Characterization of molecular mechanisms underlying VRAC activation

Multicellular organisms evolved mechanisms to accurately regulate cellular volume to counteract swelling or shrinkage which can in turn affect cell integrity. The Volume Regulated Anion Channel (VRAC) is ubiquitously found in all vertebrate cells and turned out to be a key player in the cell-intrinsic regulatory processes which tend to restore the original volume upon osmotic challenges. In combination with potassium export mechanisms, this is achieved by the swelling-induced increased VRAC activity allowing release of halide ions and organic osmolytes, that subsequently drive water efflux through the membrane, in the so-called regulatory volume decrease process. Difficulties encountered in the molecular identification of the channel greatly undermined biophysical characterization of VRAC and the mechanism underlying channel activation is not resolved. Several biochemical and mechanical events have been implicated in the capability of VRAC to respond to a hypotonic challenge. The most relevant among these include: a foreground role for reactive oxygen species (ROS) and channel oxidation; the involvement of phosphorylation with several kinases and signalling pathways leading to a differential modulation of the channel; and finally the sensing of low intracellular ionic strength. Molecular identification of genes encoding VRAC and recently solved cryo-EM structures of LRRC8A homomers revealed that the channel is a hexamer formed by members of the leucine rich repeat containing protein 8 (LRRC8) family, with the obligatory LRRC8A subunit, essential for channel functioning, and at least one of the other closely related members, which in total comprise five paralogues (LRRC8A-E). Little is known about the relevant stoichiometry, even if the high variability of the expression patterns depending on the cell type suggests that subunit composition is modified to meet physiological demand. In this project, I followed parallel strategies to obtain a deeper insight the relevant trigger events which underlie channel activation. By means of site-directed mutagenesis and electrophysiological analysis I elucidated mechanism of oxidation dependent VRAC modulation with the goal to understand which structural motifs are involved. Using oocytes as a heterologous expression system, I confirmed that oxidation sensitivity is subunit specific, a finding that was previously supported by the work of (Gradogna et al, 2017): LRRC8A-LRRC8E (8A-8E) heteromers are activated by oxidation, whereas 8A-8C heteromers are inhibited. A chimeric approach allowed to identify regions responsible of the divergent effect mediated by the application of oxidant component. Swapping the C-terminus leucine-rich repeat domains (LRRD) between 8E and 8C subunits resulted in a corresponding interchange of the respective oxidation sensitivity. Further specific targeted site-directed mutagenesis led to the identification of cysteines C424 and C448 unique to the 8E subunit, as those responsible of the dramatic increase of the current upon oxidative stimulation, with both 8A/8EC424F and 8A/8EC448S heteromers losing their capability to activate upon oxidant application. In a parallel set of experiments I investigated the role played by phosphorylation in swelling dependent VRAC activation. On this purpose I mutated several residues with a high prediction score to be target of kinases from the first intracellular loop (IL1) of LRRC8A. By means of a chimeric strategy I finally disclosed that mutating residue T169 to alanine (A) or serine (S), gave rise to channels that completely lost their ability to undergo activation upon hypotonic stimulation. Surprisingly, mutation T169S retained activation by low intracellular ionic strength. Overall, the results obtained in this thesis provide strong evidence in support of a role of oxidation in the subunit dependent VRAC modulation. In parallel, the identification of T169 as a determinant residue for channel functioning lays the groundwork to further investigation to disclose whether its importance might rely on phosphorylation or other unknown mechanisms.Multicellular organisms evolved mechanisms to accurately regulate cellular volume to counteract swelling or shrinkage which can in turn affect cell integrity. The Volume Regulated Anion Channel (VRAC) is ubiquitously found in all vertebrate cells and turned out to be a key player in the cell-intrinsic regulatory processes which tend to restore the original volume upon osmotic challenges. In combination with potassium export mechanisms, this is achieved by the swelling-induced increased VRAC activity allowing release of halide ions and organic osmolytes, that subsequently drive water efflux through the membrane, in the so-called regulatory volume decrease process. Difficulties encountered in the molecular identification of the channel greatly undermined biophysical characterization of VRAC and the mechanism underlying channel activation is not resolved. Several biochemical and mechanical events have been implicated in the capability of VRAC to respond to a hypotonic challenge. The most relevant among these include: a foreground role for reactive oxygen species (ROS) and channel oxidation; the involvement of phosphorylation with several kinases and signalling pathways leading to a differential modulation of the channel; and finally the sensing of low intracellular ionic strength. Molecular identification of genes encoding VRAC and recently solved cryo-EM structures of LRRC8A homomers revealed that the channel is a hexamer formed by members of the leucine rich repeat containing protein 8 (LRRC8) family, with the obligatory LRRC8A subunit, essential for channel functioning, and at least one of the other closely related members, which in total comprise five paralogues (LRRC8A-E). Little is known about the relevant stoichiometry, even if the high variability of the expression patterns depending on the cell type suggests that subunit composition is modified to meet physiological demand. In this project, I followed parallel strategies to obtain a deeper insight the relevant trigger events which underlie channel activation. By means of site-directed mutagenesis and electrophysiological analysis I elucidated mechanism of oxidation dependent VRAC modulation with the goal to understand which structural motifs are involved. Using oocytes as a heterologous expression system, I confirmed that oxidation sensitivity is subunit specific, a finding that was previously supported by the work of (Gradogna et al, 2017): LRRC8A-LRRC8E (8A-8E) heteromers are activated by oxidation, whereas 8A-8C heteromers are inhibited. A chimeric approach allowed to identify regions responsible of the divergent effect mediated by the application of oxidant component. Swapping the C-terminus leucine-rich repeat domains (LRRD) between 8E and 8C subunits resulted in a corresponding interchange of the respective oxidation sensitivity. Further specific targeted site-directed mutagenesis led to the identification of cysteines C424 and C448 unique to the 8E subunit, as those responsible of the dramatic increase of the current upon oxidative stimulation, with both 8A/8EC424F and 8A/8EC448S heteromers losing their capability to activate upon oxidant application. In a parallel set of experiments I investigated the role played by phosphorylation in swelling dependent VRAC activation. On this purpose I mutated several residues with a high prediction score to be target of kinases from the first intracellular loop (IL1) of LRRC8A. By means of a chimeric strategy I finally disclosed that mutating residue T169 to alanine (A) or serine (S), gave rise to channels that completely lost their ability to undergo activation upon hypotonic stimulation. Surprisingly, mutation T169S retained activation by low intracellular ionic strength. Overall, the results obtained in this thesis provide strong evidence in support of a role of oxidation in the subunit dependent VRAC modulation. In parallel, the identification of T169 as a determinant residue for channel functioning lays the groundwork to further investigation to disclose whether its importance might rely on phosphorylation or other unknown mechanisms

A phylogeny of the tinamous (Aves: Palaeognathiformes) based on integumentary characters

A cladistic analysis of the tinamous, including the 47 currently recognized species and some distinct subspecies, was conducted based on 80 integumentary characters from adult and natal plumage, ramphoteca (corneum sheath of bill), and podoteca (horny scales of legs). For the adult plumage (50 characters), we studied feather pigmentation patterns from different pterylae (feather tracts). A criterion of overlap of basic pigmentation elements was used to assign costs to the transformation between the states in most of these characters in such a way that transformations between more similar conditions were less costly. The consensus tree was almost fully resolved, and about 50% of its groups were relatively well supported. Because the only outgroup that could be used provided a poor root, two possible rootings of the ingroup subtree were considered; in both cases, only one of the two traditional subfamilies (the steppe tinamous) was recovered, and the other (the forest tinamous) appeared as paraphyletic. The results of the present analysis are compared with those from an osteological data set, using a strict supertree technique. The combined tree has a large number of nodes, indicating a high degree of congruence between the two data sets.Fil: Bertelli, Sara Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - Tucumán. Unidad Ejecutora Lillo; ArgentinaFil: Giannini, Norberto Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - Tucumán. Unidad Ejecutora Lillo; ArgentinaFil: Goloboff, Pablo Augusto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - Tucumán. Unidad Ejecutora Lillo; Argentina. Universidad Nacional de Tucumán. Facultad de Ciencias Naturales e Instituto Miguel Lillo. Instituto Superior de Entomología; Argentin

On the use of integumentary characters in bird phylogeny: the case of Tinamus osgoodi (Palaeognathae: Tinamidae) and plumage charcater coding

Los caracteres tegumentarios raramente han sido incluidos en Ornitología Sistemática, a pesar de que la mayoría de los caracteres utilizados para diferenciar especies son de plumaje, ranfoteca (cubierta córnea del pico y narinas) y podoteca (escamas de las patas), y de que muchos de estos caracteres contribuyen a las diagnosis tradicionales de los grandes grupos de Órdenes. Varios estudios recientes han utilizado caracteres tegumentarios en un contexto cladístico, en particular, una filogenia comprensiva de perdices que incluyó a los 9 géneros, todas las 47 especies actualmente reconocidas, y algunas de las subpecies claramente definidas. En este trabajo re-evaluamos la posición en dicha filogenia de la especie oscura Tinamus osgoodi. En análisis previos, esta perdiz selvática había sido recuperada junto a las especies de Crypturellus, que también tienen plumaje de colores oscuros. Todas estas especies son posiblemente melánicas, o presentan un patrón de coloración de plumaje que afecta a muchos pterylae a la vez. Discutimos la dependencia de algunos caracteres del plumaje de perdices, y re-analizamos la matriz de datos tegumentaria recodificando dichos caracteres en las especies oscuras. Utilizando pesos implicados para la reconstrucción filogenética, recuperamos la mayoría de los agrupamientos esperados, incluyendo a Tinamus osgoodi como un miembro de su género. Es interesante destacar que la posición de dicha especie no influyó en las especies oscuras de Crypturellus, las cuales podrían haber sido afectadas por el mismo esquema de codificación, y sin embargo, se agruparon junto a sus congéneres, indicando que la interacción con otros caracteres jugó un papel crucial en la recuperación de estos taxa en sus respectivos géneros. Finalmente, comentamos sobre el uso de caracteres tegumentarios y la incidencia de su codificación en Ornitología SistemáticaIntegumentary characters have rarely been included in Systematic Ornithology in spite of the fact that most characters used to differentiate species are of plumage, ramphoteca (corneous sheath of the bill and nares) and podoteca (horny scales of the legs), and many such characters contribute to higher-order groupings of traditional diagnosis. Several recent studies have used integumentary characters in a cladistic context, particularly a comprehensive phylogeny of tinamous that included the 9 genera, all 47 currently recognized species, and some distinct subspecies. Here we re-evaluate the position in that phylogeny of the uniformly dark species Tinamus osgoodi. This forest tinamou was recovered in the previous analysis as closely related to species of Crypturellus that also exhibited dark plumage coloration. All these species are possibly melanic, or alternatively, exhibit a plumage coloration pattern affecting many pterylae at once. We discuss the nonindependence of some plumage characters of tinamous, and re-analize the integumentary data set by re-coding these characters for the dark species. Using implied-weights for phylogeny reconstruction, we recover most of the expected groupings, including Tinamus osgoodi as one member of its genus. Interestingly this position did not influence the dark Crypturellus species, which could have been affected by the same coding scheme but instead grouped together with their congeners, indicating that interaction with other characters played a crucial role in the recovery of these taxa in their respective genera. Finally, we comment on the usefulness of integumentary characters and the incidence of their coding in Systematic OrnithologyFil: Bertelli, Sara Beatriz. Fundación Miguel Lillo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán; ArgentinaFil: Giannini, Norberto Pedro. Universidad Nacional de Tucumán. Facultad de Ciencias Naturales e Instituto Miguel Lillo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán; Argentin

María Teresa León: la testimonianza della memoria durante l'esilio repubblicano spagnolo

María Teresa León, autrice spagnola appartenente alla generazione del '27, vive il periodo della guerra civile schierandosi attivamente dalla parte dei repubblicani. Per questo, con la vittoria di Francisco Franco, si reca in un esilio volontario che la tenne lontana dalla sua terra per quasi quarant'anni. Questa tesi di laurea si concentra sulle opere dell'esilio, sia autobiografiche che finzionali, che riflettono la testimonianza dell'impegno sociale e politico dell'autrice circa la situazione di una Spagna che mai dimentica, e che relaziona sempre alla realtà dei paesi con cui ha modo di entrare in contatto

Phylogenetic interrelationships of living and extinct Tinamidae, volant palaeognathous birds from the New World

Tinamous, one of the earliest diverging living avian lineages, consists of a Neotropical clade of palaeognathous birds with a fossil record limited to the early Miocene–Quaternary of southern South America. Here, we conduct a comprehensive, morphology-based phylogenetic study of the interrelationships among extinct and living species of tinamous. Morphological data of fossil species are included in a matrix of 157 osteological and myological characters of 56 terminal taxa. The monophyly of most recognized genera is supported by the results of the analysis. The cladistic analysis also recovers the traditional subdivision between those tinamous specialized for open areas (Nothurinae) and those inhabiting forested environments (Tinaminae). Temporal calibration of the resultant phylogeny indicates that such a basal divergence had already taken place in the early Miocene, some 17 million years ago. The placement of the fossil species within the open-area (Nothurinae) and the forest-dwelling (Tinaminae) tinamous is also consistent with the palaeoenvironmental conditions inferred from the associated fauna.Fil: Bertelli, Sara Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación Miguel Lillo; Argentina. Natural History Museum of Los Angeles County; Estados UnidosFil: Chiappe, Luis M.. Natural History Museum of Los Angeles County; Estados UnidosFil: Mayr, Gerald. Forschungsinstitut Senckenberg; Alemani

Birds, sounds and shamans. Interdisciplinary study of an osseous musical instrument from a prehispanic settlement of the southern jungles of northwestern Argentina

Los instrumentos musicales de viento procedentes de contextos arqueológicos del noroeste de Argentina son escasos, y el conocimiento acerca de las implicaciones sociales vinculadas con su uso en tiempos prehispánicos es mínimo. En este artículo se presentan los resultados de un estudio interdisciplinario efectuado sobre un aerófono óseo hallado en el sitio Yánimas 1 (provincia de Tucumán), abordándose aspectos arqueológicos, biológicos, tecnológicos y musicológicos. Se contribuye a la comprensión de las prácticas involucradas en la producción y uso del instrumento. Asimismo, se avanza en la definición de espacialidades en una ocupación datada en ca. 900-1200 d. C.Wind instruments from archaeological sites of Northwest Argentina are scarce, and knowledge of the social implications related to their use in pre-Hispanic periods is minimal. This article presents the results of an interdisciplinary study of a wind instrument made of bone that was found in Yánimas 1 (province of Tucumán), addressing aspects of archaeology, biology, technology and musicology. It contributes to the understanding of the practices involved in the production and use of the instrument. Similarly, it aids progress in the definition and use of space in an occupation dated ca. 900-1200 A.D.Fil: Miguez, Gabriel Eduardo. Universidad Nacional de Tucumán. Facultad de Ciencias Naturales E Instituto Miguel Lillo. Instituto de Arqueología y Museo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán; ArgentinaFil: Nasif, Norma. Universidad Nacional de Tucumán. Facultad de Ciencias Naturales E Instituto Miguel Lillo; ArgentinaFil: Gudemos, Monica Lucia. Universidad Nacional de Cordoba. Facultad de Artes; ArgentinaFil: Bertelli, Sara Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación Miguel Lillo; Argentin

Mesozoic fossil vertebrates from northwestern Argentina

En la presente contribución se detalla el registro de fósiles vertebrados mesozoicos en el noroeste de Argentina. El mismo se compone de una parte triásica y otra cretácica, pero hay también un registro puntual del Jurásico. El Triásico se presenta en la cuenca de Ischigualasto-Villa Unión en las provincias de San Juan y La Rioja y en la cuenca de Marayes-El Carrizal al sur de San Juan, mientras que el Jurásico Inferior existe en la última cuenca y en la Formación Cañón del Colorado en el centro de la provincia de San Juan. El Cretácico está documentado en la Formación Los Llanos de La Rioja y en la cuenca del Noroeste en Jujuy y Salta. El Triásico Superior es el mejor representado mostrando una buena diversidad de arcosauromorfos. El mejor registro de dinosauriformesno dinosaurianos es en la Formación Chañares, mientras que los dinosaurios más antiguos del mundo aparecen ya bien diversifi cados en la Formación Ischigualasto y se vuelven abundantes en Los Colorados. Dicinodontes son componentes residuales (poco diversos y abundantes), mientras que los cinodontes son abundantes en la Formación Chañares y abundantes y más diversifi cados en la fauna de Ischigualasto. Registros antiguos detortugas existen en las formaciones Los Colorados y Quebrada del Barro, y en esta última hay también esfenodontes. En el Cretácico de la Formación Los Llanos se documentaron nidadas de huevos de saurópodos y restos fragmentarios de cocodrilos. En la cuenca del Noroeste, la Formación Las Curtiembres registra peces teleósteos, ranas pípidas, tortugas pleurodiras, mesoeucrocodilidos, dinosaurios terópodos y enantiornites; mientras queen la Formación Los Blanquitos fueron hallados dinosaurios titanosaúridos y terópodos y un diente de cocodrilo. La Formación Lecho está dominada por dinosaurios titanosaurios y también se hallaron terópodos y enantiornites. Finalmente, en la Formación Yacoraite hay peces y cocodrilos, así como también huellas atribuidas a diferentes grupos de dinosaurios.We present a synthesis of the vertebrate fossil record from northwestern Argentina. The Triassic is represented in the Ischigualasto-Villa Unión Basin in La Rioja and San Juan provinces and in the Marayes-El Carrizal Basin in the south of San Juan, whereas the Lower Jurassic crops out in the latt er basin and in the Cañón del Colorado Formation in the center of San Juan Province. Cretaceous fossils are documented in the Los Llanos Formation in La Rioja Province and in the Noroeste Basin in Jujuy and Salta provinces. The Upper Triassic is best represented in the Ischigualasto-Villa Union Basin, featuring well-diversifi ed archosauromorphs. Non dinosaur dinosauriforms are well diversifi ed in the Chañares Formation, and the world’s oldest dinosaurs are already well diversifi ed in the Ischigualasto Formation and become abundant in Los Colorados. Ancient records of terrestrial turtles exist in the Los Colorados and Quebrada del Barro formations. Sphenodontians are also represented in the latt er unit. A nesting site of sauropods and fossils of a crocodile are documented in the Cretaceous Los Llanos Formation. In the Noroeste Basin, the Las Curtiembres Formation has records of teleost fi sh, pipid frogs, pleurodire turtles, mesoeucrocodiles, theropod dinosaurs and enantiornithes birds; whereas in the Los Blanquitos Formation were found titanosaurid and theropod dinosaurs, as well as a crocodile tooth. The Lecho Formation is dominated by titanosaurid dinosaurs and also record theropod dinosaurs and enantiornithes. Finally, in the Yacoraite Formation there are fish and crocodiles, as well as tracks att ributed to diff erent groups of dinosaurs.Fil: Abdala, Nestor Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - Tucumán. Unidad Ejecutora Lillo; Argentina. University of the Witwatersrand; SudáfricaFil: Bertelli, Sara Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - Tucumán. Unidad Ejecutora Lillo; ArgentinaXX Congreso Argentino de GeologiaSan Miguel de TucumánArgentinaAsociación Geológica ArgentinaUniversidad Nacional de Tucumán. Facultad de Ciencias Naturale

Evolucion de la masa corporal en la filogenia de paleognatas actuales y fósiles

Palaeognathae es el clado basalde Neornithes, que incluye alas neotropicales Tinamidae o perdices, las ratites (las actualeskiwis, emúes, avestruces, casuares y ñandúes, y las recientementeextintas moas y aves elefantes), y otras aves fósiles comoLithornis,Palaeotisy Diogenornisdel Eoceno temprano (~55 millones de años). El tamaño corporal enel grupo varía desde grandes aves como la extinta Aepyornismaximus con un pesoestimado de 409 kgs a la perdiz enana Taoniscusnanus de 45 gramos. Eneste trabajo reconstruimos la evolución del tamaño corporal de laspaleognatas en base a la optimización de datos de masa corporalcuidadosamente curados de taxa actuales y fósiles. La estimaciónmás reciente del tamaño corporal del ancestro de paleognatas fue de 3,5-5 kgs y ese valor se hereda en el ancestro de Tinamidae. Encontraste, nuestra estimación de esta masa corporal ancestral esmayor, con numerosos cambios en los linajes de ratites y una fuertereducción en el linaje de Tinamidae.Fil: Bertelli, Sara Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - Tucumán. Unidad Ejecutora Lillo; Argentina. Fundación Miguel Lillo; ArgentinaFil: Giannini, Norberto Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - Tucumán. Unidad Ejecutora Lillo; Argentina. Universidad Nacional de Tucumán. Facultad de Ciencias Naturales e Instituto Miguel Lillo; ArgentinaXIII Reunion Argentina de Cladistica y BiogeografíaSan Miguel de TucumanArgentinaFundación Miguel LilloUniversidad Nacional de Tucumán. Facultad de Ciencias Naturale

Second record of <i>Colibri thalassinus</i> for Argentina

A Green Violetear <i>Colibri thalassinus</i> was mistnetted in <i>Alnus acuminata</i> forest at Catamarca province, northwestem Argentina. The previous record for this hummingbird was Olrog (1963) for Tucumán province. The individual captured was an adult, with neither molt nor brood patch

Structural and electrical properties of annealed Ge2Sb2Te5 films grown on flexible polyimide

The morphological, structural, and electrical properties of as-grown and annealed Ge2Sb2Te5 (GST) layers, deposited by RF-sputtering on flexible polyimide, were studied by means of optical microscopy, atomic force microscopy, X-ray diffraction, Raman spectroscopy, and electrical characterization. The X-ray diffraction annealing experiments showed the structural transformation of GST layers from the as-grown amorphous state into their crystalline cubic and trigonal phases. The onset of crystallization of the GST films was inferred at about 140 degrees C. The vibrational properties of the crystalline GST layers were investigated via Raman spectroscopy with mode assignment in agreement with previous works on GST films grown on rigid substrates. The electrical characterization revealed a good homogeneity of the amorphous and crystalline trigonal GST with an electrical resistance contrast of 8 x 10(6)