cryo-electron microscopy reconstruction of the Arx1-particle at 11.9 Å resolution reveals regions of extra densities on the pre-60S particle attributed to associated biogenesis factors, confirming the immature state of the nascent subunit. One of these densities could be unambiguously assigned to Arx1. Immuno-electron microscopy and UV cross-linking localize Arx1 close to the ribosomal exit tunnel in direct contact with ES27, a highly dynamic eukaryotic rRNA expansion segment. The binding of Arx1 at the exit tunnel may position this export factor to prevent premature recruitment of ribosome-associated factors active during translation

The initial U3 snoRNA:pre-rRNA base pairing interaction required for pre-18S rRNA folding revealed by in vivo chemical probing

The synthesis of ribosomal subunits in the nucleolus is a conserved, essential process that results in cytoplasmic ribosomes with precisely processed and folded rRNAs assembled with ribosomal proteins. It has been proposed, but never directly demonstrated, that the U3 small nucleolar RNA (snoRNA), a nucleolar component required for ribosome biogenesis, is a chaperone for pre-18S rRNA folding. To test this, we used in vivo chemical probing with dimethyl sulfate to detect changes in pre-rRNA structure upon genetic manipulation of the yeast, Saccharomyces cerevisiae. Based on changes in nucleotide reactivity, we found that the U3 snoRNA is indeed required for folding of the pre-18S rRNA. Furthermore, we detected a new essential base pairing interaction that is likely the initial anchor that recruits the U3 snoRNA to the pre-rRNA, is a prerequisite for the subsequent interactions, and is required for the small subunit processome formation. Substitution of the 5′-ETS nucleotides of the pre-rRNA involved in this initial base pairing interaction is lethal, but growth is restored when a complementary U3 snoRNA is expressed. The U3 snoRNP, via base pairing, and its associated proteins, are part of the required machinery that orchestrates the folding of pre-rRNA that results in the assembly of the small ribosomal subunit

Transcriptome-wide RNA processing kinetics revealed using extremely short 4tU labeling

Background: RNA levels detected at steady state are the consequence of multiple dynamic processes within the cell. In addition to synthesis and decay, transcripts undergo processing. Metabolic tagging with a nucleotide analog is one way of determining the relative contributions of synthesis, decay and conversion processes globally. Results: By improving 4-thiouracil labeling of RNA in Saccharomyces cerevisiae we were able to isolate RNA produced during as little as 1 minute, allowing the detection of nascent pervasive transcription. Nascent RNA labeled for 1.5, 2.5 or 5 minutes was isolated and analyzed by reverse transcriptase-quantitative polymerase chain reaction and RNA sequencing. High kinetic resolution enabled detection and analysis of short-lived non-coding RNAs as well as intron-containing pre-mRNAs in wild-type yeast. From these data we measured the relative stability of pre-mRNA species with different high turnover rates and investigated potential correlations with sequence features. Conclusions: Our analysis of non-coding RNAs reveals a highly significant association between non-coding RNA stability, transcript length and predicted secondary structure. Our quantitative analysis of the kinetics of pre-mRNA splicing in yeast reveals that ribosomal protein transcripts are more efficiently spliced if they contain intron secondary structures that are predicted to be less stable. These data, in combination with previous results, indicate that there is an optimal range of stability of intron secondary structures that allows for rapid splicing

Analysis of two human pre-ribosomal factors, bystin and hTsr1, highlights differences in evolution of ribosome biogenesis between yeast and mammals

Recent studies reveal that maturation of the 40S ribosomal subunit precursors in mammals includes an additional step during processing of the internal transcribed spacer 1 (ITS1), when compared with yeast Saccharomyces cerevisiae, even though the protein content of the pre-40S particle appears to be the same. Here, we examine by depletion with siRNA treatment the function of human orthologs of two essential yeast pre-ribosomal factors, hEnp1/bystin and hTsr1. Like their yeast orthologs, bystin is required for efficient cleavage of the ITS1 and further processing of this domain within the pre-40S particles, whereas hTsr1 is necessary for the final maturation steps. However, bystin depletion leads to accumulation of an unusual 18S rRNA precursor, revealing a new step in ITS1 processing that potentially involves an exonuclease. In addition, pre-40S particles lacking hTsr1 are partially retained in the nucleus, whereas depletion of Tsr1p in yeast results in strong cytoplasmic accumulation of pre-40S particles. These data indicate that ITS1 processing in human cells may be more complex than currently envisioned and that coordination between maturation and nuclear export of pre-40S particles has evolved differently in yeast and mammalian cells

Mapping targets for small nucleolar RNAs in yeast

Background: Recent analyses implicate changes in the expression of the box C/D class of small nucleolar RNAs (snoRNAs) in several human diseases. Methods: Here we report the identification of potential novel RNA targets for box C/D snoRNAs in budding yeast, using the approach of UV crosslinking and sequencing of hybrids (CLASH) with the snoRNP proteins Nop1, Nop56 and Nop58. We also developed a bioinformatics approach to filter snoRNA-target interactions for bona fide methylation guide interactions. Results: We recovered 241,420 hybrids, out of which 190,597 were classed as reproducible, high energy hybrids. As expected, the majority of snoRNA interactions were with the ribosomal RNAs (rRNAs). Following filtering, 117,047 reproducible hybrids included 51 of the 55 reported rRNA methylation sites. The majority of interactions at methylation sites were predicted to guide methylation. However, competing, potentially regulatory, binding was also identified. In marked contrast, following CLASH performed with the RNA helicase Mtr4 only 7% of snoRNA-rRNA interactions recovered were predicted to guide methylation. We propose that Mtr4 functions in dissociating inappropriate snoRNA-target interactions. Numerous snoRNA-snoRNA interactions were recovered, indicating potential cross regulation. The snoRNAs snR4 and snR45 were recently implicated in site-directed rRNA acetylation, and hybrids were identified adjacent to the acetylation sites. We also identified 1,368 reproducible snoRNA-mRNA interactions, representing 448 sites of interaction involving 39 snoRNAs and 382 mRNAs. Depletion of the snoRNAs U3, U14 or snR4 each altered the levels of numerous mRNAs. Targets identified by CLASH were over-represented among these species, but causality has yet to be established. Conclusions: Systematic mapping of snoRNA-target binding provides a catalogue of high-confidence binding sites and indicates numerous potential regulatory interactions

Flow cytometric mepacrine fluorescence can be used for the exclusion of platelet dense granule deficiency

Background: δ-storage pool disease (δ-SPD) is a bleeding disorder characterized by a reduced number of platelet-dense granules. The diagnosis of δ-SPD depends on the measurement of platelet ADP content, but this test is time consuming and requires a relatively large blood volume. Flow cytometric analysis of platelet mepacrine uptake is a potential alternative, but this approach lacks validation, which precludes its use in a diagnostic setting. Objectives: To evaluate the performance of platelet mepacrine uptake as a diagnostic test for δ-SPD. Patients/Methods: Mepacrine fluorescence was determined with flow cytometry before and after platelet activation in 156 patients with a suspected platelet function disorder and compared with platelet ADP content as a reference test. Performance was analyzed with a receiver operating characteristic (ROC) curve. Results: Eleven of 156 patients had δ-SPD based on platelet ADP content. Mepacrine fluorescence was inferior to platelet ADP content in identifying patients with δ-SPD, but both mepacrine uptake (area under the ROC curve [AUC] 0.87) and mepacrine release after platelet activation (AUC 0.80) had good discriminative ability. In our tertiary reference center, mepacrine uptake showed high negative predicitive value (97%) with low positive predictive value (35%). Combined with a negative likelihood ratio of 0.1, these data indicate that mepacrine uptake can be used to exclude δ-SPD in patients with a bleeding tendency. Conclusion: Mepacrine fluorescence can be used as a screening tool to exclude δ-SPD in a large number of patients with a suspected platelet function disorder

The lipid droplet coat protein perilipin 5 also localizes to muscle mitochondria

Perilipin 5 (PLIN5/OXPAT) is a lipid droplet (LD) coat protein mainly present in tissues with a high fat-oxidative capacity, suggesting a role for PLIN5 in facilitating fatty acid oxidation. Here, we investigated the role of PLIN5 in fat oxidation in skeletal muscle. In human skeletal muscle, we observed that PLIN5 (but not PLIN2) protein content correlated tightly with OXPHOS content and in rat muscle PLIN5 content correlated with mitochondrial respiration rates on a lipid-derived substrate. This prompted us to examine PLIN5 protein expression in skeletal muscle mitochondria by means of immunogold electron microscopy and Western blots in isolated mitochondria. These data show that PLIN5, in contrast to PLIN2, not only localizes to LD but also to mitochondria, possibly facilitating fatty acid oxidation. Unilateral overexpression of PLIN5 in rat anterior tibialis muscle augmented myocellular fat storage without increasing mitochondrial density as indicated by the lack of change in protein content of five components of the OXPHOS system. Mitochondria isolated from PLIN5 overexpressing muscles did not possess increased fatty acid respiration. Interestingly though, 14C-palmitate oxidation assays in muscle homogenates from PLIN5 overexpressing muscles revealed a 44.8% (P = 0.05) increase in complete fatty acid oxidation. Thus, in mitochondrial isolations devoid of LD, PLIN5 does not augment fat oxidation, while in homogenates containing PLIN5-coated LD, fat oxidation is higher upon PLIN5 overexpression. The presence of PLIN5 in mitochondria helps to understand why PLIN5, in contrast to PLIN2, is of specific importance in fat oxidative tissues. Our data suggests involvement of PLIN5 in directing fatty acids from the LD to mitochondrial fatty acid oxidation

Substrate specificity of the TRAMP nuclear surveillance complexes

During nuclear surveillance in yeast and human cells, the RNA exosome functions together with the TRAMP complexes. Here, the authors defined the protein composition of the TRAMP complexes and identified specific RNA binding sites for the different TRAMP components