Individual Professional Practice in the Company

Import 23/08/2017Cílem této bakalářské práce je popsat absolvování odborné praxe ve firmě HS Interactive s.r.o. Praxe byla zaměřena na vývoj mobilní aplikace pro operační systém Android. Aplikace je mobilním klientem pro sociální síť MatchToMe. V úvodu popisuji důvody, které vedly k výběru odborné praxe. Dále se věnuji úkolům, které mi byly zadány s jejich implementací a postupem řešení problémů, které se objevily při vývoji. Závěr práce je věnován zhodnocení získaných zkušeností a dosažených výsledků.Purpose of this bachelor thesis is to describe a professional practice in company HS Interactive s.r.o. Practice was focused on the development of mobile application for the operating system Android. The application is a mobile client for social network MatchToMe. In the introduction I describe reasons that led to the selection of professional practice. Then I describe tasks that I have been awarded with their implementations and process of solution issues that have emerged during development. The conclusion of thesis is dedicated to the evaluation of the experience gained and the results achieved.440 - Katedra telekomunikační technikyvýborn

Endogenous ATGL-1 Protein is More Abundant in AMPK-deficient Dauer Larvae.

<p><b>(A)</b> The ATGL-1 antisera recognizes a single band that migrates at approximately 70kD, which corresponds to the molecular weight of ATGL-1, and is eliminated in <i>atgl-1(RNAi)</i> animals. <b>(B)</b> Western blot analysis with <i>C</i>. <i>elegans</i> specific ATGL-1 antibody in <i>daf-2; aak(0)</i> mutant dauer larvae and control <i>daf-2</i> dauer larvae during the period of dauer entry. <b>(C)</b> mRNA Expression levels of ATGL-1 in control <i>daf-2</i> and <i>daf-2; aak(0)</i> dauers. Relative mRNA levels were analyzed by quantitative real-time PCR in dauer day 0 animals.</p

Variation in phosphatidic acid (PA) levels is associated with reduced C20PUFA abundance.

<p><b>(A)-(B)</b> PA levels are low in both the lipid droplets and cytoplasm of <i>daf-2; aak(0); cgi-58</i> <b>(A)</b> and <i>daf-2; cgi-58</i> <b>(B)</b> dauer larvae, and are not affected by dietary supplement of C20PUFAs. <b>(C)-(F)</b> Fluctuation in PA levels achieved by eliminating enzymes involved in either PA turnover (LPIN-1), or biosynthesis (ACL-1) resulted in reduced C20PUFA levels in the lipid droplets of <i>daf-2; aak(0)</i> (C), (E) and <i>daf-2</i> (D), (F) dauer larvae. LD = lipid droplet. <b>(G)</b> PA levels were unchanged in <i>CGI-58</i> MEF mutants.</p

The Major AMPK Phosphorylable Site Ser303 on ATGL-1 Is Important for Its Intestinal Expression and Proteasome Degradation.

<p><b>(A)</b> Expression patterns of ATGL-1::GFP and ATGL-1(S303A)::GFP were compared in control <i>daf-2</i> dauer larvae. Unlike ATGL-1::GFP, some ATGL-1(S303A)::GFP is expressed in hypodermis and intestine in S303A variants (white arrowheads in the insets). Scale bar = 10μm. Insets were generated by selecting the same size of frame and amplified to the same magnification. <b>(B)</b> Western blot analysis of GFP levels obtained from ATGL-1(S303A)::GFP in isolated lipid droplets (L) and cytoplasm (C) in control <i>daf-2</i> dauer day 0 larvae. <b>(C)</b> Western blot analysis of GFP levels obtained from ATGL-1::GFP and ATGL-1(S303A)::GFP in control <i>daf-2</i> dauer day 0 larvae. <b>(D)</b> Western blot analysis of GFP levels obtained from ATGL-1::GFP and ATGL-1(S303A)::GFP in control <i>daf-2</i> dauer larvae at different time points. <b>(E)</b> Quantiifcation of GFP mRNA levels in <i>daf-2; ATGL-1</i>::<i>GFP</i> and <i>daf-2; ATGL-1(S303A)</i>::<i>GFP</i> dauer day 0 larvae using semi-quantitative RT-PCR. <i>act-1</i> was used as loading control. <b>(F)</b> Immunoprecipitation of ATGL-1::GFP and ATGL-1(S303A)::GFP using GFP antibody from lysates obtained from <i>daf-2;</i> ATGL-1::GFP and <i>daf-2;</i> ATGL-1(S303A)::GFP dauer larvae was analyzed by western blot analysis using anti-ubiquitin antibody. “IP:GFP” refers to the protein lysate that was subjected to immunoprecipitation with an anti-GFP antibody. <b>(G)</b> Immunoprecipitation of ATGL-1::GFP and ATGL-1(S303A)::GFP using GFP antibody from protein lysates obtained from <i>daf-2;</i> ATGL-1::GFP and <i>daf-2;</i> ATGL-1(S303A)::GFP dauer larvae were subjected to Western analysis using PAR-5 antibody.</p

ATGL-1::GFP Expressed at a Higher Level in AMPK-deficient Animals during the Early Dauer Stage.

<p><b>(A)</b> Comparison of ATGL-1::GFP levels between control <i>daf-2</i> and <i>daf-2; aak(0)</i> animals during the early dauer stage. Dauer day 1 is defined as 72 hours after shifting to restrictive temperature (25°C) at the L1 stage (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130480#sec002" target="_blank">materials and methods</a>). Scale bar = 10μm. <b>(B)</b> Western blot analysis of GFP levels in control <i>daf-2</i> and <i>daf-2; aak(0)</i> mutant animals during early dauer stage. <b>(C)</b> Western blot analysis of endogenous ATGL-1 levels in control <i>daf-2</i> and <i>daf-2; aak(0)</i> mutant animals during early dauer stage. <b>(D)</b> Quantification of ATGL-1::GFP mRNA levels in control <i>daf-2</i> and <i>daf-2; aak(0)</i> mutant dauer day 4 animals using semi-quantitative RT-PCR. <i>act-1</i> was used as loading control.</p

ATGL-1::GFP Accumulates to Higher Levels in AMPK-deficient Animals Prior to Dauer Entry.

<p><b>(A)</b> Comparison of ATGL-1::GFP levels between control <i>daf-2</i> and <i>daf-2; aak(0)</i> animals during the dauer entry period. Images were taken with a Zeiss 510 Meta Confocal Laser Microscope at x20 magnification. All strains harbor the same <i>hjIs67[Patgl-1</i>::<i>atgl-1</i>::<i>GFP]</i> transgenic array in <b>(A)</b> and <b>(B)</b>. Scale bar = 20μm. <b>(B)</b> Western blot analysis of ATGL-1::GFP levels as measured using an anti-GFP antibody in control <i>daf-2</i> and <i>daf-2; aak(0)</i> mutant animals during the period prior to dauer entry. <b>(C)</b> Quantification of ATGL-1::GFP mRNA levels in control <i>daf-2</i> and <i>daf-2; aak(0)</i> mutant dauer day 0 animals using semi-quantitative RT-PCR. <i>act-1</i> was used as loading control.</p

Elimination of <i>cgi-58</i> rescued both triglyceride levels and excretory defects in <i>daf-2; aak(0)</i> mutant Dauer larvae.

<p><b>(A)</b> Protein alignment of the two isoforms of <i>C</i>. <i>elegans</i> CGI-58 (C37H5.a and C37H5.b) with human CGI-58. The red underlined amino acids indicate the deleted region in the <i>ok3245</i> allele. <b>(B)</b> Elimination of <i>cgi-58</i> by RNAi feeding or by using the <i>ok3245</i> allele significantly enhanced the survival of <i>daf-2; aak-1; aak-2</i> mutant dauer larvae. <i>aak-1; aak-2</i> is represented as <i>aak(0)</i> in all Figs. A Log-rank (Mantel-Cox) test was used to statistically compare the survival curves in all Figs. <b>(C)</b> Mutation of <i>cgi-58</i> in control <i>daf-2</i> animals significantly enhanced their survival during dauer stage (P = 0.0003). <b>(D)</b> <i>cgi-58</i> protects the triglyceride stores from depletion in <i>daf-2; aak(0)</i> mutant dauer larvae. Colorimetric analysis of triglyceride content in day 4 dauer larvae. ** indicates statistical significance (P<0.01) compared to all four of the other genotypes using one-way ANOVA followed by a Tukey HSD test. Error bars indicate SD of three independent experiments. The same statistical analysis was applied for all subsequent experiments performed hereafter. <b>(E)</b> Elimination of <i>cgi-58</i> protects the triglyceride stockpile in <i>daf-2; aak(0)</i> mutant dauer larvae. Oil Red O staining of day 4 dauer larvae. Arrowhead indicates the junction between the pharynx (left) and the intestine (right). Oil Red O staining intensity was evaluated by measuring optical density. * and ** indicate statistical significance (P<0.05 and P<0.01, respectively) compared to <i>daf-2; aak(0)</i> mutant dauer larvae. Error bars indicate SD of 20 animals. Scale bar = 10 μm. <b>(F)</b> Osmoregulatory defects typical of AMPK dauer larvae were corrected by reducing <i>cgi-58</i> function. <i>cgi-58</i> compromise restores osmoresistance of day 4 <i>daf-2; aak(0)</i> mutant dauer larvae following culture in varying NaCl concentrations for 24 hours at 25°C. The data represent a pool of three independent experiments. Survival of <i>daf-2</i>, <i>daf-2; aak(0) cgi-58(RNAi)</i> and <i>daf-2; aak(0); cgi-58</i> dauers is significantly higher than <i>daf-2; aak(0)</i> dauers at concentrations of both 1M and 1.5M NaCl, analyzed using one-way ANOVA followed by a Tukey HSD test.</p

CGI-58 interacts with ATGL-1 and tethers it to the lipid droplets.

<p><b>(A)</b> CGI-58::GFP was expressed in the hypodermis and the intestine at comparable levels in both control <i>daf-2</i> and <i>daf-2; aak(0)</i> mutant dauer larvae. Both strains carry the same <i>ex[Pcgi-58</i>::<i>cgi-58</i>::<i>GFP; rol-6(gf)]</i> transgene (See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005284#sec010" target="_blank">Materials and Methods</a>). Scale bar = 20 μm. <b>(B)</b> CGI-58::GFP localizes to the surface of the lipid droplets at dauer day 0 (48 hours after being shifted to 25°C at the L1 stage) in both <i>daf-2</i> and <i>daf-2; aak(0)</i> mutant dauers. Both strains carry the same <i>ex[Pcgi-58</i>::<i>cgi-58</i>::<i>GFP; rol-6(gf)]</i> transgene. Scale bar = 10μm. Insets (B' and B") were generated by selecting the same size of frame for each image followed by proportionate amplification to the same magnification. <b>(C)</b> Optimal ATGL-1 lipase activity requires <i>cgi-58</i>. ATGL-1-dependent lipase activity was determined in <i>daf-2; aak(0)</i> mutant dauer larvae with wild type or compromised <i>cgi-58/atgl-1</i> function. ** indicates statistical significance (P<0.01) compared to all three of the other genotypes. Error bars indicate SD of three independent experiments. <b>(D)</b> Elimination of <i>cgi-58</i> resulted in the dissociation of ATGL-1 from the lipid droplets. Both strains carry the same <i>hjIs67[Patgl-1</i>::<i>atgl-1</i>::<i>GFP]</i> transgene. Scale bar = 10μm. Insets (D' and D") were generated by selecting the same size of frame for each image followed by proportionate amplification to the same final magnification. <b>(E)</b> ATGL-1 association with the lipid droplets is dependent on appropriate CGI-58 levels. Immunoblot analysis was used to determine the levels of ATGL-1 in isolated lipid droplets (L) and cytoplasm (C) obtained from total day 0 dauer extracts of each genotype. Protein concentration was measured and 30μg of total protein was loaded in each sample lane. Actin was used as a loading control for the total protein level. <b>(F)</b> CGI-58 does not contribute to ATGL-1 stability in AMPK mutant dauers. ATGL-1 levels were determined by immunobloting using anti-ATGL-1 antisera in lysates obtained from AMPK mutants with or without <i>cgi-58</i>. <b>(G)</b> CGI-58 physically interacts with ATGL-1 in vivo in both control and AMPK mutant dauers. Co-immunoprecipitations were performed with <i>daf-2</i> and <i>daf-2; aak(0)</i> day 0 dauer larvae carrying the same <i>ex[Pcgi-58</i>::<i>cgi-58</i>::<i>GFP;rol-6(gf)]</i> transgene using anti-ATGL-1 serum for pull down and blotted with anti-GFP serum.</p

AMPK-Mediated Phosphorylation of ATGL-1 Enhances Ubiquitin-Mediated Degradation via the Proteasome.

<p><b>(A)-(C)</b> ATGL-1 protein levels are regulated by AMPK by enhancing ubiquitin-mediated proteasomal degradation. Individual proteasome components were compromised through RNAi, of which many, but not all, increase the levels of ATGL-1 in control <i>daf-2</i> dauer larvae. <b>(D)</b> Ubiquitylated ATGL-1 intermediates accumulate in <i>daf-2; aak(0)</i> mutant dauer larvae. Immunoprecipitation of ATGL-1 from lysates obtained from both control <i>daf-2</i> and <i>daf-2; aak(0)</i> mutant dauer larvae was analyzed by western blot analysis using anti-ubiquitin antibody. High molecular weight ubiquitin-conjugated entities are seen in immunoprecipitates obtained from <i>daf-2; aak(0)</i> but are more prominent in the <i>daf-2</i> lysates. More lysate was loaded for control <i>daf-2</i> animals to equalize the amount of ATGL-1 protein loaded with that of <i>daf-2; aak(0)</i> animals. “IP:ATGL-1” refers to the protein lysate that was subjected to immunoprecipitation with our anti-ATGL-1 polyclonal antibody. “Input” refers to the total protein lysate before performing the immunoprecipitation step.</p

ATGL-1 Compromise Causes Enlarged Lipid Droplets in <i>C</i>. <i>elegans</i> Dauer Larvae.

<p><b>(A)-(F)</b> Disruption of ATGL-1, but not HSL function in both control <i>daf-2</i><b>(A)</b>, <b>(C)</b> and <b>(E)</b> and <i>daf-2; aak(0)</i><b>(B)</b>, <b>(D)</b> and <b>(F)</b> dauer day 0 animals (48 hours after shifting to restrictive temperature since L1 stage) caused increase in lipid droplet size. Animals were stained with C₁-BODIPY-C₁₂. These and all subsequent images were taken with a Zeiss 510 Meta Confocal Laser Microscope at x40 magnification using identical microscope settings, unless specified otherwise. Scale bar = 10μm. Insets were generated by selecting the same size of frame on each image and amplified to the same magnification. <b>(G)</b><i>atgl-1</i>(RNAi) feeding reduces endogenous ATGL-1 protein substantially. A Western blot probed with an anti-ATGL-1 polyclonal antibody raised against endogenous ATGL-1 was used to quantify ATGL-1 levels in control <i>daf-2</i> animals and <i>daf-2</i> animals subjected to feeding <i>atgl-1(RNAi)</i>. <b>(H)-(I)</b> Quantification of the volume <b>(H)</b> and number <b>(I)</b> of C₁-BODIPY-C₁₂-stained lipid droplets using AxioVision (Zeiss) software. ** indicates statistical significance (P<0.0001) and ns indicates not significant using the unpaired t test compared to control <i>daf-2</i> and <i>daf-2; aak(0)</i> animals respectively. Error bars indicate SD of three independent experiments.</p