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

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

Proteomic Analysis of the Ubiquitin Landscape in the <i>Drosophila</i> Embryonic Nervous System and the Adult Photoreceptor Cells

<div><p>Background</p><p>Ubiquitination is known to regulate physiological neuronal functions as well as to be involved in a number of neuronal diseases. Several ubiquitin proteomic approaches have been developed during the last decade but, as they have been mostly applied to non-neuronal cell culture, very little is yet known about neuronal ubiquitination pathways <i>in vivo</i>.</p><p>Methodology/Principal Findings</p><p>Using an <i>in vivo</i> biotinylation strategy we have isolated and identified the ubiquitinated proteome in neurons both for the developing embryonic brain and for the adult eye of <i>Drosophila melanogaster</i>. Bioinformatic comparison of both datasets indicates a significant difference on the ubiquitin substrates, which logically correlates with the processes that are most active at each of the developmental stages. Detection within the isolated material of two ubiquitin E3 ligases, Parkin and Ube3a, indicates their ubiquitinating activity on the studied tissues. Further identification of the proteins that do accumulate upon interference with the proteasomal degradative pathway provides an indication of the proteins that are targeted for clearance in neurons. Last, we report the proof-of-principle validation of two lysine residues required for nSyb ubiquitination.</p><p>Conclusions/Significance</p><p>These data cast light on the differential and common ubiquitination pathways between the embryonic and adult neurons, and hence will contribute to the understanding of the mechanisms by which neuronal function is regulated. The <i>in vivo</i> biotinylation methodology described here complements other approaches for ubiquitome study and offers unique advantages, and is poised to provide further insight into disease mechanisms related to the ubiquitin proteasome system.</p></div

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

<i>ipis2-</i> and <i>ipis3-</i> contribute to blood-stage growth efficiency during <i>P</i>. <i>berghe</i>i infection.

A) The appearance of ipis3- parasites in the blood following sporozoite injection is delayed in comparison to wild-type. C57BL/6 mice were infected with 1,000 sporozoites of either ipis2-, ipis3- or wild-type parasites, and blood-stage infections were monitored starting from day 3 post-infection with Giemsa-stained blood smears. Statistics: Log-rank (Mantel-cox) test; n = 5 (ipis2-), n = 10 (ipis3-). B) The growth of ipis2- and ipis3- parasites in the liver does not differ significantly from wild-type P. berghei. The relative parasite burden in the liver was measured by qPCR of RNA extracted from livers forty-two hours after intravenous injection of either ipis2-, ipis3-, or wild-type sporozoites. Levels of Pb18s rRNA were normalized to levels of mouse GAPDH RNA in each infected liver. Statistics: unpaired two-tailed t-test; n = 5; ns: not significant. C) The ipis2- and ipis3- lines do not grow as efficiently as wild type in the blood of co-infected animals. Mice were infected with equal amounts of wild-type P. berghei-infected erythrocytes and ipis2-[mCherry; PyrS] or ipis3-[mCherry; PyrS] infected erythrocytes. Parasitemia was measured by flow cytometry between days 4–7 after infection. * p ipis2- line, a statistically significant increase in mCherry signal was detected; however the extent was negligible. * p <0.05, unpaired two-tailed t-test.</p

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

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

Mass-spec results of immunoprecipitation experiments.

(XLSX)</p

Immunoprecipitation of <i>P</i>. <i>berghei</i> proteins from infected red blood cells identifies proteins that co-precipitate specifically with IBIS1-mCherry and EXP2-mCherry.

A) The indicated mCherry-tagged proteins were immunoprecipitated from lysates of late stage P. berghei using RFP-specific nanobodies and co-precipitating proteins were identified by LC-MS/MS. B) P. berghei proteins that were detected in the IBIS1-mCherry sample in at least two of the experimental replicates are depicted in the heatmap, which indicate the mean normalized LFQ intensities of detected proteins of three independent experiments. Each row represents a protein detected in the IBIS1-mCherry eluates. The columns represent the immunoprecipitates prepared from the mCherry-expressing parasite lines indicated in (A) (1: IBIS1-mCherry, 2: mCherry, 3: PEXEL-mCherry, 4:EXP2-mCherry). Proteins that were enriched in the IBIS1-mCherry pulldown relative to at least two of the three controls (top 10% of the normal distribution) are annotated. Those in bold were enriched relative to all three controls. Proteins marked with a pink dot were followed up further in this study. C) P. berghei proteins that were detected in the EXP2-mCherry sample in at least two of the experimental replicates are depicted in the heatmap, which indicate the mean normalized LFQ intensities of detected proteins of three independent experiments. Each row represents a protein detected in the EXP2-mCherry eluates. The columns represent the immunoprecipitates prepared from the different mCherry-expressing parasite lines indicated in (A). Proteins marked with dark blue dots are confirmed to play roles in PTEX-mediated export, and those marked with light blue dots were previously found biochemically associated with components of the PTEX complex [33,34].</p

IPIS2 contributes to efficient sequestration of schizonts from the peripheral circulation.

A) ipis2- or ipis3- infection increases spleen weight more than wild-type P. berghei infection. Mice were infected with sporozoites of either wild type (n = 10), ipis2-[GFP-Luc; PyrS] (n = 4), or ipis3-[GFP-Luc; PyrS] (n = 10) lines, and 8 days after infection the spleens were dissected and weighed. The spleen weights were plotted as ratios to mouse body weight.* p P. berghei during synchronous infections. Mice were infected with P. berghei schizonts (wild-type, ipis2-[GFP-Luc; PyrS] or ipis3-[GFP-Luc; PyrS]) and the parasite stages detected in peripheral blood smears were quantified at the indicated time points following infection. n = 5 mice in three experimental replicates. Statistical annotations (* p P. berghei schizonts (wild-type Pb GFP-Luc, ipis2-[GFP-Luc; PyrS] or ipis3-[GFP-Luc; PyrS]). Values are normalized to organs from wild-type-infected mice.** p P. berghei do not bind efficiently to CD36-expressing CHO cells. Schizonts were incubated with CHO cells and the number of infected-red blood cells bound per cell quantified. The background binding to control CHO cells was subtracted, and the values were normalized to wild-type-infected red blood cell binding. The depicted data comprises three experimental replicates with three technical replicates for each parasite line and CHO cell line. ** p < 0.01, ns: not significant, unpaired two-tailed t-test.</p