Overview Of Data Over Digital Subscrieber Line In Czech Republic.

Import 22/07/2015Tato bakalářská práce je zaměřena na rozbor technologie ADSL a to, jak je využívána a nabízena vybranými poskytovateli v České republice k užívání veřejným sektorem. V teoretické části pojednává o technologii ADSL a jejích vývojových verzích. Dále se zabývá řešením přípojky a vlivů rušení na vedení těchto přípojek. Hlavní částí práce je část praktická, ta se zabývá průzkumem poskytovatelů v České republice. Konkrétně využitím digitálních účastnických smyček poskytovateli, jejich nabídkou ADSL služeb pevného připojení k internetu a také doplňkových služeb a poplatků s pevným připojením spojených. Poslední částí práce je analýza možností připojení těchto služeb na konkrétní přípojku. Obsah této práce má napomoci běžným uživatelům k pochopení dané problematiky spojené se zřízením a výběrem ADSL služeb. Prostředky k řešení praktické části práce jsou především komunikace s poskytovateli ADSL služeb a získávání informací.This bachelor thesis is focused on the analysis of the technology ADSL as it is used in the public sector and offered by chosen providers in Czech Republic. In the theoretical part, it refers to the technology ADSL and its developmental versions. Furthermore, it examines the connection solution and the influence of interruption on conducting these connections. The main part of the thesis is the practical part which undertakes the research of providers in Czech Republic. Namely, it explores use of digital subscriber loops by providers, theirs ADSL service offers of the stable internet connection and its complementary services and charges. The final part of the thesis is the connection analysis of these complementary services on the specific connection. The content of this thesis should provide help to common users to understand given issues related to the selection and setting up ADSL services. Tools used for the practical part are particularly communication with ADSL service providers and acquiring appropriate information.440 - Katedra telekomunikační technikyvýborn

<i>SCAFI</i> allele status and its effect on supercomplex assembly in <i>Bcs1l</i> mutant mice (G/G) of different genetic backgrounds.

<p>Wild-types (A/A) and <i>Bcs1l</i> mutant mice (G/G) of 129/Sv:C57BL/6 mixed background (MB) and C57BL/6 background (BC) were analyzed and a wild-type of the 129/Sv strain (129/Sv) was used as a control. (A) Agarose gel electrophoresis of SCAFI genotyping PCR products showing the long (<i>SCAFI</i> long) and short (<i>SCAFI</i> short) alleles in mice of different genetic backgrounds. (B) Western blot analysis of SCAFI protein and ETFAα (electron transfer flavoprotein alpha) as loading control in liver tissue lysate showing reduced level of SCAFI protein in mixed background (MB) mice and no detectable protein in C57BL/6 mice (BC). (C) Respiratory chain complexes and supercomplexes were separated on BNGE and detected by Western blot using the antibodies indicated. Molecular weights are indicated on the right. Note that the COX1 antibody required an extended exposure time for detection in supercomplexes. ETFAα was used as a loading control. NDUFV1 antibody shows in mutant (G/G) mice abundant free CI, in all mice CI/CIII_2, and in wild-type MB and 129/Sv that this is overlapping with the band for CI/CIII₂/CIV. Core1 shows pre-CIII₂ and CIII₂, and RISP fully assembled CIII₂ indicating presence of CIII in all supercomplexes of 129/Sv and wild-type MB animals, but mainly in supercomplex CI/CIII₂ in mutant (G/G) mice of both backgrounds. CIV antibody shows a clear band in respirasome in 129/Sv mouse, but of varying intensity in MB wild-type mice in relation to the loading control (ETFAα).</p

<i>SCAFI</i> genotype and age at end-stage disease (age at sacrificing) of homozygous (<i>Bcs1l</i>^<i>G/G</i>) mice in different genetic backgrounds.

<p><i>SCAFI</i> genotype and age at end-stage disease (age at sacrificing) of homozygous (<i>Bcs1l</i>^<i>G/G</i>) mice in different genetic backgrounds.</p

2D-BNGE with Western blot detection showing supercomplex composition in mitochondria of the three mouse strains studied.

<p>Antibodies against CIII (RISP) and CIV (COX1) subunits were initially used and after stripping the antibody against Core1. After a second stripping the antibody against CI (NDUFA9) was used. Supercomplexes are indicated by vertical boxes. (A) In the 129/Sv wild-type mouse, CIV was present in two supercomplexes. (B) Both CIV containing supercomplexes were present in wild-type mice (<i>Bcs1l</i>^<i>A/A</i>) of 129/Sv:C57BL/6 mixed background strain (MB with <i>SCAFI</i>^{<i>long/short</i>}). In mutant (<i>Bcs1l</i>^<i>G/G</i>) mice, CIV was decreased in the respirasome CI/CIII₂/CIV and more abundant in the formation of pre-CIII₂/CIV. (C) In backcrossed (BC) C57BL/6 congenic strain (with <i>SCAFI</i>^{<i>short/short</i>}), CIV was very faintly present in supercomplexes even when exposure time was increased compared to MB. In homozygotes (G/G) of both backgrounds, a CI₂/CIII₂ supercomplex was found and free CI was more abundant than in wild-type (assessed with densitometry: 26% in 129/Sv, 27% vs. 49% in MB and 48% vs. 56% in BC wild-type and homozygotes, respectively), as also shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168774#pone.0168774.g002" target="_blank">Fig 2</a>.</p

BNGE analysis.

<p>Comparison of isolated liver mitochondria from <i>Bcs1l</i> mutant homozygotes (G/G) and wild-types (A/A) of 129/Sv:C57BL/6 mixed background (MB), C57BL/6 backcrossed (BC) strain and the 129/Sv strain. Four supercomplex bands (SC) are visible in wild-type animals in MB and 129/Sv strains, one of the bands not found in C57BL/6 animals. In homozygotes (G/G) in both MB and BC strains, free complex I (CI) is more abundant than in wild-type animals (A/A), and free complex III (CIII) is only present as a pre-complex (PreCIII₂).</p

Quantitation of <i>DmGfrl</i> null female fertility and oogenesis phenotype.

<p>(A) <i>delDmGfrl</i> females displayed markedly reduced fertility. The absolute fertility of <i>delDmGfrl/Df1</i> transheterozygous females was reduced by ∼60% as compared to heterozygous control females. Their fecundity (measured as the average number of progeny produced by individual females in a given time) was reduced to ∼10% of the fecundity of control females. (B) Morphology of eggs laid by control, <i>delDmGfrl</i> and rescue females. Uppermost row shows the morphology of heterozygous (<i>delDemGfl/+</i>) control eggs. Second row exemplifies the morphology of eggs laid by <i>DmGfrl</i> null females (<i>delDmGfrl/Df1</i>). The eggs were small and often translucent (not visible in this image), and ∼60% of them display lack of or abnormal dorsal appendages (arrows). <i>LacZ</i> transgene under the <i>daughterless</i> (<i>da)</i> driver (3^rd row) did not rescue the egg morphology, whereas eggs laid by females expressing the <i>DmGfrl</i> transgene under the <i>da</i> driver in <i>DmGfrl</i> null background (4^th row) were almost fully wild-type by appearance. (C) Quantitation of the size of eggs laid by control, <i>delDmGfrl</i> and rescue females. Average egg length was reduced from 0.528 mm in heterozygous control eggs (female genotype <i>delDmGfrl/+)</i> to 0.466 mm in eggs laid by homozygous <i>delDmGfrl</i>/<i>delDmGfrl</i> females and to 0.450 mm in eggs laid by <i>delDmGfrl/Df1</i> females (1^st to 3^rd bars). <i>DmGfrl</i> transgene (UAS-Tg), but not <i>LacZ</i> transgene (UAS-LacZ), partially rescued the dumpless-like phenotype (4^th and 5^th bars). Statistical significance from Tukey’s post hoc test after one-way ANOVA are shown with asterisk (*) with respect to the <i>del/+</i> genotype and hash (#) with respect to the transgene rescue genotype (<i>del da-G4/del UAS-DmGfrl</i>). (D) Quantitation and rescue of the malformed egg phenotype. The percentage of malformed eggs laid by <i>DmGfrl</i> null females, displaying either dumpless-like phenotype or malformed dorsal appendages or both, was ∼60–70% depending on the genetic background (2^nd, 4^th and 5^th bars). Expression of DmGfrl under the <i>da-GAL4</i> driver diminished the percentage of malformed eggs from 63% (driver only, 4^th bar) to ∼3% (driver and transgene, 6^th bar). Expression of <i>LacZ</i> transgene in the same background did not rescue the egg phenotype (59%, 5^th bar). Asterisk (*) represent statistical significance obtained from Dunn’s post hoc test after non-parametric Kruskal-Wallis ANOVA with respect to the <i>del/+</i> genotype. The error bars represent standard deviation in graphs A, C, and D. Two asterisks correspond to p-values of <0.01 and three asterisk to p-values of <0.001.</p

<i>In situ</i> hybridization analysis of <i>DmGfrl</i> expression during embryogenesis.

<p>Cellular blastoderm (B) and early stage embryos up to stage 10 (C) do not express <i>DmGfrl</i>. Expression (blue staining) first appeared in the seven abdominal segments in the ventral nerve cord (D, vnc) and in unidentified ganglia in the head region at about stage 13. The expression became more widespread in the central and peripheral nervous system through the later stages of embryogenesis (E, F). <i>DmGfrl</i> was expressed in single cells or clusters of a few cells in the head sensory ganglia (E, sg), ventral nerve chord (D–F) and lateral sensory ganglia (E, arrowheads). At stage 15, dorsal vessel (dv) also expressed <i>DmGfrl</i> (E). Control hybridization with a sense probe did not show specific staining (A). (G–L) Following whole mount in situ hybridization, the embryos were subjected to immunoperoxidase staining (brown color) with neuronal and glial marker antibodies. Co-staining for the neuronal marker FasII showed that <i>DmGfrl</i>-expressing cells are localized within the VNC, along the longitudinal axon bundles (G). A lateral view on the VNC shows that the <i>DmGfrl</i> signal did not colocalize with the nuclear staining for REPO, a glial cell marker (H, arrowheads show <i>DmGfrl</i>+ cells). A ventral view of the VNC also shows the paired <i>DmGfrl</i>+ cells in each segment do not co-localize with REPO (I, large arrows show <i>DmGfrl</i>+ cells). However, in the more lateral and dorsal cells there may be some overlap in the signals for DmGfrl and REPO (I, small arrows). The <i>DmGfrl</i>+ cells localized posteriorly from the dMP2 interneurons (J, brown staining) in late-stage embryos, indicating that they are not dMP2 neurons and likely not vMP2 neurons either. Futsch/22C10 staining visualizes that the lateral axonal projections along which the <i>DmGfrl</i>+ cells were located (K, arrows). Staining for cut, a sensory neuron marker showed that the <i>DmGfrl</i>-expressing cells were within the external sensory organ cell clusters and likely all positive for cut (L, arrows). Original magnification was 400X in h, i and l, and 200X in all other images.</p

Drug-induced ER stress upregulates <i>DmManf</i> expression.

<p>A–B) In Schneider 2 (S2) cells, ER stress was induced by thapsigargin (TG), tunicamycin (TM) and dithiothreitol (DTT). DMSO was used as a control treatment. A) The mRNA levels of <i>DmManf</i> and <i>Hsc3</i> were analysed by qPCR, values were normalised to control treatment (DMSO). B) RT-PCR and agarose gel electrophoresis analysis revealed two transcripts of <i>Xbp1</i>, unspliced (<i>Xbp1</i>^<i>u</i>) and spliced (<i>Xbp1</i>^<i>s</i>). <i>RpL32</i> was used as a loading control. C–D) qPCR analysis of <i>Hsc3</i> and <i>Xbp1</i> expression in <i>DmManf</i> mutant (C) and <i>DmManf</i> overexpressing (D) larvae. Expression of <i>Hsc3</i> was not altered but <i>Xbp1s</i> mRNA level was increased in response to overexpression of <i>DmManf</i>. The overexpression of <i>DmManf</i> resulted in 165-fold increase in <i>DmManf</i> mRNA level (±23, P<0.001, not shown). <i>Xbp1t</i>, total amount of <i>Xbp1</i>; <i>Xbp1s</i>, spliced-specific transcript of <i>Xbp1</i>; <i>Xbp1 s</i>:<i>t</i>, proportion of <i>Xbp1s</i> out of <i>Xbp1t</i>. OE, overexpression. Average ± standard deviation. *, P<0.05; **, P<0.01; ***, P<0.001 versus control, Student’s t-test.</p

Amino acid sequence of DmGfrlA and schematic structures of the predicted DmGfrl protein isoforms identified in this study.

<p>(A) In the amino acid sequence of DmGfrl protein isoform A the signal sequence is marked with solid double underlining. The amino acid sequence of isoform B is identical to that of isoform A except for 26 N-terminal amino acids preceding the D0 domain, which reads MLKPFAVIIGIFYLGSTIKGVVAILN in DmGfrlB. V5 after the signal sequence denotes the site (between Q31 and G32) where a sequence encoding the V5 tag (GKPIPNPLLGLDST) was inserted in some of the expression constructs. The GFRα-like domains 0 to 3 (D0 to D3) are marked with solid underlining and the sequence (MKKCDRI) similar to mammalian heparin binding sites with bold underlining. The three predicted N-glycosylation sites are marked with N above the amino acid sequence and the mucin-type glycosylation sites with asterisks (*). A predicted low-score (big-PI Predictor) GPI anchoring site (G1008, underlined) is marked with ‘GPI’. It precedes a C-terminal hydrophobic region typical of GPI anchored proteins (dash underlining). (B) Schematic structures of four predicted DmGfrl isoforms and comparison to human GFRα1 and predicted <i>C. elegans</i> Gfr-like protein. Several insect genomes encode Gfr-like proteins that are approximately double the size of vertebrate Gfrα proteins <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051997#pone.0051997-Airaksinen2" target="_blank">[30]</a>. Most of this size difference is due to the long sequence between the fourth GFRα-like domain and the plasma membrane anchor in the insect proteins.</p

Overview of genetic interactions between <i>DmManf</i> and selected ER- and UPR-related genes.

<p>A) UAS-RNAi lines were crossed to MS1096-GAL4 and 69B-GAL4 driver lines in wild type and <i>DmManf</i>-overexpressing backgrounds. The observed phenotypes of knockdown flies in <i>DmManf</i>-overexpressing background (OE vs. wt) were compared to the phenotype of knockdown flies in wild type background. Yellow (stronger phenotype) represents affected phenotypes. Light gray (no phenotype in either background), gray (phenotype similar in both backgrounds) and dark gray (lethal phenotype in both backgrounds) represent cases where the overexpression of <i>DmManf</i> did not affect the phenotype caused by knockdown of target gene. As a comparison, results from our previously published microarray analysis (MAA) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151550#pone.0151550.ref020" target="_blank">20</a>] are presented; red and blue indicate up- and down-regulation of the target gene, respectively. Mutant larvae stands for zygotic <i>DmManf</i>^<i>Δ96</i> mutant larvae, OE larvae for 69B-GAL4>UAS-<i>DmManf</i>^L3 larvae, and mutant embryos for maternal and zygotic <i>DmManf</i>^{<i>Δ96mz</i>} mutant embryos. B) Overexpression of DmManf by UAS-DmManf^L5 with either semi-ubiquitous 69B-GAL4 or with wing driver MS1096-GAL4 did not result in any obvious phenotypes in adult flies. In MS1096-GAL4 line, we detected a weak GAL4 expression in CNS as well presenting a probable reason for the lethal phenotypes we observed in our knockdown experiments. However, for screening we only monitored the adult wing phenotype. ER, endoplasmic reticulum; ERAD, ER associated degradation; ERSS, ER stress sensor protein; MAA, microarray analysis; OE, overexpression.</p