Design local e desenvolvimento: um espaço para coordenação entre design, artesanato e território

The creation of the Centro Metropolitano de Diseño (Design Metropolitan Center) was the resolution emerging from a strategy and a methodological search which allowed to contact the design and its main actors with the new context phenomena which appeared by the end of the 90s in Argentina, such as the social crisis, unemployment, the devaluation and subsequent revaluation of national production, the industry collapse and recovery, the exponential growth in the number of design professionals, the emergence of new entrepreneurs, etcetera. Several experiences, programs and activities have been developed during the 10 years of this institution and, perhaps, the greatest contribution has been to clearly propose an agenda linking design activities to local development strategies in the City of Buenos Aires, through a systemic model which covered each of its initiatives. Hence, recurrent and many times irreductible conflicts concerning apparently antagonistic positions, which are usually frequent in academic and professional debates of the Latin American design world, were channeled from practices exceeding reductionisms and diving into the complexity brought by the work with multiple actors who are part of aproject settled in the territory. This work explores, based on one of the projects carried out by the CMD and other particular experiences still in progress, the outstanding aspects observed in a process of mutual transfer between craftsmen and designers located in different territories: one being an urban and compact territory, and the other being the vast puna of the Argentine Northwest. Likewise, it intends to mention the multiplicity of actors and factors operating in the design-craft relation as part of a local development system, which contains and conditions it. Finally, some methodological recommendations are proposed to advance in the design of programs which allow to articulate these two universes.Key words: design, craft, local development, territory, research.A criação do Centro Metropolitano de Diseño (Design Metropolitan Center) surgiu a partir da resolução de uma estratégia e de uma pesquisa metodológica que permitiu entrar em contato com o projeto e seus principais atores. Assim como, com os fenômenos do novo contexto, que surgiu no final dos anos 90 na Argentina , como a crise social, desemprego, desvalorização e revalorização subsequente da produção nacional, o colapso da indústria e de recuperação, o crescimento exponencial do número de profissionais de design, o surgimento de novos empreendedores, etc. Várias experiências, programas e atividades foram desenvolvidas durante os 10 anos desta instituição e, talvez, a maior contribuição foi a de propor uma conexão clara na agenda entre as atividades de design para estratégias de desenvolvimento local na Cidade de Buenos Aires, através de um modelo sistêmico, que abrangeu cada uma de suas iniciativas. Por isso, de forma recorrente muitos conflitos irredutíveis sobre posições aparentemente antagônicas, presentes em muitos debates acadêmicos e profissionais, foram canalizados pelas práticas e reducionismos diante da complexidade trazida pelo trabalho com múltiplos atores que fazem parte de um projeto comum estabelecido no território. Este trabalho explora, baseado em um dos projetos realizados pelas experiências CMD e outros ainda em curso, os aspectos de um processo de transferência mútua entre artesãos e designers localizados em territórios diferentes: diferenças que se definem por um território ser urbano e compacto, e sendo o outro um vasto território do noroeste argentino. Da mesma forma, pretende-se mencionar a multiplicidade de atores e fatores operacionais na relação de design artesanal como parte de um sistema de desenvolvimento local, que contém e condiciona. Finalmente, algumas recomendações metodológicas são propostas para avançar na elaboração de programas que permitem articular esses dois universos.Palavras-chave: design, artesanato, desenvolvimento local, território de pesquisa

Laboratorio de Sabores: Diseño de experiencias innovadoras con productores de alimentos

Laboratorio de sabores of Esquel, Argentina, is an experiential space of co-creation in a real environment, where producers together with experts from different disciplines–who articulate practices and knowledge from the public, academic and private sector–develop, deploy and test new technologies, processes, designs and products. El Laboratorio de sabores de Esquel, Argentina, es un espacio experiencial de cocreación en un entorno real, donde los productores junto a expertos de diferentes disciplinas, que articulan prácticas y conocimiento provenientes del sector público, académico y privado, desarrollan, despliegan y prueban nuevas tecnologías, procesos, diseños y productos

Chemical Synthesis and Expression of the HIV-1 Rev Protein

The HIV-1 Rev protein is responsible for shuttling partially spliced and unspliced viral mRNA out of the nucleus. This is a crucial step in the HIV-1 lifecycle, thus making Rev an attractive target for the design of anti-HIV drugs. Despite its importance, there is a lack of structural, biophysical, and quantitative information about Rev. This is mainly because of its tendency to undergo self-assembly and aggregation; this makes it very difficult to express and handle. To address this knowledge gap, we have developed two new highly efficient and reproducible methods to prepare Rev in large quantities for biochemical and structural studies: 1) Chemical synthesis by using native chemical ligation coupled with desulfurization. Notably, we have optimized our synthesis to allow for a one-pot approach for the ligation and desulfurization steps; this reduced the number of purification steps and enabled the obtaining of desired protein in excellent yield. Several challenges emerged during the design of this Rev synthesis, such as racemization, reduced solubility, formylation during thioester synthesis, and the necessity for using orthogonal protection during desulfurization; solutions to these problems were found. 2) A new method for expression and purification by using a vector that contained an HLT tag, followed by purification with a Ni column, a cation exchange column, and gel filtration. Both methods yielded highly pure and folded Rev. The CD spectra of the synthetic and recombinant Rev proteins were identical, and consistent with a predominantly helical structure. These advances should facilitate future studies that aim at a better understanding of the structure and function of the protein

Regulation of influenza a virus mRNA splicing by CLK1

Influenza A virus carries eight negative single-stranded RNAs and uses spliced mRNAs to increase the number of proteins produced from them. Several genome-wide screens for essential host factors for influenza A virus replication revealed a necessity for splicing and splicing-related factors, including Cdc-like kinase 1 (CLK1). This CLK family kinase plays a role in alternative splicing regulation through phosphorylation of serine-arginine rich (SR) proteins. To examine the influence that modulation of splicing regulation has on influenza infection, we analyzed the effect of CLK1 knockdown and inhibition. CLK1 knockdown in A549 cells reduced influenza A/WSN/33 virus replication and increased the level of splicing of segment 7, encoding the viral M1 and M2 proteins. CLK1-/- mice infected with influenza A/England/195/2009 (H1N1pdm09) virus supported lower levels of virus replication than wild-type mice. Screening of newly developed CLK inhibitors revealed several compounds that have an effect on the level of splicing of influenza A gene segment M in different models and decrease influenza A/WSN/33 virus replication in A549 cells. The promising inhibitor KH-CB19, an indole-based enaminonitrile with unique binding mode for CLK1, and its even more selective analogue NIH39 showed high specificity towards CLK1 and had a similar effect on influenza mRNA splicing regulation. Taken together, our findings indicate that targeting host factors that regulate splicing of influenza mRNAs may represent a novel therapeutic approach

Thrombospondin-1-N-Terminal Domain Induces a Phagocytic State and Thrombospondin-1-C-Terminal Domain Induces a Tolerizing Phenotype in Dendritic Cells

In our previous study, we have found that thrombospondin-1 (TSP-1) is synthesized de novo upon monocyte and neutrophil apoptosis, leading to a phagocytic and tolerizing phenotype of dendritic cells (DC), even prior to DC-apoptotic cell interaction. Interestingly, we were able to show that heparin binding domain (HBD), the N-terminal portion of TSP-1, was cleaved and secreted simultaneously in a caspase- and serine protease- dependent manner. In the current study we were interested to examine the role of HBD in the clearance of apoptotic cells, and whether the phagocytic and tolerizing state of DCs is mediated by the HBD itself, or whether the entire TSP-1 is needed. Therefore, we have cloned the human HBD, and compared its interactions with DC to those with TSP-1. Here we show that rHBD by itself is not directly responsible for immune paralysis and tolerizing phenotype of DCs, at least in the monomeric form, but has a significant role in rendering DCs phagocytic. Binding of TSP-1-C-terminal domain on the other hand induces a tolerizing phenotype in dendritic cells

Mechanism of the Interaction between the Intrinsically Disordered C-Terminus of the Pro-Apoptotic ARTS Protein and the Bir3 Domain of XIAP

ARTS (Sept4_i2) is a mitochondrial pro-apoptotic protein that functions as a tumor suppressor. Its expression is significantly reduced in leukemia and lymphoma patients. ARTS binds and inhibits XIAP (X-linked Inhibitor of Apoptosis protein) by interacting with its Bir3 domain. ARTS promotes degradation of XIAP through the proteasome pathway. By doing so, ARTS removes XIAP inhibition of caspases and enables apoptosis to proceed. ARTS contains 27 unique residues in its C-terminal domain (CTD, residues 248–274) which are important for XIAP binding. Here we characterized the molecular details of this interaction. Biophysical and computational methods were used to show that the ARTS CTD is intrinsically disordered under physiological conditions. Direct binding of ARTS CTD to Bir3 was demonstrated using NMR and fluorescence spectroscopy. The Bir3 interacting region in ARTS CTD was mapped to ARTS residues 266–274, which are the nine C-terminal residues in the protein. Alanine scan of ARTS 266–274 showed the importance of several residues for Bir3 binding, with His268 and Cys273 contributing the most. Adding a reducing agent prevented binding to Bir3. A dimer of ARTS 266–274 formed by oxidation of the Cys residues into a disulfide bond bound with similar affinity and was probably required for the interaction with Bir3. The detailed analysis of the ARTS – Bir3 interaction provides the basis for setting it as a target for anti cancer drug design: It will enable the development of compounds that mimic ARTS CTD, remove IAPs inhibition of caspases, and thereby induce apoptosis

Specific Recognition of p53 Tetramers by Peptides Derived from p53 Interacting Proteins

Oligomerization plays a major role in regulating the activity of many proteins, and in modulating their interactions. p53 is a homotetrameric transcription factor that has a pivotal role in tumor suppression. Its tetramerization domain is contained within its C-terminal domain, which is a site for numerous protein-protein interactions. Those can either depend on or regulate p53 oligomerization. Here we screened an array of peptides derived from proteins known to bind the tetrameric p53 C-terminal domain (p53CTD) and identified ten binding peptides. We quantitatively characterized their binding to p53CTD using fluorescence anisotropy. The peptides bound tetrameric p53CTD with micromolar affinities. Despite the high charge of the binding peptides, electrostatics contributed only mildly to the interactions. NMR studies indicated that the peptides bound p53CTD at defined sites. The most significant chemical shift deviations were observed for the peptides WS100B(81–92), which bound directly to the p53 tetramerization domain, and PKCα(281–295), which stabilized p53CTD in circular dichroism thermal denaturation studies. Using analytical ultracentrifugation, we found that several of the peptides bound preferentially to p53 tetramers. Our results indicate that the protein-protein interactions of p53 are dependent on the oligomerization state of p53. We conclude that peptides may be used to regulate the oligomerization of p53