Liquid Galaxy POIs Controller

Per tal de contextualitzar la proposta del TFG, al llarg d'aquesta memòria, presentarem el programa Google Summer of Code (durant la memòria també referenciat com a “GSoC”). Així doncs, introduirem els conceptes de GSoC, algunes dades històricament significatives, l’estructura que el composa, la seva organització, el procediment per a escollir els membres que en formaran part i la meva relació amb el programa. També explicarem breument què és el projecte Open Source Liquid Galaxy (d’aquí en endavant anomenat també com “LG”), en què consisteix, la relació amb els dos projectes que vam presentar al GSoC 2015 (Liquid Galaxy POIs Controller, el qual va ser finalment escollit, i Wikipedia Liquid Galaxy Mashup) i algunes de les seves característiques. Posteriorment, essent el nucli d’informació més rellevant d’aquesta memòria, expliquem les tasques realitzades prèviament a l’inici del projecte com són l’aprenentatge de tecnologies i llenguatges nous, la presa de requeriments i riscs, l’establiment dels objectius del projecte i el càlcul de la línia de temps que guia aquest. Definim l’estructura de la base de dades de l’aplicació i exposem exemples del seu funcionament, expliquem el disseny i l’estructura de l’aplicació, tant en aspectes a nivell d’usuari com a nivell d’implementació, detallem l’evolució de l’aplicació juntament amb els problemes sorgits i les corresponents solucions i finalment determinem les tasques que s’han de realitzar per finalitzar la implementació de l’aplicació juntament amb una avaluació del treball realitzat

Expression and Function of the Lipocalin-2 (24p3/NGAL) Receptor in Rodent and Human Intestinal Epithelia

<div><p>The lipocalin 2//NGAL/24p3 receptor (NGAL-R/24p3-R) is expressed in rodent distal nephron where it mediates protein endocytosis. The mechanisms of apical endocytosis and transcytosis of proteins and peptides in the intestine are poorly understood. In the present study, the expression and localization of rodent 24p3-R (r24p3-R) and human NGAL-R (hNGAL-R) was investigated in intestinal segments by immunofluorescence and confocal laser scanning microscopy, immunohistochemistry and immunoblotting. r24p3-R/hNGAL-R was also studied in human Caco-2 BBE cells and CHO cells transiently transfected with r24p3-R by immunofluorescence microscopy, RT-PCR and immunoblotting of plasma membrane enriched vesicles (PM). To assay function, endocytosis/transcytosis of putative ligands phytochelatin (PC₃), metallothionein (MT) and transferrin (Tf) was assayed by measuring internalization of fluorescence-labelled ligands in Caco-2 BBE cells grown on plastic or as monolayers on Transwell inserts. The binding affinity of Alexa 488-PC₃ to colon-like Caco-2 BBE PM was quantified by microscale thermophoresis (MST). r24p3-R/hNGAL-R expression was detected apically in all intestinal segments but showed the highest expression in ileum and colon. Colon-like, but not duodenum-like, Caco-2 BBE cells expressed hNGAL-R on their surface. Colon-like Caco-2 BBE cells or r24p3-R transfected CHO cells internalized fluorescence-labelled PC₃ or MT with half-maximal saturation at submicromolar concentrations. Uptake of PC₃ and MT (0.7 µM) by Caco-2 BBE cells was partially blocked by hNGAL (500 pM) and an <i>EC</i>_<i>50</i> of 18.6 ± 12.2 nM was determined for binding of Alexa 488-PC₃ to PM vesicles by MST. Transwell experiments showed rapid (0.5-2 h) apical uptake and basolateral delivery of fluorescent PC₃/MT/Tf (0.7 µM). Apical uptake of ligands was significantly blocked by 500 pM hNGAL. hNGAL-R dependent uptake was more prominent with MT but transcytosis efficiency was reduced compared to PC₃ and Tf. Hence, r24p3-R/hNGAL-R may represent a high-affinity multi-ligand receptor for apical internalization and transcytosis of intact proteins/peptides by the lower intestine.</p> </div

Immunohistochemical labeling of r24p3-R in rat intestine.

<p>Very weak r24p3-R immunoreactivity was observed at the apical brush border of duodenum (<b>A</b>) and jejunum (<b>B</b>). Stronger labeling was observed in the apical brush border of ileum (<b>C</b>). Diffuse intracellular labeling was also observed in the ileum and colon (<b>D</b>). No labeling was detectable in any segment following pre-incubation of r24p3-R antibody with the immunizing peptide as demonstrated in ileum (<b>E</b>) or colon (<b>F</b>). Scale bar = 10µm. (<b>G</b>) Immunoblots of homogenates (H) and the membrane fraction (M) of different rat intestinal segments (D = duodenum; J = jejunum; I = ileum; C = colon) were performed with α-CT-24p3-R. A specific double band at ~60 kDa (<i>arrows</i>) was detected in ileum and colon, in accordance with immunohistochemical staining of rat intestine. For loading controls, the same membranes were reprobed with antibodies to β-actin.</p

NGAL-R mediates apical-to-basolateral transcytosis of PC3, MT and Tf in confluent Caco-2 BBE cell monolayers.

<p>Transcytosis of fluorescence-labelled ligands through confluent monolayers of Caco-2 BBE cells plated on Transwell culture inserts. Fluorescent ligands of hNGAL-R were added to the apical compartment at a final concentration of 700 nM and the apical decrease as well as the basolateral increase of the concentration of the fluorescent ligands was recorded over a period of 8 h (<b>A</b>, <b>C</b> and <b>E</b>). Confluent colon-like Caco-2 BBE monolayers display rapid NGAL-R dependent apical-to-basolateral transcytosis of A488-PC3 (<b>A</b>), A546-MT (<b>C</b>) and A546-Tf (<b>E</b>), which saturates at 4-8 h. Means ± SEM of 9-14 experiments are shown. Data are fitted to a one-phase exponential decay function. For further details, see Experimental Procedures. Experiments with or without 500 pM hNGAL in the apical compartment were also performed to determine the contribution of hNGAL-R to transcytosis (<b>B</b>, <b>D</b>, <b>F</b> and <b>G</b>). To obtain a quantitative estimate of the apical decrease and basolateral delivery of the fluorescent ligands due to transcytosis and to determine the impact of hNGAL on this process experimental data were integrated over a period of 8 h and expressed as “area under the curve” (AUC) (<b>B</b>, <b>D</b> and <b>F</b>). hNGAL significantly reduces the apical decrease of fluorescent ligand concentration. Means ± SEM of 9-14 experiments are shown; * <i>P</i><0.05; ** <i>P</i><0.01. A plot of the ratio of basolateral to apical AUC of fluorescent PC₃, MT and Tf, as shown in B, D and <b>F</b>, provides an estimate of transcytosis efficiency as well as the proportion of intracellular ligand trapping in the absence or presence of 500 pM hNGAL (<b>G</b>). In controls, intracellular A546-MT trapping is significantly increased compared to A488-PC₃ or A546-Tf, but not with hNGAL. The data are expressed as a percentage of apical AUC. Means ± SEM of 9-14 experiments are shown; * <i>P</i><0.05; ** <i>P</i><0.01; *** <i>P</i><0.001.</p

Expression of hNGAL-R in Caco-2 BBE cells.

<p>RT-PCR for hNGAL-R and GAPDH in colon-like Caco-2 BBE cells (<b>A</b>). A PCR product of 296 bp is amplified from colon-like Caco-2 BBE cell cDNA using specific primers for human NGAL-R and reverse transcriptase (+RT), but not in the control reaction without reverse transcriptase (-RT). The housekeeping gene human GAPDH is used as a control. A 326 bp PCR product is only amplified in the presence of reverse transcriptase (+RT). Immunoblotting of colon-like Caco-2 BBE cell homogenate (Ho) and plasma membranes (PM) (<b>B</b>). Specific signals are detected in PM of colon-like Caco-2 BBE cells with antibodies against hNGAL-R (α-CT-24p3-R; 1:500) and the α1-subunit of Na⁺,K⁺-ATPase (1:500). Live immunofluorescence staining of non-permeabilized colon- and duodenum-like Caco-2 BBE cells (<b>C</b> and <b>D</b>). Immunofluorescence staining with α-NT-24p3-R (1:100) reveals hNGAL-R expression (red fluorescence) at apical (asterisks) and lateral plasma membranes (arrows) of colon-like Caco-2 BBE cells (<b>C</b>). No staining for hNGAL-R is detected in duodenum-like Caco-2 BBE cells (<b>D</b>).</p

Distal Renal Tubules Are Deficient in Aggresome Formation and Autophagy upon Aldosterone Administration

<div><p>Prolonged elevations of plasma aldosterone levels are associated with renal pathogenesis. We hypothesized that renal distress could be imposed by an augmented aldosterone-induced protein turnover challenging cellular protein degradation systems of the renal tubular cells. Cellular accumulation of specific protein aggregates in rat kidneys was assessed after 7 days of aldosterone administration. Aldosterone induced intracellular accumulation of 60 s ribosomal protein L22 in protein aggregates, specifically in the distal convoluted tubules. The mineralocorticoid receptor inhibitor spironolactone abolished aldosterone-induced accumulation of these aggregates. The aldosterone-induced protein aggregates also contained proteasome 20 s subunits. The partial de-ubiquitinase ataxin-3 was not localized to the distal renal tubule protein aggregates, and the aggregates only modestly colocalized with aggresome transfer proteins dynactin p62 and histone deacetylase 6. Intracellular protein aggregation in distal renal tubules did not lead to development of classical juxta-nuclear aggresomes or to autophagosome formation. Finally, aldosterone treatment induced foci in renal cortex of epithelial vimentin expression and a loss of E-cadherin expression, as signs of cellular stress. The cellular changes occurred within high, but physiological aldosterone concentrations. We conclude that aldosterone induces protein accumulation in distal renal tubules; these aggregates are not cleared by autophagy that may lead to early renal tubular damage.</p></div

Primary antibodies used in the study.

<p>Rb  =  rabbit; Mo  =  mouse; Gt  =  goat; Ch  =  chicken.</p

Analysis of the colocalization between proteasome 20 s aggregates and HDAC6.

<p>A) Triple fluorescence labeling for proteasome 20 s (red), aggresome (HDAC6, green), and calbindin-D_28K (blue) merged with the corresponding DIC image. B) Magnification of tubular structures in the same image. C) Quantitation of the co-localization of proteasome 20 s immunoreactive punctae and the aggresome transfer protein HDAC6 (n = 5, n.s.).</p

LC MS/MS analysis of proteins pulled down by H95 preferentially in samples from aldosterone treated rats but not detected in IP without beads or without antibody.

<p>Ctrl: control, Aldo: aldosterone treated, n.d.: not detected.</p

Aldosterone administration increases proteasome numbers and labeling intensity in distal renal tubules.

<p>A) Double labeling for proteasomes (proteasome 20 s, green) and a marker for DCT (NCC, red) in renal cortex from control rats. B) Similar fluorescence labeling in renal cortex from aldosterone treated rats. C) Quantitation of the mean number of proteasome-containing punctae, the mean area of these, and the mean proteasome 20 s immunoreactivity in the control and aldosterone treated groups in DCT (Con and Aldo, as indicated, n = 5). D) Double labeling of proteasomes (proteasome 20 s, red) and a marker for CNT (calbindin-D_28K, blue) in renal cortex from control rats. E) Similar fluorescence labeling in renal cortex from aldosterone treated rats. F) Quantitation of the mean number of proteasome-containing punctae, the mean area of these, and the mean proteasome 20 s immunoreactivity in the control and aldosterone treated groups in CNT (Con and Aldo, as indicated, n = 5). * indicates statistical significance.</p