La gestión del agua y su influencia en la construcción del territorio

Programa Oficial de Doutoramento en Arquitectura e Rehabilitación. 501V01[Resumen] El agua, fuente de vida, es el elemento más importante para comprender los paisajes culturales. Su necesaria movilización mediante el empleo de la gravedad conforma territorios mediante un sabio manejo. El ser humano ha sido capaz de generar paisajes habitables y revertir las originales condiciones negativas naturales en positivas, planteando técnicas de gestión y de funcionamiento muy próximas al ciclo del agua y a la naturaleza. Estudiar estas técnicas descubre las aldeas como “espacios hidráulicos” que manejan el agua de su territorio. Se pone de relieve un sistema complejo, absolutamente medido y construido según la estricta disciplina del agua, gestionado en “man común” desde el “casal”, célula base de la comunidad. Las distintas unidades hidráulicas se articulan entre sí, siempre con la preminencia de las situadas aguas arriba de manera que las situadas aguas abajo se verán beneficiadas de los “reboses”, en un conjunto coherente donde los distintos elementos, presas y levada se acoplan entre sí de manera que el agua está en continuo movimiento y uso: “el agua no duerme”. Con esta lógica se han creado sistemas que permiten adaptarse a la variabilidad de la naturaleza utilizando el recurso hídrico como un proceso en el que interviene el ser humano. Lecturas que permiten no solo entender históricamente un modo de ocupación, sino que revelan un conocimiento riquísimo de las condiciones locales y dan información para el desarrollo de nuevas técnicas que incorporen estos sabios principios.[Resumo]A auga, fonte de vida, é o elemento máis importante para comprender as paisaxes culturais. A sua necesaria movilización mediante o empleo da gravedade conforma territorios mediante un sabio manexo. O ser humano foi capaz de xerar paisaxes habitables e revertir as orixinais condicións negativas naturais en positivas, planteando técnicas de xestión e de funcionamento moi próximas ó ciclo da auga e á natureza. Estudiar estas técnicas descubre as aldeas como “espacios hidráulicos” que manexan a auga do seu territorio. Ponse de relevo un sistema complexo, absolutamente medido e construido segundo a estricta disciplina da auga, xestionado en “man común” desde o “casal”, célula base da comunidade. As distintas unidades hidráulicas articulanse entre sí, sempre coa preminencia das situadas augas arriba de maneira que as situadas augas abaixo veranse beneficiadas dos “reboses”, nun conxunto coherente onde os distintos elementos, presas e levada acoplanse entre sí de maneira que a auga está en continuo movemento e uso: “a auga non durme”. Con esta lóxica crearonse sistemas que permiten adaptarse á variabilidade da natureza utilizando o recurso hídrico como un proceso no que interven o ser humano. Lecturas que permiten non só entender históricamente un modo de ocupación, senon que revelan un coñecemento riquísimo das condicións locais e dan información para o desenrolo de novas técnicas que incorporen estes sabios principios.[Abstract] Water, source of life, is the most important element for understanding cultural landscapes. Its necessary mobilization through the use of gravity creates territories through a wise management. The human being has been able to generate inhabitable landscapes and revert negative natural original conditions into positive conditions by proposing management and functioning techniques very close to the water cycle and nature. By studying these techniques, it is possible to discover villages such as the “Hydraulic spaces” that manage water within its territory. This fact empathizes a complex system, which is absolutely measured and constructed according to the strict discipline of water, managed in “man común” from the “casal”, which is the base cell of the community. The different hydraulic units are articulated amongst themselves, always with the preminence of those located upstream so that those located downstream will benefit from “overflows” in a coherent set where the different elements, dams and levada are coupled together so water is in ceaselessly ongoing and use: “water does not sleep”. With this logic, a system that allows to adapt to the variability of nature using water resources as a process in which human being intervenes has been created. An interpretation that not only allows to historically understand a mode of occupation, but reveals a rich knowledge of local conditions and provides information for the development of new techniques that incorporate these wise principles

Site-Specific Noncovalent Interaction of the Biopolymer Poly(ADP-ribose) with the Werner Syndrome Protein Regulates Protein Functions

Werner syndrome is a premature aging disorder that is caused by defects in the Werner protein (WRN). WRN is a member of the RecQ helicase family and possesses helicase and exonuclease activities. It is involved in various aspects of DNA metabolism such as DNA repair, telomere maintenance, and replication. Poly­(ADP-ribose) polymerase 1 (PARP1) is also involved in these processes by catalyzing the formation of the nucleic-acid-like biopolymer poly­(ADP-ribose) (PAR). It was previously shown that WRN interacts with PARP1 and that WRN activity is inhibited by PARP1. Using several bioanalytical approaches, here we demonstrate that the enzymatic product of PARP1, <i>i.e.</i>, PAR, directly interacts with WRN physically and functionally. First, WRN binds HPLC-size-fractionated short and long PAR in a noncovalent manner. Second, we identified and characterized a PAR-binding motif (PBM) within the WRN sequence and showed that several basic and hydrophobic amino acids are of critical importance for mediating the PAR binding. Third, PAR-binding inhibits the DNA-binding, the helicase and the exonuclease activities of WRN in a concentration-dependent manner. On the basis of our results we propose that the transient nature of PAR produced by living cells would provide a versatile and swiftly reacting control system for WRN’s function. More generally, our work underscores the important role of noncovalent PAR-protein interactions as a regulatory mechanism of protein function

Phenotypes of <i>ipis2</i>- and <i>ipis3</i>- in mosquito and blood infection stages.

(A) Midguts of mosquitoes were harvested 14 days after an infectious blood meal and microscopy was used to determine the presence or absence of oocysts. (B) The number of sporozoites per mosquito were calculated at least 17 days following infected blood meal. (C) Liver stage ipis2- and ipis3- parasites grow similarly to wild-type parasites in vitro. HepG2 cells infected with either wild-type, ibis2-[GFP-Luc;mCherry], or ibis3-[GFP-Luc;mCherry] sporozoites were fixed 48 hours after infection, and the parasites were stained with antibodies against PbHSP70. Exo-erythrocytic forms were imaged with the fluorescence microscope, and their size was determined using FIJI. ns, not significant, unpaired t-test. (D) Parasitemia in the blood of mice following infection with 1000 sporozoites was quantified by microscopy from Giemsa-stained blood smears. *, P ipis2- and ipis3- are less pathogenic in mice than wild-type P. berghei. Survival of mice after infection with 1000 sporozoite. n = 5 (ipis2-), n = 10 (ipis3-). ns, not significant **, P (TIF)</p

Generation of <i>ipis2-</i>[GFP-Luc;mCherry] and <i>ipis3</i>-[GFP-Luc;mCherry] was achieved through double homologous recombination and confirmed by PCR.

(A) Homology regions (labeled as the 5’ and 3’ genomic regions) flanking the gene of interest were cloned into the GOMO GFP-Luciferase vector. The resulting parasites lack either IPIS2 or IPIS3, express both mCherry and GFP-luciferase, and contain a DHFR-γFCU drug resistance cassette. (B) Successful integration of the construct was verified by PCR amplification of the products indicated in grey in panel A. Produ cts were sequenced to confirm the genomic editing. (C) Excision of the mCherry and drug resistance expression cassette from the recycled lines was confirmed by the presence of product using the mCherry excision (mCherry exc) and absence of the 3’ integration (3’) product. (TIF)</p

Primers used in this study.

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Localization of mCherry tagged PBANKA_0524300 (<i>IPIS2</i>), PBANKA_0623100 (<i>IPIS3</i>), and PBANKA_1400700 in the liver stage of infection.

(A) Hepatoma cells were infected with transgenic parasites expressing the indicated proteins fused to the fluorescent protein mCherry. After fixation at the time points indicated, the infected cells were labelled with an anti-RFP antibody to amplify the mCherry signal. Scale bars, 10 μm. (B) P. berghei expressing both GFP- and mCherry-tagged proteins following cross fertilization were imaged by confocal microscopy 48 hours after infection of hepatoma cells. Scale bars, 10 μm (TIF)</p

mCherry tagged PBANKA_0524300 (IPIS2), PBANKA_0623100 (IPIS3) colocalize with IBIS1-GFP at the liver-stage parasitophorous vacuole.

A) Protein features of candidate proteins that coprecipitated with IBIS1. B) Hepatoma cells were infected with sporozoites after the cross-fertilization of IBIS1-GFP and either PBANKA_0524300-mCherry, PBANKA_0623100-mCherry or PBANKA_1400700-mCherry transgenic parasite lines. Cells were fixed at indicated time points and the mCherry and GFP signals were amplified by using anti-RFP and anti-GFP antibodies. Scale bars, 10 μm.</p

PBANKA_0524300 (IPIS2), PBANKA_0623100 (IPIS3), PBANKA_1400700 and putative <i>P</i>. <i>vivax</i> orthologs localize to discrete structures in the cytoplasm of infected erythrocytes.

A) PBANKA_0524300-mCherry, PBANKA_0623100-mCherry, and PBANKA_1400700-mCherry colocalize with the IBIS marker IBIS1-GFP in the erythrocyte cytoplasm. Infected cells were fixed and labeled with anti-GFP and anti-mCherry antibodies to amplify the fluorescent signal. Scale bars, 5 μm. B) Erythrocytes infected with P. berghei expressing the HA-tagged P. vivax proteins PvTRAg8 or PvTRAg2, which are putative orthologs of IPIS2 and IPIS3, respectively, were fixed and labeled with anti-HA antibodies. Scale bars, 5 μm.</p

Integration of IPIS3-mCherry and IPIS2-mCherry expression into the <i>ipis2</i>- and <i>ipis3</i>- lines, respectively.

(A) ibis3-[GFP-Luc; PyrS] or ibis2-[GFP-Luc; PyrS] were used as recipient lines for transfection of IPIS2 and IPIS3 B3D+mCherry vectors, respectively. (B) Successful integration of the plasmid was confirmed by PCR. The absence of product for the 5’ and 3’ integration PCRs in the wild type, ipis2-[GFP-Luc;PyrS], and ipis3-[GFPLuc;PyrS] were negative controls. The PCR reactions labeled “IPIS2” or “IPIS3” amplified a region in the IPIS2 or IPIS3 genes, which are absent from the recipient knockout lines. (TIF)</p

Phylogeny of TrpThr domain-containing proteins.

Neighbor joining tree representing the phylogeny of TryThr domain containing proteins based on multiple sequence alignment of amino acid sequences using MUSCLE(ref). The tree was vizualized and annotated using iTOL (ref). For better representation branch length were ignored. The color strip indicates the Plasmodium strains from which the proteins originate. Proteins used in this study are highlighted in blue for Plasmodium berghei and in light red for Plasmodium vivax, respectively. (TIF)</p