MOLECULAR INSIGHTS IN ION ACCESS, DEPENDENCE AND SELECTIVITY IN THE NSS/SLC6 TRANSPORTERS KAAT1 AND GAT1.

In this thesis we have investigated some aspects of the molecular physiology of the amino acid transporter KAAT1 and of the GABA transporter GAT1 both belonging to the Neurotransmitter:Sodium Symporter (NSS) family. Wild type as well mutants of these proteins were investigated after expression in Xenopus laevis oocytes and functionally analyzed by radiochemical and electrophysiological assays. In the first chapter a general overview of the biological importance of membrane proteins is provided jointly to a description of the Xenopus oocytes as expression system for this kind of proteins. The second part of the chapter is presenting the general physiological features of KAAT1 and GAT1 while the last part collects information regarding the thermodynamic aspects of secondary active transport as well as the characteristics of the bacterial amino acid transporter LeuT and of the insect dopamine transporter DAT, the two structural models of the NSS family. The last part of the chapter presents one of the most intriguing and cutting edge aspects in the field of membrane transporters: the role of symmetry in the process of ferrying substrates through the lipid bilayer. In chapter 2 are reported the results of our investigation of a highly conserved glycine triplet in KAAT1; this sequence is conserved at the Extracellular Loop 1 (EL1) in almost all the members of the NSS family but the data available at the beginning of our research for GAT1 and for the serotonin transporter SERT were not exhaustive and not in full agreement one to each other. We found that in KAAT1 the flexibility that these amino acids provide to the EL1 is of fundamental importance for the cation access to the extracellular vestibule of the protein. The role that we propose could justify the high degree of conservation showed by this stretch of residues in order to allow the driver ion to get access to its binding site. Different aspects of KAAT1 molecular physiology are addressed in the chapter 3. With our experiments we were able to link the potassium selectivity that characterizes KAAT1 to the polarity of Na1 site and to the dimensional flexibility that is provided in Na2 site by a KAAT1 specific residue of glycine. Beside cation selectivity, we explored the weak chloride dependence of KAAT1 providing new evidences of the fact that its interaction with chloride occurs in an almost unique fashion. The analysis of the 3D homology model of KAAT1 allowed us to identify Thr67 as a residue that we proved experimentally to be a key molecular hinge for the coupling mechanism of ion and substrate flux realized by KAAT1. Furthermore this residue is involved in the initial stereochemical selection that the transporter operates on its substrates as well in the chloride dependence of the transport mechanism. Chapter 4 will briefly resume some preliminary data concerning the possibility to combine the insect Sf9 cell line from Spodoptera frugiperda with the highly efficient Baculovirus expression system with the aim of obtaining the adequate amount of purified protein for KAAT1 crystallization. The last chapter gathers the results of our analysis regarding the influence of internal chloride in the reverse operational mode of the GABA transporter GAT1. Our results provides a link between the oscillations of the intracellular concentration of this anion with the calcium independent GABA release that is described in different pathological and physiological conditions

Threonine 67 is a key component in the coupling of the NSS amino acid transporter KAAT1

The crystallizations of the prokaryotic LeuT and of the eukaryotic DAT and SERT transporters represent important steps forward in the comprehension of the molecular physiology of Neurotransmitter: Sodium Symporters, although the molecular determinants of the coupling mechanism and of ion selectivity still remain to be fully elucidated. The insect NSS homologue KAAT1 exhibits unusual physiological features, such as the ability to use K+ as the driver ion, weak chloride dependence, and the ability of the driver ion to influence the substrate selectivity; these characteristics can help to define the molecular determinants of NSS function. Two non-conserved residues are present in the putative sodium binding sites of KAAT1: Ala 66, corresponding to Gly 20 in the Na2 site of LeuT, and Ser 68, corresponding to Ala 22 in the Nal site. Thr 67 appears also to be significant since it is not conserved among NSS members, is present as threonine only in KAAT1 and in the paralogue CAATCH1 and, according to LeuT structure, is close to the amino acid binding site. Mutants of these residues were functionally characterized in Xenopus oocytes. The T67Y mutant exhibited uptake activity comparable to that of the wild type, but fully chloride-independent and with enhanced stereoselectivity. Interestingly, although dependent on the presence of sodium, the mutant showed reduced transport-associated currents, indicating uncoupling of the driver ion and amino acid fluxes. Thr 67 therefore appears to be a key component in the coupling mechanism, participating in a network that influences the cotransport of Na+ and the amino acid

ARTIFICIAL INTELLIGENCE AND CULTURAL HERITAGE: DESIGN AND ASSESSMENT OF AN ETHICAL FRAMEWORK

The pioneering use of Artificial Intelligence (AI) in various fields and sectors, and the growing ethical debate about its application have led research centers, public and private institutions to establish ethical guidelines for a trustworthy implementation of these powerful algorithms. Despite the recognized definition of ethical principles for a responsible or trustworthy use of AI, there is a lack of a sector-specific perspective that highlights the ethical risks and opportunities for different areas of application, especially in the field of Cultural Heritage (CH). In fact, there is still a lack of formal frameworks that evaluate the algorithms’ adherence to the ethical standards set by the European Union for the use of AI in protecting CH and its inherent value. Because of this, it is necessary to investigate a different sectoral viewpoint to supplement the widely used horizontal approach. This paper represents a first attempt to design an ethical framework to embody AI in CH conservation practises to assess various risks arising from the use of AI in the field of CH. The contribution presents a synthesis of the different AI applications to improve the preservation process of CH. It explores and analyses in depth the ethical challenges and opportunities presented by the use of AI to improve CH preservation. In addition, the study aims to design an ethical framework of principles to assess the application of this ground-breaking technology at CH

Justice and Corporate Governance: New Insights from Rawlsian Social Contract and Sen’s Capabilities Approach

By considering what we identify as a problem inherent in the ‘nature of the firm’—the risk of abuse of authority—we propound the conception of a social contract theory of the firm which is truly Rawlsian in its inspiration. Hence, we link the social contract theory of the firm (justice at firm’s level) with the general theory of justice (justice at society’s level). Through this path, we enter the debate about whether firms can be part of Rawlsian theory of justice showing that corporate governance principles enter the “basic structure.” Finally, we concur with Sen’s aim to broaden the realm of social justice beyond what he calls the ‘transcendental institutional perfectionism’ of Rawls’ theory. We maintain the contractarian approach to justice but introduce Sen’s capability concept as an element of the constitutional and post-constitutional contract model of institutions with special reference to corporate governance. Accordingly, rights over primary goods and capabilities are (constitutionally) granted by the basic institutions of society, but many capabilities have to be turned into the functionings of many stakeholders through the operation of firms understood as post-constitutional institutional domains. The constitutional contract on the distribution of primary goods and capabilities should then shape the principles of corporate governance so that at post-constitutional level anyone may achieve her/his functionings in the corporate domain by exercising such capabilities. In the absence of such a condition, post-constitutional contracts would distort the process that descends from constitutional rights and capabilities toward social outcomes

Proyecto, investigación e innovación en urbanismo, arquitectura y diseño industrial

Actas de congresoLas VII Jornadas de Investigación “Encuentro y Reflexión” y I Jornadas de Investigación de becarios y doctorandos. Proyecto, investigación e innovación en Urbanismo, Arquitectura y Diseño Industrial se centraron en cuatro ejes: el proyecto; la dimensión tecnológica y la gestión; la dimensión social y cultural y la enseñanza en Arquitectura, Urbanismo y Diseño Industrial, sustentados en las líneas prioritarias de investigación definidas epistemológicamente en el Consejo Asesor de Ciencia y Tecnología de esta Universidad Nacional de Córdoba. Con el objetivo de afianzar continuidad, formación y transferencia de métodos, metodología y recursos se incorporó becarios y doctorandos de los Institutos de investigación. La Comisión Honoraria la integraron las tres Secretarias de Investigación de la Facultad, arquitectas Marta Polo, quien fundó y María del Carmen Franchello y Nora Gutiérrez Crespo quienes continuaron la tradición de la buena práctica del debate en la cotidianeidad de la propia Facultad. Los textos que conforman las VII Jornadas son los avances y resultados de las investigaciones realizadas en el bienio 2016-2018.Fil: Novello, María Alejandra. Universidad Nacional de Córdoba. Facultad de Arquitectura, Urbanismo y Diseño; ArgentinaFil: Repiso, Luciana. Universidad Nacional de Córdoba. Facultad de Arquitectura, Urbanismo y Diseño; ArgentinaFil: Mir, Guillermo. Universidad Nacional de Córdoba. Facultad de Arquitectura, Urbanismo y Diseño; ArgentinaFil: Brizuela, Natalia. Universidad Nacional de Córdoba. Facultad de Arquitectura, Urbanismo y Diseño; ArgentinaFil: Herrera, Fernanda. Universidad Nacional de Córdoba. Facultad de Arquitectura, Urbanismo y Diseño; ArgentinaFil: Períes, Lucas. Universidad Nacional de Córdoba. Facultad de Arquitectura, Urbanismo y Diseño; ArgentinaFil: Romo, Claudia. Universidad Nacional de Córdoba. Facultad de Arquitectura, Urbanismo y Diseño; ArgentinaFil: Gordillo, Natalia. Universidad Nacional de Córdoba. Facultad de Arquitectura, Urbanismo y Diseño; ArgentinaFil: Andrade, Elena Beatriz. Universidad Nacional de Córdoba. Facultad de Arquitectura, Urbanismo y Diseño; Argentin

Practical Identity and Open Cooperation

This chapter explores the notion of open cooperation in political life in connection with a particular form of practical identity. This form of cooperation takes shape when the zero-sum games produced by the familistic and tribal forms of cooperation melt away, and the relationships based on loyalty and trust are generalized to a collectiveness, i.e., a multitude of strangers who are willing to establish non-zero-sum relationships. This process originates from a specific relational motivation: the act of giving trust, construed as the decision to entrust oneself, according to one's choice, to an interpersonal situation of risk within cooperation. The hypothesis is made that good cooperation occurs only by virtue of a specific form of individualism that includes the concept of individual responsibility. For it is only through the permanent internalization of ethics of individual responsibility that the strategies of trust among strangers can work effectively and prevail over both the traditional tribal solidarity and the localist and nepotistical systems. Open cooperation originates from the individuals’ capacity to be psychologically autonomous, viz., to understand their own responsibilities, to choose new ones, and to remain faithful to them

Chloride interaction in the SLC6 amino acid cotransporter KAAT1

The SLC6 family of solute transporters groups eukaryotic, Cl--dependent proteins as the serotonin transporter SERT (1) and the GABA transporter GAT1 (2) and prokaryotic, Cl--independent proteins, as the family model LeuT (3, 4). The main role of the anion in the transport cycle appears to be related to the neutralization of the positive charges of sodium ions transported with the substrate. KAAT1 is a lepidopteran SLC6 amino acid cotransporter, activated by Na+ and K+, but characterized by a weak chloride dependence (5, 6). In recent years, taking advantage of the special features of KAAT1, we have investigated the structural/functional relationships within the SLC6 family. By site-directed mutagenesis and functional expression in Xenopus laevis oocytes, we have identified residues involved in Na+ and K+ interaction and in amino acid translocation (7,8,9). The aim of this study has been the identification of the molecular determinants of chloride interaction of KAAT1 to obtain insights in the transport mechanism of SLC6 members. Comparison of KAAT1 sequence with SERT, GAT1 and LeuT has revealed some differences in residues forming the putative anion binding site but, among theme, only T339 seemed to be relevant for chloride dependence: T339S and T339E mutant transport activity became indeed almost completely Cl--dependent. The mutation of a further residue (T67), conserved only in KAAT1 and in the other weakly Cl--dependent transporter of the family CAATCH1, affected KAAT1 activity: T67Y mutant was fully chloride independent whereas T67S and T67A showed an enhancement in chloride dependence. In order to confirm the role of T67, we built the reciprocal mutant of KAAT1 T67Y in GAT1 transporter: Y60T, interestingly, showed a reduced chloride dependence compared to the wt. These data suggest that T67 and T339 influence KAAT1 interaction with chloride. 1. Forrest L.R. et al., Proc Natl Acad Sci U S A. 2007; 104:12761-6. 2. Zomot E. et al., Nature. 2007 Oct 11; 449: 726-30. 3. Yamashita A. et al., Nature. 2005; 437: 215-23 4. Ben-Yona A. et al., J Biol Chem. 2011; 286: 2826-33 5. Castagna M. et al., Proc Natl Acad Sci U S A.; 95: 5395-400 6. Bett\ue8 S. et al., Channels (Austin). 2008; 2: 358-62 7. Mari et al., Cell. Mol. Life Sci. 2004, 61: 243-56. 8. Miszner et al., J. Physiol. 2007, 58: 1899-1913. 9. Castagna et al., Am. J. Physiol. Cell. Physiol. 2007, 293: C1286-C129