Caída libre de los cuerpos (Simulación)

En este artículo se muestra la simulación por computador y sus resultados tanto gráficos como numéricos de la posición, velocidad y aceleración de uno o dos cuerpos que pueden ser lanzados hacia arriba o hacia abajo en forma simultánea o con tiempos diferentes, en el mismo planeta o en planetas diferentes, desde la misma posición o posiciones diferentes y en el mismo sentido o sentidos opuestosAbstract : In this article is shown the simulation by computer and their results graphic as well as numerical of the position, velocity and aceleration when an object or two objects are launched to up or to down simultaneous or times diferents in the same planet or diferents planets, from the same position or diferents positions and same sense or opposite senses

Knockdown of <i>Cep164</i> causes S-phase delay.

<p>(A–D) Endogenous <i>Cep164</i> knockdown leads to block in S-phase and is rescued by inducible human wild type <i>CEP164</i> but not by human mutant <i>CEP164</i>. Under thymidine synchronization, <i>Cep164</i> knockdown cells (black) were lagging in the transition from S to G₂/M phase in comparison to the control siRNA treated cells (white) in all IMCD3 <i>N-GFP-CEP164-WT</i> (A) IMCD3 <i>N-GFP-CEP164-Q525X</i> (B) and IMCD3 <i>N-GFP-CEP164-R93W</i> (C) cell lines. Upon doxycycline induction of wild type human CEP164 construct IMCD3 <i>N-GFP-CEP164-WT</i> (light grey) cells were rescued from arrest in S-phase (A). In contrast, overexpression of the cDNA clone that represents the human truncating mutant <i>N-GFP-CEP164-Q525X</i> cells did not rescue the S-phase lag. In addition, overexpression of <i>N-GFP-CEP164-Q525X</i> (dark grey) caused an increase of cells in S-phase, indicating a dominant negative effect of the human truncating mutant (B). Cells were not rescued by <i>N-GFP-CEP164-R93W</i> (C) from the S-phase lag upon doxycycline induction. P values were calculated using two-way ANOVA and Bonferroni multiple comparison test. (n = 3, *p<0.05). (D) Western blot showing expression of N-GFP CEP164 WT and R93W by CEP164 antibody and N-GFP CEP164 Q525X by GFP antibody upon doxycycline induction. β-actin is used as loading control. (E) Lysates of IMCD3 cells were made 1 hour after 10J UV exposure. Western blot was performed for Cep164 and loading control β-actin. Stabilization of Cep164 is visible after DNA damage induction (n = 3). (F) Lysates of RPE cells were made 56 hours after knockdown of <i>CEP164</i> with 20 nM siRNA. Upregulation of DNA damage marker γH2AX is shown on western blot with loading controls H2AX and β-actin (n = 3). (G) Lysates of thymidine synchronized IMCD3 cells were made 56 hours after knockdown of <i>Cep164</i> with 20 nM siRNA. Upregulation of DNA damage marker γH2AX is shown on western blot with loading control H2AX and β-actin 6 hours after release of the thymidine block (n = 3). (H) Urine derived renal cells stained for γH2AX (green) and CEP164 (magenta) of NPHP patient and unaffected control. γH2AX intensities were quantified by ZEN2011 software. T-test reveals statistical difference (***p<0.001) (n = 15). (I) S-phase marker PCNA expression is increased in RPE and IMCD3 cells transfected with siCep164/CEP164p and –i compared to control transfected cells. β-actin was used as loading control. APH exposure (18 hour, 400 nM) enhanced the PCNA expression levels in both cell lines.</p

CEP164 regulates cell cycle progression and proliferation.

<p>(A) RPE-FUCCI cells and their daughter cells were tracked by live cell imaging for 72 hours (hr) after transfection. <i>CEP164</i> depleted cells have a quicker cell cycle (∼35 hr) compared to control cells (∼48 hr) (>25 cells and their daughter cells per position (n = 3) per experimental condition per experiment (n = 3), One-way ANOVA (Dunnett's post hoc) (*p<0.05). Error bars represent SEM. (B) Each cell cycle stage in siControl and si<i>CEP164</i> transfected cells was measured. S-phase took significantly longer in si<i>CEP164</i> transfected cells and their daughter cells (8.6 hour) compared to control (5.7 hour) (**p<0.01). G₁-phase is significantly shorter in si<i>CEP164</i> transfected cells (10.7 hour) compared to control (20 hr) (*p<0.05). G₂- and M-phase were almost significantly shorter in si<i>CEP164</i> transfected cells (7.9 and 6.8 hr respectively) compared to control (11.6 and 11.1 hour respectively; both p = 0.06) (>25 cells and their daughter cells per position (n = 3) per experimental condition per experiment (n = 3), ± represent SEM, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004594#pgen.1004594.s001" target="_blank">Figure S1E</a> for details. (C) Fluorescence images of RPE-FUCCI cells expressing mKO2-hCdt1 (30/120) (magenta) and mAG-hGem (1/110) (green) constructs were pulsed for 30 minutes with EdU (10 µM) and stained with Alexa anti-EdU-647 to visualize cells in early S-phase (white) and DAPI to visualize the nuclei (blue). Cells expressing both constructs also show EdU incorporation (white). Scale bar represents 10 µm. (D) Quantification of a time series of serum released RPE-FUCCI cells after 24 hour serum starvation. Cell cycle stage and ciliation were quantified and correlated. Both decreased ciliary frequency and increased cell cycle entry were observed in <i>CEP164</i> depleted cells. Error bars represent SEM. (E) Quantification of RPE and IMCD3 cell proliferation using the CyQUANT NF Cell Proliferation Assay Kit. Fluorescence intensities of quadruplicate samples after 72 hours after transfection were measured. A significant reduction in cell number is visible after respectively <i>CEP164</i> and <i>Cep164</i> knockdown (n = 3, **p = 0.007, ***p = 0.001). Error bars represent SEM.</p

Schematic overview of signaling cascade involved in development of PKD and NPHP.

<p>Mutations in ciliary genes cause ciliary defects. Ciliary defects result in proliferation defects, loss of planar cell polarity (PCP) and deregulated cellular signaling. This causes cystic kidney disease. When the gene mutated is also involved in the DNA damage response (DDR) or DNA replication, impairments of these processes cause apoptosis, epithelial-to-mesenchymal transition (EMT) and consequently fibrosis in the NPHP patients.</p

Loss of Cep164 induces EMT.

<p>(A) Relative gene expression levels of epithelial marker E-cadherin as measured by RT-QPCR in IMCD3 cells, normalized to RPL27. Total RNA was isolated 6 days after transfection with siControl or siCep164 oligos with or without TGFβ incubation (5 ng/mL) in serum-free medium. After 6 days of transient transfection (siRNA transfection occurred at day 0 and day 3) E-cadherin mRNA levels are significantly decreased. (**p<0.01; n = 4, error bars represent SEM). (B) Relative gene expression levels of mesenchymal marker Snail as measured by RT-QPCR in IMCD3 cells, normalized to RPL27. Total RNA was isolated 6 days after transfection (siRNA transfection occurred at day 0 and day 3) with siControl or siCep164 oligos with or without TGFβ incubation (5 ng/mL) in serum-free medium. After 6 days of transient transfection Snail mRNA levels (*p<0.05) are increased. (n = 4, error bars represent SEM). (C) Representative scratch healing images of IMCD3 cells taken by light microscope before and 18 h after a scratch was made in a confluent monolayer of cells. The white solid lines represent the wound edges at t = 0 h and the white dashed lines indicate the edges at t = 18 h. Scale bar represents 200 µm. (D) Quantification of absolute distance (µm) of cell migration after 18 hours after a scratch. IMCD3 <i>Cep164</i> depleted cells migrate (*p<0.05) more than siControl cells 48 hour after transfection. TGFβ incubation (5 ng/mL) in serum-free medium enhances this effect (n = 4, error bars represent SEM). P-values were calculated using two-way ANOVA and Bonferroni multiple comparison test. (E–F) Lysates of IMCD3 <i>N-GFP-CEP164-Q525X</i> cells treated with doxycycline were made 24 hours after addition to the culture medium. Western blot was performed for Snail and γH2AX and loading controls β-actin and H2AX respectively. Upregulation of both Snail and γH2AX are visible after induction of dominant negative allele <i>N-GFP-CEP164-Q525X</i> (n = 3). Quantification of the protein expression of Snail (Student's t-test *p<0.05) and γH2AX (**p<0.01) in lysates of IMCD3 <i>N-GFP-CEP164-Q525X</i> cells normalized to loading control β-actin and H2AX respectively was performed using Image Lab software (n = 3, error bars represent SEM).</p