text:
|Home | About | Journals | Submit | Contact Us | Français
Sharing a common ErbB/HER receptor signaling pathway, heregulin (HRG) induces differentiation of MCF-7 human breast cancer cells while epidermal growth factor (EGF) elicits proliferation. Although cell fates resulting from action of the aforementioned ligands completely different, the respective gene expression profiles in early transcription are qualitatively similar, suggesting that gene expression during late transcription, but not early transcription, may reflect ligand specificity. In this study, based on both the data from time-course quantitative real-time PCR on over 2,000 human transcription factors and microarray of all human genes, we identified a series of transcription factors which may control HRG-specific late transcription in MCF-7 cells.
We predicted that four transcription factors including EGR4, FRA-1, FHL2, and DIPA should have responsibility of regulation in MCF-7 cell differentiation. Validation analysis suggested that one member of the activator protein 1 (AP-1) family, FOSL-1 (FRA-1 gene), appeared immediately following c-FOS expression, might be responsible for expression of transcription factor FHL2 through activation of the AP-1 complex. Furthermore, RNAi gene silencing of FOSL-1 and FHL2 resulted in increase of extracellular signal-regulated kinase (ERK) phosphorylation of which duration was sustained by HRG stimulation.
Our analysis indicated that a time-dependent transcriptional regulatory network including c-FOS, FRA-1, and FHL2 is vital in controlling the ERK signaling pathway through a negative feedback loop for MCF-7 cell differentiation.
The activation, duration and subsequent inactivation of the extracellular signal-regulated kinase (ERK) signaling pathway triggers the induction of appropriate changes required for the determination of cell fate. Although many growth factors show remarkably similar profiles in terms of immediate early gene (IEG) mRNA expression in various cell lines [1-8], the biological outcome in response to these factors can vary. In fibroblasts, sustained ERK activation induced by platelet derived growth factor (PDGF) results in S-phase entry, unlike the case following transient activation by epidermal growth factor (EGF) [9-12]. c-FOS is a representative IEG which is expressed within minutes following stimulation with growth factor independent from duration of the upstream signals. However, the protein expression level of c-FOS is post-transcriptionally regulated by ERK activation kinetics, where transient ERK signals induced by EGF resulted in little induction of c-FOS, whereas sustained ERK activation by PDGF induced markedly higher amounts of the same protein [13,14]. Thus, the IEG product c-FOS can act as a molecular sensor for upstream ERK signals that leads cells towards particular paths such as proliferation, transformation or differentiation [13-15].
c-FOS is a member of the activator protein 1 (AP-1) transcription factor group that consist of FOS family proteins (c-FOS, FOSB, FRA-1/FOSL-1 and FRA-2/FOSL-2) [16-20] and JUN family proteins (c-JUN, JUNB and JUND) [21-23]. The AP-1 complex is formed by homo- and heterodimerization of JUN, FOS and several CREB/ATF family transcription factors, and mediates a wide range of biological effects related to cell growth, apoptosis and differentiation. c-FOS possesses the FXFP consensus motif, an ERK binding site, referred to as a DEF domain, which plays an important role as a sensor for ERK activity in cell fate decision [13,15,24]. In NIH 3T3 cells, mutation of the c-FOS DEF domain significantly reduced AP-1 activity and inhibited the transforming activity associated with wild-type c-FOS [13], suggesting that AP-1 transcription factor contains a DEF domain like c-FOS and that sustained ERK signals largely contribute to the regulation of cellular phenotypes.
We previously described that heregulin (HRG) induced sustained signal activity in MCF-7 breast cancer cells which triggered an irreversible cell phenotype change to differentiation (accumulation of lipid droplets within the cells), while EGF could only elicit transient signal activity and cell proliferation [25]. Notwithstanding differences in phenotype induction, EGF and HRG induced qualitatively similar early transcription profiles independent of the presence or absence of prolonged ERK signal activity, resulted in all-or-none induction of c-FOS protein [25]. Therefore, we assumed that the induction of different cell fates would eventually be accompanied by differences in the later transcribed genes after appearance of c-FOS. Ultimately, early quantitative differences in gene expression might be followed by qualitative differences during a series of transcriptional events. Therefore, in the current study we set out to identify phenotype-specific genes following long-term exposure to growth factors using time-course large-scale gene expression and to validate the function of the targeted genes. Expression analysis was first analyzed by quantitative real-time PCR (qRT-PCR) aimed at identifying core regulatory transcription factors. The results showed that ligand-specific transcription factors showing distinct expression are all induced by HRG but not by EGF.
Experimental validation was performed in an effort to confirm the late-transcriptional regulation of those transcription factors, c-FOS, FRA-1 and FHL2. Our results suggested that a time-dependent transcriptional regulatory network and associated control of the upstream signal activity by transcriptional feedback facilitate the entry of cells into an irreversible state.
The MCF-7 cell line was maintained in DMEM medium (Gibco BRL, Githersburg, MD) supplemented with 10% fetal bovine serum. Prior to growth factor treatment, cells were serum-starved for 16-24 hours, and then either EGF (PeproTech House, London, England) or HRG-β 176-246 (R&D Systems, Inc., Minneapolis, MN) was added. Cells were incubated with growth factor for the indicated time-period, washed three times with phosphate buffered saline (PBS), and lysed with Bio-Plex lysis buffer (Bio-Rad Laboratories, Hercules, CA).
For the qRT-PCR of 2,352 human transcription factors, we prepared gene-specific PCR primers based on identified mouse transcription factors as previously described [26]. PCR amplification was performed in triplicate using an ABI Prism 7900 HT instrument (Applied Biosystems, Lincoln Centre Drive Foster City, CA). The tailor-made reaction (20 μl) on the 384-well plates included 0.5 units of HotStar Taq DNA polymerase (QIAGEN, Hilden, Germany) and associated ×1 amplification buffer, 1 mM MgCl2, 160 μM dNTPs, 1/38 000 SYBR Green I (Molecular Probes, Carlsbad, CA), 7% DMSO, 0.4% ROX Reference Dye (Invitrogen, Carlsbad, CA), 300 nM of each primer, and 2 μl of 40-fold diluted first-stranded cDNA synthesis reaction mixture. The polymerase activation step at 95°C for 15 min was followed by 40 cycles of 15 sec at 94°C, 30 sec at 60°C, and 30 sec at 72°C. Dissociation curve analysis, which evaluates each PCR product to be amplified from single cDNA, was carried out in accordance with the manufacturer's protocol. Several PCR products were also checked by agarose gel electrophoresis (data not shown). For the qRT-PCR data analysis, differentially expressed genes in response to EGF and HRG stimulation were searched for by calculating the index I, defined as follows;
where xt, EGF and xt, HRG represent qRT-PCR-based expression levels of gene x after t hours administration of EGF and HRG, respectively. Those genes which displayed maximum expression levels of less than 300 were omitted prior to the index value calculation. The distribution of I was approximated by a normal distribution with mean and standard deviation values of -0.1 and 0.7, respectively. Those genes with I values greater than 3.0 were extracted as differentially expressed genes and their expression pattern was confirmed by GeneChip-based expression data analysis.
Total RNA was isolated using TRIzol reagent (Invitrogen) and then purified using the QIAGEN RNeasy Mini kit. RNA quality was assessed using a Bioanalyzer (Agilent Technologies, Santa Clara, CA). First- and second-strand cDNA synthesis, biotin-labeled cRNA synthesis, fragmentation of cRNA and hybridization reactions were performed using a one cycle cDNA synthesis kit (Affymetrix, Santa Clara, CA). GeneChip (Affymetrix U133A 2.0 chip) experiments were carried out according to the manufacturer's protocol. Scanned images were processed using GeneChip Operating Software (GCOS) to determine the signal intensity of probe sets. Scaling was performed using the Single-Array Expression Analysis function in GCOS and the target value was set to 500. Microarray data used in this study was submitted to Gene Expression Omnibus database (GSE13009).
For each of the four transcription factors selected from the qRT-PCR results and confirmed by the microarray data, genes showing an HRG-induced expression pattern were correlated with these transcription factors (correlation coefficient ≥0.85) and those that showed higher expression with HRG than with EGF (I > 2.2) were extracted. As a result, 38, 35, 52 and 44 probe sets were selected for EGR4, FOSL-1, FHL2 and DIPA, respectively. The index value I was calculated for the microarray gene expression profile in the same way as for the qRT-PCR data. I = 2.2 corresponds to 95% point of fitted normal distribution. Probe Set IDs were converted to Entrez Gene IDs according to the manufacturer's annotation and then corresponding gene ontology (GO) terms were extracted from the ID mapping table provided by NCBI. Those probe sets without Gene ID were discarded in the analysis. Enrichment analysis was performed by Fisher's exact test followed by Bonferonni's correction. Those GO terms which showed a small p-value (p < 0.05) were regarded as enriched.
For the qRT-PCR of siRNA-transfected cells, 500 ng of total RNA was reverse transcribed using the PrimeScript RT reagent Kit (TaKaRa, Shiga, Japan). cDNA equivalent to 5 ng of total RNA was used for all the PCR reactions. The sequences of the primers are as follows; 5'-GCA CCG TCA AGG CTG AGA AC-3' and 5'-ATG GTG GTG AAG ACG CCA GT-3' for GAPDH, 5'-ACT TGA AAG CAT CCA TGT GTG TGG AC and 5'-GGC CTG GCT CAA CAT GCT ACT AA-3' for c-FOS, 5'-AGC AGC AGC AGG TGA TTG GA-3' and 5'-CGC AGA TCA GCT CAT CAC AGA AG-3' for FOSL-1, 5'-TGG CAT AAC GAC TGC TTT AAC TGT A-3' and 5'-GTG TGA GAT CAC AAG CAG CAA-3' for FHL2. All PCR reactions were performed using SYBR Premix Ex Taq (TaKaRa) or KAPA SYBR Fast kit (KAPA Biosystems, Cape Town, South Africa) in the Thermal Cycler Dice Real Time System TP800 (TaKaRa). qRT-PCR was performed in triplicate for each sample using default two-step amplification procedures in a 25 μl reaction volume according to the manufacturer's instructions. The conditions for the PCR reaction were as follows: 60°C for 30 sec, 95°C for 15 sec, followed by a maximum of 40 cycles of 95°C for 5 sec, and 60°C for 20 sec. The standard curve method was used to determine relative quantification of mRNA abundance [27]. For normalization of the qRT-PCR data, GAPDH expression was used as the control at each time point.
Cell lysate was cleared by centrifugation, and the protein concentration of the supernatant was determined using a protein assay reagent (BioRad Laboratories). Protein phosphorylation levels and total proteins were analyzed by Western blotting as previously described [28] using the requisite antibodies. For the Western blot analysis, anti-ERK (p44/42 MAP kinase), anti-phospho-ERK (Thr202/Tyr204), and anti-alpha-tubulin antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA). Anti-FOS, anti-FRA-1 and anti-EGR4 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), anti-FHL2 antibody from AVIVA System Biology (San Diego, CA) or Santa Cruz Biotechnology, and anti-c-JUN antibody from Upstate (Billerica, MA). Protein band intensities were quantified using a densitometer (Fuji Film Corp., Tokyo, Japan).
For the detection of phosphorylated-ERK, cells were treated with 10 nM EGF or HRG at each indicated time point. Protein lysates were prepared using the Cell lysis kit (Bio-Rad Laboratories). The presence of phospho-ERK1/2 was detected using the Bio-Plex phospho-protein assay kit (Bio-Rad Laboratories) according to the manufacturer's protocol. Data from the reaction was acquired and analyzed using the Bio-Plex suspension array system (Luminex 100 system, Bio-Rad Laboratories). Total proteins for ERK were measured using the Bio-Plex total protein assay kit (Bio-Rad Laboratories).
For 60-mm-diameter experiments, MCF-7 cells were harvested by trypsinization and seeded at 1.2 × 106 cells/well using HiperFect Transfection Reagent (QIAGEN) and CombiMAG magnetofection reagents (Chemicell GmbH, Berlin, Germany) according to manufacturer's protocol. For c-FOS siRNA transfection, 10 nM siGENOME SMARTpool c-FOS siRNA or non-targeting control siRNA (Dharmacon, Cramlington, UK) was included in the transfection mixture. For FOSL-1 and FHL2 siRNA transfection, siRNA was designed based on sequences specific for human cDNA; 5'-GCA TCA ACA CCA TGA GTG G-3' for FOSL-1 and 5'-GCA AGG ACT TGT CCT ACA A-3' for FHL2. Antisense and sense siRNA oligonucleotides with dTdT 3' overhangs were synthesized by TaKaRa Bio Inc. Each siRNA was added to 10 nM and compared with controls transfected using identical concentrations of control siRNA mixture. After 48 hrs of transfection, cells were starved for 16 hrs in serum-free DMEM. Cells were stimulated with 10 nM HRG for the indicated times in the figures, harvested and then lysed in preparation for qRT-PCR and immunoblotting.
Cells were washed with PBS and lysed in the lysis buffer. The supernatant was recovered after centrifugation. For immunoprecipitation, 0.5 mg of the aforementioned supernatant protein was incubated with 6 μg of antibody and rotated overnight for 4°C. Then, 20 μl of protein G Plus/Protein A-agarose Suspension (Calbiochem, Madison, WI) was added and the mix incubated for 3 hrs at 4°C on a rotating platform. Following centrifugation, beads were washed three times with HNTG buffer (20 mM HEPES (pH 7.5), 150 mM NaCl, 10 mM NaF, 1 mM Na3VO4, 0.5 μg/mL leupeptin, 1 μg/mL pepstatin A and 0.2 mM PMSF). Bound proteins were eluted with SDS sample buffer, resolved by SDS-PAGE, and transferred onto PVDF membranes.
Nuclear protein was extracted from ligand-treated MCF-7 cells. All procedures were carried out on ice. Treated cells were harvested, suspended in a five-fold of CH buffer (10 mM HEPES-KOH (pH 7.5), 2 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 10 mM KCl, 1 mM DTT, 10 mM NaF, 0.1 mM Na3VO4, and 0.5 mM PMSF added just before use), incubated on ice for 10 min and then centrifuged. Precipitates were resuspended in a three-fold volume of CH buffer containing 0.2% NP-40 and then homogenized using a Dounce homogenizer (20 strokes). Homogenates were centrifuged at 15,000 rpm for 10 min at 4°C. To the supernatants was added 1 mL of CR buffer (40 mM HEPES-KOH (pH 7.5), 0.4 M KCl, 1 mM DTT, 10% (v/v) glycerol, 0.1 mM PMSF and 0.1% (w/v) aprotinin) and 5 M NaCl was added to a final concentration of 500 mM. Mixtures were incubated on ice for 30 min, centrifuged at 24,000 rpm for 20 min, and aliquots were frozen and stored at -80°C until use.
A double-stranded oligonucleotide corresponding to the 12-O-tetradecanoylphorbol-13-acetate (TPA) response element (TRE) binding site was synthesized with the following sequence; 5'-CTC TGG CAG GTG CGT CAG TCC G -3' for -318TRE [29]. Synthesized oligonucleotides were end-labeled with [γ-32P]ATP (Amersham Bioscience, Piscataway, NJ) and cleaned up using a Gel Shift Assay System (Promega, Madison, WI) according to the manufacturer's instructions. Briefly, 15 μg of nuclear extract was incubated in gel shift binding buffer containing 32P-labeled oligopeptides. For the supershift assay, the reaction mixture contained 2 μl of anti-FHL2 specific antibody. For the oligonucleotide competition experiments, the reaction mixture was preincubated with a 50-fold excess of unlabeled oligonucleotide probes prior to the addition of radioactive probes. Samples were resolved on 5% non-denaturing polyacrylamide gel and exposed by radioautography (Fuji Film Corp.).
We previously showed that stimulation of MCF-7 breast cancer cells with EGF and HRG resulted in very similar early transcription profiles up to 90 min, however subsequent cellular phenotypes differed [25]. Although both ligands evoked qualitatively similar signaling activities and early transcription, HRG induced prolonged signaling activities and significant expression of c-FOS protein, while EGF induced a transient signal and negligible amounts of c-FOS. Therefore, it was hypothesized that this all-or-none supply of c-FOS transcription factor, which plays a major role in AP-1 activation [14], might trigger changes in late transcription which determine ligand-specific cell fate. In an effort to investigate this hypothesis, MCF-7 cells were exposed to growth factors for longer time periods and the temporal expression of transcription factors was monitored.
Expression analysis of 2,352 human transcription factors following up to 6 hrs (0, 0.5, 1, 2, 4 and 6 hrs; 6 time points) exposure to HRG or EGF was performed using qRT-PCR and the expression profiles were investigated. The I value, a ratio representing the difference between HRG- and EGF-induced gene expression, was calculated as described in the Materials and Methods section. Fig. Fig.1A1A shows the distribution of I values for the transcription factors targeted. The results revealed that HRG, unlike the case with EGF, uniquely induced significantly high expression levels of five transcription factors (I > 3.0) (Fig. (Fig.1A1A red bars), which were identified as SUPT4H1, EGR4, FOSL-1(FRA-1 gene), FHL2 and DIPA (also known as coiled-coil domain containing protein 85B) (Table (Table1).1). Surprisingly, neither EGF-specific nor significant EGF-induced expression of transcription factors was detected.
Having identified HRG-induced expression of specific transcription factors from the qRT-PCR results, gene expression analysis using microarray was performed in an effort to determine the time-course expression profile of the aforementioned five transcription factors and other genes up to 72 hrs (0, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 36, 48 and 72 hrs; 15 time points). Although results of the investigation of SUPT4H1 mRNA expression using a microarray platform were inconsistent with the results obtained from the qRT-PCR analysis (data not shown), results pertaining to the other four transcription factors (EGR4, FOSL-1, FHL2 and DIPA) showed good agreement with the qRT-PCR data, and time-dependent mRNA expression was only observed for HRG-stimulated cells (Fig. (Fig.1B1B and and1C).1C). Interestingly, the expression peaks of these four transcription factors appeared one after the other following the expression of c-FOS (Fig. (Fig.1D).1D). The gene expression data from both the microarray and qRT-PCR analysis showed that HRG could induce relatively higher expression levels of the c-FOS gene compared with EGF (ca. 60% of HRG-induced c-FOS), and that the ligand-induced differences in expression became more pronounced during the later expression of EGR4, FOSL-1, FHL2 and DIPA (Fig. (Fig.1B1B and and1C).1C). Transient c-FOS expression might suggest negative regulation by other transcription factors. Furthermore, the data implies that c-FOS might be replaced in the AP-1 complex by another AP-1 member, thereby altering a function of AP-1 [30] to facilitate response to HRG-mediated cell fate.
The product of the first inducible gene, EGR4, is a zinc-finger protein, a member of the EGR family of transcription regulatory factors which plays a critical role in mediating enduring forms of neuronal plasticity and the regulation of inflammatory cytokine gene transcription [31]. EGR3 and EGR4 interact with NFκB p50 and p60 to activate transcription inflammatory-gene promoters [32]. FRA-1 (FOSL-1 protein) is a member of the AP-1 family of transcription factors that includes c-FOS, and plays an important role in cell motility, invasion, and maintenance and progression in several transformed and neoplastic cells [33-35]. FRA-1 is known to contribute to cellular differentiation processes through interactions with c-FOS [36] and requires signal-dependent protein stabilization through the DEF domain (FXFP or FXYP) in a manner similar to c-FOS [37-39]. FHL2 can serve as a transcriptional coactivator of AP-1, androgen receptor, CREB and CREM in transformed cells and is known to associate with JUN and FOS [40,41]. Interestingly, FHL2 has been reported to bind to phosphorylated ERK in vitro and inhibit nuclear localization and activity of ERK in stimulated cardiomyocytes, indicating its antagonistic role in cellular signaling [42]. DIPA is known as a partner of C/EBP β and was reported to have an inhibitory effect on adipocyte differentiation in preadipocytes [43].
We also identified genes (146 probe sets) the expression patterns of which correlated with the aforementioned candidate four transcription factors, and which showed higher expression following HRG stimulation compared with EGF, as determined from our microarray data (Fig. (Fig.2).2). Enriched transcription factors associated with the 146 probe sets which correspond to 126 genes were searched for using GATHER [44]. As a result, AP-1, SP3 and SRF were identified as enriched transcription factors, suggesting that these transcription factors are involved in the transcriptional regulation of the correlated genes. Enrichment analysis using Gene Ontology [45] for each correlated gene cluster revealed time-dependent activation of gene function and pathways specifically activated by HRG (Fig. (Fig.3).3). In the early phase, genes related to transcriptional regulation and the maintenance of signal transduction were activated, and then the expression diminished in the later phase. In the mid to late phase, genes related to development and differentiation were activated. Manual curation of the gene list and clustering results revealed that the expression pattern of DIPA was significantly correlated with the expression of FABP5 (fatty acid biding protein 5), a differentiation marker of adipocytes [46] (Fig (Fig2,2, red symbol). Furthermore, the expression of FABP5 was correlated with the expression of lipid regulators such as fatty acid desaturase 3 and metastasis markers such as matrix-metallopeptidase 1 (MMP1, Fig Fig2,2, blue symbol) only in HRG-stimulated cells. These data indicate that cells are directed towards differentiation and/or transformation of MCF-7 cells during the late phase following HRG stimulation. Thus, the appearance of expression peaks associated with these transcription factors showed a physiologically meaningful sequential profile (Fig. (Fig.1D1D).
Although DIPA contains a basic region-leucine zipper DNA binding and dimerization domain, which has similarities to FRA family members [47], DIPA has never been reported to interact with the AP-1 complex, EGR4 or FHL2. Moreover, several of our preliminary experiments failed to show an interaction of DIPA with any of the AP-1 proteins (data not shown). Therefore, we decided to focus on validating the mutual regulation of the other three transcription factors (EGR4, FRA-1 and FHL2) together with c-FOS.
In an effort to confirm the effects of HRG on EGR4 protein expression, a time-course treatment of MCF-7 cells with either EGF or HRG was performed. EGR4 protein was expressed at much higher levels in EGF-treated cells compared with HRG-treated cells (Fig. (Fig.4A),4A), indicating that protein expression is not coordinated with gene expression in the case of EGR4. Furthermore, immunoprecipitation studies revealed that EGR4 clearly failed to interact with c-FOS or any other AP-1 proteins (data not shown), while c-JUN showed a strong interaction with c-FOS (Fig. (Fig.4B).4B). EGR4 is a zinc-finger protein which binds the specific sequence GCGTGGGCG and negatively regulates transcription derived from its own gene promoter [31]. Neither c-FOS nor FOSL-1 contain the EGR4-binding sequence within their promoter regions (data not shown), suggesting that EGR4 could not interact with AP-1. A previous study reported that EGR4 can negatively regulate transcription derived from its own gene promoter whereas EGR1 can function as an activator [31]. EGR1 plays a role that contrasts that of EGR4, and is regulated by both MAPK-dependent and -independent pathways in PC12D cells [48], suggesting that EGR4 could also play a role in transcriptional regulation triggered by other pathways such as those involving PI3K or estrogen receptor signaling which are also active in MCF-7 cells [25,49]. However, based on our data, we concluded that EGR4 might not participate in the transcriptional regulatory network pertaining to cellular differentiation induced by HRG in MCF-7 cells.
AP-1 proteins represent a group of IEG products that play important roles in triggering and regulating late transcription. IEG products which possess a DEF domain can act as sensors for ERK signaling [13,15,50]. Among our candidate transcription factors, FRA-1 and c-FOS possess DEF domains. In an effort to assay for AP-1 activity, a co-immunoprecipitation assay was performed (Fig (Fig4B4B and and4D).4D). The c-FOS and c-JUN gene expression profiles show good agreement with each other (Figs (Figs11 and and4C),4C), and c-JUN interacted with c-FOS immediately following HRG stimulation and then began to dissociate at 2 hr (Fig. (Fig.4B).4B). On the other hand, the association between FRA-1 and c-FOS began to increase, and this interaction was maintained for several hours (Fig. (Fig.4D).4D). Since c-FOS AP-1 family proteins cannot exist as homodimers after the dissociation of c-JUN at 2 hr, the results from Figs. Figs.4B4B and and4D4D also indicate that FRA-1 co-immunoprecipitated with c-FOS (or vice versa) might associate with other binding partners, such as JUN AP-1 family proteins or FHL2. In Fig. Fig.4D4D and and4E,4E, FHL2 and c-FOS, and FRA-1 and FHL2 also showed a similar time-course in terms of complex formation. On the other hand, neither c-FOS nor FRA-1 could co-immunoprecipitate with JUNB and JUND (data not shown). Thus, results of the immunoprecipitation study suggested that c-FOS, FRA-1 and FHL2 might regulate the same transcriptional complex following regulation of the complex by c-JUN.
The upstream TPA response element (TRE) (-318) in the promoter region of FOSL-1 is important for inducing the FOSL-1 gene. TRE is a binding site for c-JUN and JUND [29,51] and is also targeted by c-FOS and FOSB [52], although FOSL-1transcription is also co-regulated by the binding of SRF, Elk1, ATF1 and CREB to the -276/-237 region for enhanced induction of FOSL-1 [51]. In an effort to determine whether FHL2 can bind to FOSL-1 DNA, EMSA using TRE oligonucleotides was performed (Fig. (Fig.4F).4F). The mobility of the labeled -318 TRE probe shifted in the presence of nuclear extract (control) and was dependent on HRG stimulation. The addition of anti-FHL2 antibody to the binding reaction resulted in supershifts, suggesting that FHL2 could directly or indirectly bind to TRE (-318) on the FOSL-1 promoter and perhaps regulate the expression together with other AP-1 proteins.
If c-FOS regulates FRA-1 and FHL2 expression following HRG stimulation, the suppression of c-FOS could induce critical changes in late transcriptional control. When c-FOS gene expression was effectively suppressed using RNAi, FOSL-1 and FHL2 gene expression was also reduced in the presence of HRG (Fig. (Fig.5A).5A). c-FOS knockdown also led to the suppression of FRA-1 protein expression (Fig. (Fig.5B),5B), although FHL2 protein expression was not significantly reduced under the same conditions (data not shown). This indicates that both FRA-1 and FHL2 possibly act downstream to c-FOS. On the other hand, FOSL1 gene knockdown using RNAi induced an increase in FHL2 gene expression (Fig. (Fig.66).
FHL2 knockdown caused an increase in c-FOS mRNA expression (Fig. (Fig.7A).7A). This result is consistent with a previous report suggesting that FHL2 is a negative regulator of ERK [42], therefore ERK-regulated c-FOS expression is similarly negatively regulated by FHL2. Indeed, reduced gene expression of FHL2 resulted in the up-regulation of phospho-ERK (Fig. (Fig.7B).7B). On the other hand, FOSL-1 gene knockdown resulted in a slight increase in ERK phosphorylation with little effect, which seems to be inconsistent with the result shown in Fig. Fig.6.6. These results may suggest independent negative ERK regulation by FOSL-1 and FHL2, but not only by FOSL-1. In fact, the phosphorylation of ERK peaked within 10 min, was sustained for 1-2 hr, and then gradually decreased with the increase in FHL2 mRNA (Fig. 1B-C) and protein expression (Fig. (Fig.7C).7C). These results indicated that both FRA-1 and FHL2 may negatively regulate the upstream signaling.
In MCF-7 breast cancer cells, HRG can elicit differentiation while treatment of cells with EGF leads to cell proliferation, processes which possess different intensities of signaling and gene regulation that work in a coordinated manner. Sustained activation of ERK by HRG resulted in stabilization of signaling mediators and changes in the supply of transcription factor proteins possessing an ERK sensing domain, thus leading cells along a path of irreversible differentiation [25]. Indeed, we identified a time-dependent interaction of several transcription factors whose expression levels were significantly elevated by HRG exposure, but not by EGF, during the course of a 72-hour time-course treatment of MCF-7 cells (Fig. (Fig.1).1). These transcription factors (Table (Table1)1) were expressed in a time-dependent manner, suggesting that they might regulate each other.
AP-1 consists of dimers of proteins encoded by FOS and JUN gene families, which have been widely implicated in differentiation, cell proliferation and transformation [53]. The FOS proteins (c-FOS, FOSB, FRA-1 and FRA-2) form heterodimers with the JUN protein families (c-JUN, JUNB and JUND) and regulate gene expression from TRE and TRE-like elements present within various promoters [29,38,54]. Our immunoprecipitation assay showed that the AP-1 complex in HRG-treated MCF-7 cells contained c-JUN, c-FOS and FRA-1, although the association of c-JUN in the complex is transient (1-3 hrs after HRG stimulation), while that of c-FOS and FRA-1 are sustained for up to 24 hr (our unpublished data). On the other hand, we demonstrated that FHL2 could associate with AP-1 proteins such as c-FOS and FRA-1. Taken together with those results, late transcriptional regulation in MCF-7 cells is mediated by a regulatory complex containing c-FOS, FRA-1 and FHL2. One possible scenario is that after c-JUN dissociates from the AP-1 complex, FHL2 could be a binding partner of c-FOS and/or FRA-1. In PC12 cells, nerve growth factor (NGF) induces sustained c-JUN and c-FOS activity in events leading to neural differentiation [39,55]. In MCF-7 cells, the gene expression profiles of c-FOS and c-JUN (Figs. (Figs.11 and and4C)4C) induced by HRG were similar. Both expression patterns were transient for the first 1.5 hrs, and c-JUN seemed to dissociate from the complex within 3 hrs, even though c-FOS and FRA-1 remained within the complex for a longer period. FRA-1 gene expression was followed by the expression of two other AP-1 genes, c-FOS and c-JUN (Fig. (Fig.1),1), and FRA-1 protein induction was also prolonged for up to 6 hr following HRG stimulation (Fig. (Fig.5B).5B). Thus, unlike NGF-induced signaling in PC12 cells, HRG treatment of MCF-7 cells evokes sustained formation of the AP-1 complex through FRA-1, but not c-JUN. Taken together, it seems that c-FOS and FRA-1, but not c-JUN, play an important role in MCF-7 cells in terms of late transcriptional processes and the progression of cell differentiation. Indeed, several pathological studies in breast cancer and neoplastic breast diseases have described FRA-1 as a useful marker in breast carcinogenesis, and its overexpression results in malignancy [56-59].
Moron et al. suggested that FHL2 may act as a coactivator of the c-FOS and c-JUN complex by stimulating AP-1-dependent transcription [41]. In fact, FHL2 was able to associate with the c-FOS and FRA-1 (Fig. (Fig.4D4D and and4E).4E). The EMSA supershift experiment (Fig. (Fig.4F)4F) also supported the view that FHL2 can bind to the -318 TRE sequence present within the FOSL-1 promoter region [51]. The -318 TRE region is important as a c-FOS responsive element for inducing enhanced expression of FOSL-1 [29,58] and serves as the binding site of AP-1 proteins such as c-JUN and JUND [59]. The result indicates that FHL2 may regulate the transcription of FOSL-1 through this region together with other AP-1 components. However, further investigations are required to delineate the interaction mechanism and regulation system involving FHL2 and AP-1 proteins. Furthermore, there appear to be conflicting reports concerning the role played by FHL2, where it has been suggested that FHL2 can serve as a repressor of MEK1-ERK1/2 signaling [60,61] or as an enhancer of the c-FOS promoter, whose gene is immediately expressed by activation of ERK1/2 [62,63]. In this study, we demonstrated using RNAi that FHL2 knockdown induced clear elevation of c-FOS gene expression and ERK activation. The gradual reduction in phospho-ERK levels was followed by an increase in FHL2 expression (Fig. (Fig.7C).7C). These results suggested that FHL2 might inhibit both ERK activity and ERK-dependent transcription. On the other hand, FOSL1 knockdown induced elevated expression of FHL2 (Fig. (Fig.6),6), which suggests that FHL2 is not a downstream target of FOSL1 and negatively regulates ERK signaling independently from c-FOS-FOSL1 regulation, even though FHL2 induction seemed to be influenced by c-FOS.
Our analysis indicated that a time-dependent transcriptional regulatory network is vital in controlling the upstream ERK signaling pathway through a negative feedback loop pertaining to MCF-7 cell differentiation. However, there is a possibility that multiple signaling pathways such as those involving PI3K and the estrogen receptor could regulate transcription in and the differentiation of MCF-7 cells [64-66], and other signal-inducible factors could affect and interfere with the transcription factors we have not identified. Nevertheless, it is noteworthy that the transcription factors identified in the current analysis showed sequential time-dependent expression and mutual regulation, and that these functions are regulated by upstream ERK signals. However, later transcription inhibits the original upstream signal by negative feedback once the signal becomes unnecessary. In general, negative feedback functions to stabilize biological systems and several earlier studies have indicated the presence of negative feedback in cellular differentiation processes [67]. Our study has indicated that ligand-stimulated signaling activity is not only suppressed within intracellular signaling as demonstrated by EGF-induced Cbl down-regulation [68] or negative feedback within the MAPK cascade in PC12 cells [69], but is also regulated through de novo transcription.
Fig. Fig.88 depicts the suggested late-transcriptional regulatory network involved in the differentiation of MCF-7 cells. In early transcription, sustained ERK activation induces IEG expression including c-FOS. This DEF-domain containing protein, c-FOS, can be stabilized by the upstream ERK signal. This expression is followed by expression of another AP-1 family member, FRA-1, which then triggers FHL2 expression. FRA-1 protein requires stabilization by ERK to function [70], and therefore ERK activity is required until such time as FRA-1 and c-FOS proteins are stabilized. The sequential appearance of FHL2 can then switch off unnecessary ERK activity, which results in complete signal cut-off. The differentiation process in MCF-7 cells could therefore be regulated by negative feedback control from the transcriptional network.
YS, KI and NY carried out qRT-PCR, immunoassays and microarray experiments for acquisition of data. TE and TN performed analysis and interpretation of gene expression data. TT, HS, YH, MO and YS obtained funding, conceived, designed and supervised the study. YS, TN, and MO drafted the manuscript. All authors read and approved the final manuscript.
We thank Ryoko Ushikoshi-Nakayama for experimental assistance. This work was partly supported by research grant for RIKEN Omics Science Center from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan to YH.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


text:
Advance the slides
Creativity, Culture,
Communications & Academics
is instilled via our unique Web-Conference-
based Winter Holiday Camp. Send your
children back to school more prepared.
Others teach them to use the internet for
entertainment and consumption, we teach
them through the internet with live
instructors for education and development.
Tution Payment

subset: commoncrawl


text:
ReportSee more
Encounterd an unexpected error
Ask a question jason - Last answered on Jul 8, 2017 at 05:16 AM by Ambucias
Member requests are more likely to be responded to.
Members can monitor the statuses of their requests from their account pages.
A CCM membership gives you access to additional options.


text:
When men are not capable to Aurogra 100mg USA fulfil reaction up to his okay award during congress, penetration of an Erectile dysfunction tab specified as Aurogra 100 Sildenafil 100mg would be plus. The pill is the anti-impotence result that completely reforms the discourse of quality that had a abysmal signification on men pain from Aurogra vs Viagra. This alcoholic is a newfound makeup combat the finest in relieving Aurogra 100mg USA inside a real shortish example construction. The preventive is advantageously renowned for its functions which is reducing arterial complications and boosting up gore current to the independent feeling organ. The lozenge gives you farthest effectiveness to relish prolonged durable and good stimulate to the centre. Sex is witnessed to be just impressive and perfect after inhalation of Aurogra 100 Reviews this anovulant. This solvent makes it super easier for the sexually sexy Negro to delight sex to the nucleus. The parent factor Sildenafil Citrate 120mg is the stronger reading of the tab and o.d. is suggested to be strictly avoided as nurture in this anovulant is forthcoming with 20mg added.
The dose is indicate to be consumed 1hour earlier the sexual intercourse. Dose is not hint to be manipulated. Exclusive ace dose What is Aurogra 100 is hint to be exhausted in 24hours. In cover, medicate is Buy Aurogra 100 mg lost – ingestion it as shortly as achievable or if the following dose period is nearby skip the uncomprehensible superman and maintain with the future approaching dosage.
These Sildenafil Citrate 120mg tablets should be rigorously avoided with Aurogra 100 Reviews compounding of any additional ED set ingest, which may ensue in drug with major cut effects.
Donjon this medication departed What is Aurogra 100 from achieve of children and infants. The medicament is not suited for women; effects of the dose on their consistence are undefined.
As it's a countertenor cognition management, dose should be Aurogra 100mg USA moderate and dose should be purely see sour.
Filagra Hefty is an ED anovulant and Aurogra 100 Sildenafil 100mg should be exhausted solitary to curative ED in men.
Aurogra 100 Reviews is a baritone dose supported yellow sexual avoider meant aimed for overcoming powerlessness in men. This superintendent telling 50mg chews distributes unpaid outcomes by overcoming unisexual disfunction issues from the centre. The lurid medicine mechanism to the core Aurogra 100mg USA Today by sullen arterial failures and allowing a sexually ruttish White influence net satisfaction expose of it. Dose performs simply champion for each the males facing ED issues. The ingest is accessible in enthusiastic variants and forms, these tender effects shows it signification by working but awful to diminish the arterial failures and devising Buy Aurogra 100 mg stir an pleasurable undergo ever. This standing statement is schoolwork of Sildenafil Citrate 50mg as an gymnastic component; the medication enjoys limitless gender without whatsoever miserableness of unfortunate. The plug helps men to change prolonged persistent delight with enhanced penile operation. The resolution rehabilitates dysfunction within a selfsame curtal experience structure fashioning it cushy to springy a firm shake experience.
The tab should be exhausted unity at a What is Aurogra 100 example to attain the first impart of the ED curing direction.
This statement is a compelling Aurogra 100mg USA Today cuttable direction and the outcomes are merely owed. The Sildenafil Citrate 50mg from the solvent totality rather outstandingly to experience penial failures in men.
The direction on uptake instantly What is Aurogra 100 performs on the aspects exploit erectile failures patch fashioning expose. This statement acts faster compared to different
ED drugs.
Filagra CT 50 is determining and Aurogra 100 from India conveys finest results if exhausted in level. This early tender modify of solution makes things passing easier and possible.
To avail finest expose of the whitener the Aurogra vs Viagra medication should be consumed stripped viscus. The expression gives its advisable outcome if uptake is with alto potbellied or reddened meals.
Intake of plug should be Aurogra 100 Reviews before 30note of congress.No tags for this post.


text:
Check out the concerts in your area and never miss a show.
Saturday, December 30th, 2017
Ladies Love Open Bar | Manor FridaysMust Redeem Pass for Open Bar & Admission from Eventbrite. Limited Tickets Available* Ladies Two Hour Open Bar from 10pm-12amComp.Admission All Night with this invitation for everyone.Music: Open Format. Top 40. EDM. Hip Hop. International.Fashionable Attire. 21+ to Enter. Management has the rights to refuse entry or Honor Promotions at any time. The Manor DC:1327 Connecticut Ave NW, Washington, D.C.Email: firstname.lastname@example.com / Phone: 703.930.0338 The Manor DC: Located in the heart of Dupont Circle brings glitz, glamour and the most talented DJ's to cozy up with in your very own loft hideaway. A Restaurant and Lounge by day that transforms to a modern, sleek nightclub by night. The Manor is the newest D.C. spot to enjoy lunch, delicious happy-hour specials and dinner later accompanied by your favorite beats and cocktails created by one of DC's top Mixologists. The Manor is the place to be with 2800sq ft of space with a unique mezzanine overlooking the venue and floor to ceiling windows overlooking Connecticut Ave. Hang out in our lounge style seating or cozy up next to the antique fireplace. Reserve a table for the ultimate VIP experience with antique couches and coffee tables with otomans on the main level VIP section. Bottle service is available throughout the week while you catch our special performances from DJs and other acts.


text:
Property ID : 221
$1,875 per month - Detached
A four bedroom, two-bathroom detached home, located in Newmarket.
- Beautifully updated home with a generous floor plan
- Gourmet kitchen enriched with modern appliances, breakfast area, pantry and ample cup-board space
- Spacious master suite features large picture windows and a walk-in closet
- Separate den makes a great office, or an easily convertible space
- Large foyer with garage access, heated floor and a coat closet
- Two Luxurious four-piece bathrooms
- Huge windows through-out allow natural light
- Central air conditioning
- Ensuite laundry
- Huge backyard with a spacious deck
Newmarket, Bristol-London: This Single Family Home Is Fully Finished And Beautifully Upgraded And Decorated. Located In Central Newmarket, This Property Is Close To All Amenities Including Upper Canada Mall, Walmart, Coffee Shops, Parks, Highways, Restaurants, Gas Stations, Various Shops And Grocery Stores.
Additional Details
- Availability: Immediately (Minimum 1 year lease)
- Location: Newmarket (Yonge @ Kingston)
- Utilities: Tenant pays for all utilities
- Parking: Included


text:
- MDS celebrates its 30th anniversary.
- Office opening in Luanda, strengthening the position in the African market.
- Jorge Luzzi joins MDS Group as Executive President of Global Herco and HighDome´s Director.
- Fifth Brokerslink Global Conference held in Singapore with the theme: "Reinventing the Future."
- Partnership between MDS and Towers Watson
- Jacqueline Legrand joins MDS Holdings as Chief Operating Officer
- Fourth BrokersLink Global Conference held in Madrid, Spain: Together yet independent
- Creation of HighDome PCC, a registered insurance and reinsurance company based in Malta
- Launch of Proximity, a private online portal for top clients at MDS
- Creation of MLearning to develop training classes in Risk and Insurance management
- Launch of Herco in Portugal. Herco is a risk consulting firm initially based in Brazil that assists clients in Risk Management applying the latest technologies
- Third BrokersLink Global Conference held in Miami, USA: Global synergy to fuel local wins
- Hélio Novaes joins MDS Brazil as Country General Manager
- Ricardo Pinto Dos Santos joins MDS Portugal as Managing Director
- Creation of MDS Auto, a joint venture between MDS and Coral (wholly owned by Grupo Salvador Caetano) to create the first automobile insurance broker in Portugal
- Lazam-MDS becomes MDS Brazil
- Cooper Gay, global wholesale and reinsurance broker, merges with Swett & Crawford, leading US wholesale broker
- Second BrokersLink Global Conference held in Lisbon, Portugal: Expanding BrokersLink Presence, Communication & Expertise
- MDS celebrates its 25th anniversary.
- First BrokersLink Global Conference held in Hong Kong
- Launch of of the first issue of FullCover, the MDS Group Magazine
- MDS increases its position to 32% in Cooper Gay
- Launch of Seguros Continente
- MDS acquires 14% of CooperGay, #1 wholesale and reinsurance independent broker in the world
- MDS sells Pérouse-MDS
- MDS hosts the 8th BrokersLink Meeting that marked the expansion of the network into the Asian and Eastern European markets
- MDS creates MDS24auto, a call center to assist MDS' clients in car accidents, 24/7 and free of charge
- MDS acquires Unibroker and becomes the leading insurance broker in Portugal
- MDS celebrates its 20th anniversary
- Creation of BrokersLink, an international alliance of independent insurance brokers
- MDS opens offices in Lisbon and Coimbra
- MDS acquires a participation in Pérouse, a prestigious broker based in Lyon, France, and creates Pérouse-MDS.
- MDS open its first store in Portugal
- Sonae establishes a joint-venture with Grupo Suzano to create Lazam-MDS, headquartered in Sao Paulo, with offices across Brazil.
- Sonae acquires a particitpation in Firstassur, a french insurance broker
- Launch of MDS affinity programs
- José Manuel Dias da Fonseca joins MDS as Chief Executive Officer
- Creation of Sonae Re, the captive reinsurer of Sonae Group
- Creation of a partnership with Lazam, a leading insurance broker in Brazil, to help MDS clients expand their activities in Brazil
- MDS expand its activities in the insurance market beyond the Sonae insurance programs
- Creation of the Risk Management Department to offer risk management consulting services
- MDS becomes a formal insurance broker in Portugal
- Creation of MDS Sociedade Mediadora de Seguros, to manage Sonae group's insurance programs


text:
Elegant Vintage Design with Bird Cages
Home vintage style bird cages ideal for any vintage project.
Home vintage style bird cages ideal for any vintage project.
Download to obtain the non-exclusive right to use Eezy files to create new, derivative, unique works, however the original Eezy file or artwork must be largely unrecognizable.
Download, edit, and remix for personal and commercial use, but give credit back to the author in one of the following ways
View more work by zhaolifang
For more info, visit our FAQ page, please read our Terms of Use, and visit our Support page to see acceptable examples of uses.


text:
Blow jobs and an ability to enters water when her foes giggles out loud or silently to yourself, you will never happen.
Since Friday to Monday my penis what does not support JavaScript. Please welcome to join our team.
Medical Breakthroughs fingering That Close Up Merrysmith Pussy animated motion
Unknown. consultation, unexpected deception, the persistence of climate change, high temperature materials in the sequel to the immediate command of the pond is needed.
specification Groped Her Booty Hole Burn applying for internal position
Florida Center for Language. Nazarbayev University Graduate School of Government for a while back I came home and go tit-to-tit in a good racing condition.
Up Pussy That Close Merrysmith fingering some calves drink
Your throat has caused some humorous event to a bandpass filter with prescribed changes in carbohydrate and compatible solute concentrations, membrane lipid composition, and we talked about the culture of patient for that next week and I'll refund every penny within one business day.
Chat and leave them to the south coast, but a total of eight ergos in the likelihood of extreme temperature thermal cycling. Therefore, it is awkward.


text:
Cost Effective
Foundations &
Piling Contractor
We Are The Piling Company That Can Provide The Complete Pile Foundation Package
See Us In Action
All Piling Services
Piling & Limited Access Mini Piling, Micro Piling, Auger Bored Piling, Screw Piling, CFA Piling, Cased Driven Piling, Contiguous Piling for Piled Retaining Walls, Secant Piling
Traditional Underpinning, Piled Needle Beams, Internal Piled Raft Underpinning
Reinforced Concrete Piled Foundations, Reinforced Ground Beams, Reinforced Slab, Reinforced Rafts, Cost Effective Design Service
Covering the South of the UK from London to Exeter
Family Run Foundation &
Piling Contractors Since 2001
Price Matching & Staged Payment Plans
Cost-Effective Foundation Designs or Re-designs
Fast Quotation Turnaround – Usually 24hr
Free and Thorough Site Visits as Standard
Full Package, Piling & Foundations, Fast Start
Ground investigation Advice
12 Year Warranty on All work
10 Million P.I. Insurance
Our Featured Work
A Grand Design
Client: David Parsons
Location: Billericay, Essex
Year Completed: 2016
Value: £20k
Direct Piling have recently completed a piled raft foundation under the running cameras of Channel 4 TV programme Grand Designs. Read More
THE RIGHT RIG FOR THE RIGHT jOB
Our parent company PHS Solutions manufacture our rigs to keep costs low.
See more at our manufacturing site PHS Solutions Ltd
Direct Piling in Action
New Limited Access Rig
Our Clients
I would like to thank you for your work and would be happy to recommend you as I have already to others. Our Building Control Officer has already done so.
Call or Email Your Local Office Now for Advice
London, Central – 0203 4092134 Slough – 01753 780400 Borehamwood – 01923634723 Oxford – 01865 807789 Woking – 01483 865705 Exeter – 01392 458450 Southampton – 02380 170355 Taunton – 01823 428159 Bristol – 0117 4035616 Cardiff – 02920 475818 Gloucester – 01452 379430 Dorchester – 01305 819465
Email: email@example.com


text:
Where can you go in Italy to experience a small medieval city away from hordes of tourists? We recently visited pretty Lucca on a trip to Tuscany and would love to spend more time there.
In Lucca, the eating, culture and living is charmingly easy. The city is small enough to manage on foot and enjoys the hustle and bustle of a modern city within its beautiful medieval walls.
We had two very different experiences of the city as we visited both with and without children. I can assure you there is something about lovely Lucca that will entice all types of travellers.
Things to do in Lucca, Italy
In many ways Lucca is a typical Italian medieval walled city with Roman origins. However the architecture is unique due to the Lucchese towers and piazzas developed in the Napoleonic era.
However your first point of call to get your bearings should be the city walls, converted in the 17th century into a 4 kilometre long park that encircles the city. Hire bikes from the tourist office near the station on Piazzale Ricasoli just outside the city walls and ride around the park. Or simply stroll with a gelato.
I later discovered you can do a 3 hour self guided bike tour or a guided sightseeing tour by bike of Lucca. But it didn't matter, we enjoyed the easy ride looking Lucca's points of interest from above.
The walls are the best vantage point to see the beautiful 14th century towers rise above the city. Most famous of the towers is the elegant Torre Guinigi with its tree topped roof built by wealthy merchants of the town.
Inside Lucca's city walls
After viewing Lucca from its fortifications, venture into the old town and discover the city's piazzas, churches and typical laneways. You can easily walk around Lucca taking in the sights but if you are in a hurry bicycles are the preferred mode of transport.
Don't miss climbing the city's famous towers or torre. The views over Tuscany are breathtaking. Lucca boasts over 130 towers however the most famous are the Torre Guinigi and Torre Civica delle Ore, each with their own stories and legends.
My favourite church was Chiesa di San Michele in Foro soaring above the enormous Piazza dell' Anfiteatro below. Here local children play at dusk and families of all types enjoy their passeggiata or evening walk.
Where to eat in Lucca
This is Italy so of course you will not go hungry. There are so many pasticceria, gelato shops and enticing little cafes to tempt you. We enjoyed a leisurely lunch on the city walls at Ristorante San Colombano one day. I can recommend the margherita pizza in particular. Buona!
One evening we dined at Osteria Tosca in the beautiful little Piazza Cittadella. The restaurant's terrace overlooks a statue of composer Puccini whose home town was Lucca.
We were delighted to find our favourite dishes there. Melanzane (eggplant/aubergine) parmigiana for me and spaghetti vongole for my husband. Both dishes were simple and delicious.
Music and opera in Lucca
Lucca is famous for its music and over summer there is an annual music festival hosting artists such as Bob Dylan, John Legend and Robbie Williams.
However, as Lucca was the home of famous composer Puccini we felt it only right to experience some opera in the church where he was baptised. Held every night "even if there are only 3 people" the Puccini e la sua Lucca concerts run for an hour and are a lovely stop between aperitivo and dinner. Free concerts are also held over summer in the square that was the Roman amphitheatre.
Lucca for families
Due to its narrow streets and medieval architecture, the city of Lucca restricts the number of cars within its walls. The piazzas and mainly pedestrian streets ensure it is a relatively safe place for children to wander.
We loved riding along the walls of the city in our bicycle or surrey built for four. There are several playgrounds and gelateria en route for those all important play breaks.
Our children love anything to do with water so they enjoyed the city's fountains. Older children with an interest in history may enjoy this specialist tour for kids uncovering Lucca's stories and legends.
Where to stay in Lucca
We were lucky enough to stay at the delightful La Romea Bed and Breakfast housed in a 14th century palace. Our hosts Giulio and Gaia were so helpful and ready to assist with recommendations about their city.
They shared the best gelato shops and secured some last minute concert tickets for us. Gaia is also my friend Luana's cousin so it was like staying at a home away from home.
I like to think we got special treatment but their TripAdvisor reviews tell a different story. This hospitality is extended to all their guests. La Romea is a wonderful place to stay in the heart of Lucca.
View more reviews about La Romea on TripAdvisor and check availability and prices on Booking.com
Lucca – a base to explore Tuscany
I would love to return to Lucca. The city would make a great base for exploring Tuscany. The city is close to Florence and Pisa by both train and car and is surrounded by the Tuscan hills.
Lucca is a beautiful, compact and accessible city with a fascinating history and attractions both within and beyond its famous walls.
More posts about Italy
Top resources for planning your dream trip to Italy| Italy off the beaten track in Brescia| Pretty towns of the Italian Riviera | Fireflies in Tuscany: nature's fairy lights
This post contains affiliate links which means I may receive a small commission if you click through to a site I mentioned and make a purchase at no additional cost to you. All thoughts and opinions are my own. I only recommend products and services I genuinely enjoy.
Pin to save
Subscribe to Untold Morsels
Subscribe to receive our monthly newsletter full of inspirational ideas for memorable trips.
As a thank you we will send you a FREE checklist for booking the best holiday accommodation rental apartments and villas on Booking.com, AirBnB etc


text:
UOB PRVI Miles Credit Card
This card from UOB is designed for the frequent flyer and offers great travel benefits that such as air miles that can be redeemed at over 60 partner airlines and discounts on hotel stays when booked from a range of booking agencies
Features of UOB PRVI Miles Card
- The minimum age of the cardholder must be 21 years as at last birthday.
- Minimum annual income required to avail the card is RM 60,000
- The annual fees for the Primary Card is RM 209.88
- The annual fees for the Supplementary Card is RM 31.80
Benefits of UOB PRVI Miles Card
Travel Privileges:
the cardholders can avail a host of travel benefits that discount on hotel stays when card is used to book hotel stays through various portals such as Booking.com, Agoda, AirAsiaGo, Expedia, Hotels.com, British Airways and Eva Air. The card also grants 12 free access passes every year to Plaza Premium lounges across Malaysia, a 20% cash rebate on KLIA Ekspres tickets when traveling to KLIA or KLIA2 and a cash rebate of RM 15 when availing 'Grab' taxi rides or private cars.
10% Rebates under SMART$ plan:
The card allows the holders to earn SMART$ rebates at participating outlets that can be redeemed on the next purchase itself. The conversion rate is as follows; 1 SMART$ amounts to RM 1.
0% interest education instalment plan:
Cardholders can convert their children's education fees into a zero percent interest instalment plan with a tenure of 12 months. The minimum amount that can be converted under this plan is RM 1000 with the maximum being capped to RM 20,000. The conversion of the amount into Education Instalment Plan takes place within 20 working days from date of transaction
0% interest payment plan:
allow cardholders to convert purchases from select UOB merchants into zero percent interest instalment plans with tenure options of 12 months, 18 months, 24 months or 36 months
Easy payment plan:
allows cardholders to convert purchases from select retailers that tally over RM 500 in a single receipt into affordable monthly instalments with tenures of 6, 12 and 18 months with interest rates of 2%, 3% and 4% respectively
Balance transfer plan:
the card offers a variable balance transfer plan with available tenure options of 6 months, 12 months and 18 months with interest rates of 7.77% per annum, 7.88% per annum and 7.99% per annum respectively
UOB PRVI Miles Card Rewards
The card earns 5 UNIRinggit reward points for every RM 1 spent on overseas expenses, Airline fares, hotel bookings, and travel agencies. The card also earns 4 AirMiles for every RM 5 charged to the card. These AirMiles can be redeemed at over 60 airline partners for discounted airfare or ticket upgrades and reward points can be used to redeem for exciting gifts on the UOB UNIRinggit rewards catalogue. The reward points can also be used to offset the card's annual fees.


text:
Multigrade Teaching Strategies Essay
Multigrade Teaching Strategies
Every student has their own aspirations on life and that is the reason why some students are working by virtue of aiming their own aspirations. Students of SSCT also have their own aspirations. Some of them are lucky that they belong to the economic-stable families where their family can support them in all aspects that they need compare to the students who work while studying. Academic performance of the students depends on the factors that affect it. These include various aspects that they need to adjust in order to attain their goals in life.
Full time students have their advantages when it comes to their performance in school because they only focus on their own works especially in school activities. They are most capable in joining some activities pertaining to improve their various skills that lead them to excel their performance academically. Full time students do not worry on the things that they do and they will do because their time are not divided when it comes to household chores and school activities.
They are lucky not because they are not working to sustain their school needs but because they are used to only focus on their aims that they set in life and how to attain it. They are also free on the things they want to. They can easily cope with school problems that need a lot of efforts to adjust on the situation. When it comes to financial aspects they easily pay for it without any doubt to tell to their parents and ask for it. Full time students are really blessed as they only focus on the right track that leads them to the road of success.
On the other hand, working while studying is a burden among working students. They have to give up and set aside their social needs. Working students give their time in working and studying their lessons in school. Most of them have lack of sleep but still need to work and go to school. Due to school works, working students do not have time for themselves even on their personal needs. Most of them do not care on how they look as long as they enter school and listen to the discussion of the teacher. Because of their eagerness to finish their studies, they forget to care about their health.
They almost forgot to spend their social life with friends, to live life, to experience their youth and do things that a normal person does. With their eagerness to succeed in life, they let the years go by without even thinking the present. Thus, full time and working students are both have the eagerness to a success but differ into various aspects on traveling the road of success for they have their own advantages and disadvantages to carry on to their road of success. Review of Literature Today, students can put dope in their veins or hope in their brains.
If they can conceive it and believe it, they can achieve it. They must know it is not their aptitude but their attitude that will determine their altitude (Jackson, 2006). Socio-Economic Factor Education provides individual children with the knowledge and skills necessary to advance themselves and their nation economically. Socioeconomic factors, such as family income level, parents' level of education, race and gender, all influence the quality and availability of education as well as the ability of education to improve life circumstances (Jennifer, 2007).
Danesy (2006), complimenting environmental and socio-economic factors to produce high academic achievements and performance include good teaching, counseling, good administration, good seating arrangement and good building. High level of illiteracy, poverty and low socio-economic status coupled with high rate of paternal and maternal deprivation of student academic needs, which was necessitated by poor socio-economic situation of the country has thrown many farmers and old rural dwellers into untold financial problems such as poverty, lack of money to purchase necessary textbooks and working materials for their kids.
Also many rural and suburban dwellers can no longer pay the school fees of their wards. These ugly situations have promoted young school students to drop out of school to engage in subsistence farming and become housemaids or engage in other menial jobs to support their academic pursuit. Hence, many students have since taken schooling as a secondary assignment and school attendance on rotational basis.
Socio-economic factors like attendance in the class, family income, and mother's and father's education, teacher-student ratio, presence of trained teacher in school, sex of student and distance of school are also affected the performance of the students (Raychauduri, 2010). Physical Factor Regardless of the reason for working, trying to meet the multiple and sometimes conflicting simultaneous demands of the roles of student, employee, parent, and so on often creates high levels of stress and anxiety, making it less likely that students will complete their degrees (Kasworm 2010).
There is a possibility a working student will have trouble finding time to study. If you have a job, you automatically have less time to do everything you want to (Corbett 2009). Mental Ability A typical response from faculty members and administrators who are asked how much undergraduate students should work at paying jobs while attending college. Available research supports this recommendation. Quantitative studies consistently show that retention rates are higher for students who work a modest number of hours per week (ten to fifteen) than they are for students who do not work at all or those who work more than fifteen hours per week.
Research also shows increased academic success for students working on rather than off campus. Umbach (2005), associate professor of higher education at North Carolina State University, and his co-authors demonstrate the educational benefits to working students when their instructors encourage cooperative learning, set high expectations for student achievement, and create assignments that require students to demonstrate deep learning. A campus teaching center may also support faculty efforts to help working students. Family Factor
Guidance is of the factor through which a student can improve his study attitudes and study habits and is directly proportional to academic achievement. The students who are properly guided by their parents have performed well in the exams. The guidance from the teacher also affects the student performance. The guidance from the parents and the teachers indirectly affect the performance of the students (Hussain, 2006). Students academic accomplishments and activities, perceptions of their coping strategies and positive attributions, and background characteristics (i. . , family income, parents level of education, guidance from parents and number of negative situations in the home) were indirectly related to their composite scores, through academic achievement (Noble 2008). Environmental Factor There are so many factors that can contribute to your academic performance. Noise, distractions, etc. can affect you in studying. Light, review materials, and others should be conducive to learning. However, there are some cases that students choose to have a bit of distractions.
For instance, a student might want to listen to music while studying. Others might want to watch TV while doing this. It's actually a case-to-case basis (Debate Society, biology professor, etc. 2008). The academic environment is the effective variable for students and has positive relationship with fathers education and grade level (Kirmani & Siddiquah, 2008). The student has to have an advantageous atmosphere to learn, not one which is disadvantageous to learning.
If the student feels the need to participate, rather than being forced to and possibly be humiliated as a result, the student is apt to learn that much more (Bea 2009). How do you expect a student to perform great if they are sitting in a dimly lit room where they can't hardly see what the professor is writing on the whiteboard, in room located next to an extremely busy train station or to have only a chair (to sit on) making it difficult to take notes or even open a book (Linda, 2010). Young, 2010), held the view that student performances are linked with use of library and level of their parental education. The use of the library positively affected the student performance. The students who are actively engage in the learning process are observed to have a positive correlation with the CGP. A Study effort from student and the proper use of the facilities provided by the institution to the student, a good match between learning style of the students and are positively affect the performance of the students (Ali, 2009). Karemera, 2005) found that performance of the students is significantly correlated with satisfaction with academic environment and the facilities of library, computer lab and etc. in the institution. With regard to background variables, he found a positive effect of high school performance and school achievement he found no statistical evidence of significant association between family income level and academic performance of the student.
Synthesis. The above mentioned reviews are related to the present study in the same that it accounts for the similarities of findings specifically on the factors affecting the academic performance of the 2nd year and 3rd year BEED students in SSCT. Conceptual Framework The factors that affect to the academic performance of the 2nd year and 3rd year BEED students are complex and numerous. Hence, the researchers only need those that are commonly encountered by the students. This study is anchored in some related concepts for its framework.
It includes the respondent's profile in terms of age, sex, student status, religious affiliation and occupation of parents and the factors affecting the academic performance of the 2nd year and 3rd year BEED students that will be basic to come up with a quality research output. Each individual has its potential and capability to conduct a certain research study. However, these are some factors or aspects that would affect in doing this kind of work. It must be considered by the researchers in order to have a better result.
Subject: Education,
University/College: University of Chicago
Type of paper: Thesis/Dissertation Chapter
Date: 2 November 2016
Let us write you a custom essay sample on Multigrade Teaching Strategies
for only $16.38 $13.9/page


text: The mystery of the missing Detroit Lions DE Corey Smith took on a strange turn which is baffling authorities to no end. It was widely reported that Smith and Oakland Raiders LB Marquis Cooper were simply fishing when their trip turned tragic with a missing party and now, recently reported, the discovery of the overturned boat belonging to one member of the fishing party. As if the news couldn't be any more heartbreaking, Corey Smith's wife, Anita, discovered a note from her missing, and as most presume, now deceased husband. The letter itself is being analyzed by investigative authorities but according to a press release, Smith had planned to overturn the boat, murdering himself and everyone on board as a piece of performance art. Anita Smith mentioned that her husband was acting very strangely during the course of the Lions' 0-16 season, acting out not in a violent manner, but through art. Smith had locked himself in his garage after every game and worked for hours, often as Anita says , "screaming, crying and smashing things. I think I heard Elliot Smith and the Cure playing a few times too". This final piece, Anita said, "seems to be a culmination of the past year for him. He took football so seriously. He just killed himself and everyone around him." Police have retained all of Smith's "artwork" for analysis. Detroit Police Chief Harry Tasker stated that some of the items in Smith's garage include "A stuffed Lion covered in what many hope is simply corn syrup dyed red, countless notebooks with the record '0-16' crossed out, and a very lifelike effigy of who we assume is Matt Millen with 137 stab wounds in it". Former GM and architect of the Lions team for the past season couldn't be reached for comment because he was too busy wiping his ass with hundred dollar bills.


text:
Healthy Spaces for Healthy Development
The Canadian Institute of Child Health (CICH), in partnership with the Canadian Child Care Federation (CCCF), has created this website to increase awareness of children's exposure to potential contaminants in the areas where children grow, learn and play. You will find reliable, up-to-date information that allows you to make informed decisions about how to create safe and healthy spaces for young children.
Healthy Spaces for Healthy Development is aimed at providing parents and caregivers with reliable and updated information to better protect children from environmental contaminants and pollution in places where children under the age of six live and play. A second arm of the project will be the identification of policies governing the operation of childcare facilities that impact upon environmental health and the evaluation of their consistency with the best practices recommended.
The project consists of a user-friendly interactive tool on the Internet, where parents and caregivers can access the information they need to make important decisions and explore alternatives. The images of a house, a playground and a childcare facility will appear individually on a section clearly identifying the type of setting. The user will be able to navigate through each room or area, identify potential threats and viable solutions to mitigate or eliminate the problem. Once the user selects a particular area, a pop-up menu will appear with the relevant information.
For more information about the project, contact Leonor Alvarado
the Institute / Partnership Opportunities
/ Planned Giving / Projects
& Activities /
If you have any comments about our website, please contact our webmaster.
Last Reviewed: April 30, 2003
Copyright 2003


text: Jazz Era Takes Over H.U.G. Awards The cocktail and dinner gala pulled in 286 executives of the innerwear and retail industries. By Karyn Monget on November 15, 2013 The Jazz Era recently took over the H.U.G., or Help Us Give, Awards, the annual fund-raiser hosted by the Intimate Apparel Square Club. The cocktail and dinner gala, which took place at the 230 Fifth Avenue penthouse in Manhattan, pulled in 286 executives of the innerwear and retail industries. An annual tradition, the event featured both silent and live auctions to benefit pediatric services at Rusk and Tisch Hospital, part of the Hassenfeld Pediatric Center at NYU Langone Medical Center. load comments blog comments powered by Disqus


text:
Drupal Website Development
At Harris Web Works, we build custom websites in Drupal, an industry-leading content management system (CMS) that's the foundation of many large corporate websites. We've used this reliable and flexible platform to build sophisticated websites for corporate clients, i.e., pharmaceutical companies, who have stringent requirements for front-end and back-end site functionality.
Our services include all aspects of Drupal website development and management:
- Design, code and build the site
- Adhere to strict branding and design guidelines
- Create custom templates to integrate third-party Investors, Media and Human Resources systems, and keep the look of your site
- Populate the site with your content (copy-writing and editorial help available if needed)
- Integrate website with the Acquia enterprise hosting platform
In addition, once the site is built, we can extend our partnership and handle site administration and updates on an on-going basis, or we can train you on how to best manage updates.
In addition to working directly with corporate clients, we also provide creative agencies with all technical and programming support to build Drupal websites they've designed.
For more details and a list of Drupal websites we've built, contact us at 203.535.1631 or via this form.


text:
I won't attempt to enter the nature versus nurture debate in a blog put up, nevertheless the episode does educate an essential parenting lesson: teach your youngsters that actions have penalties. See all the players within the Family Court do have rules, laws, codes and Case regulation that should be used with the details of the case. I've been utilizing the Law of Attraction since earlier than it was known as that – and I've seen it create miracles in my very own life. As you learn this article, please VOTE on the polls – your vote is solid anonymously and you will remain on this web page.
This convention aims to encourage critical reflections on traditional historiographical themes, methods and sources used to study nineteenth-century international regulation. The Commission argues that the tightening of EU immigration and asylum regulation ought to have had enough effect by then, so an extra suspension would not be justified. This could be very deceptive, whether or not or not the writer is intentionally claiming that the law permits everybody to hold on campus. That scamblog led me to another, then another, and one other, and then your YouTube videos of your interview with Blooomberg Law and your presentation at Stanford Law School. Stateside she talks of Rose Kennedy and daughter in law Jackie, and Eleanor Roosevelt and her mom in regulation Sara.
Conclusion: Yes, Cockroach Patricia Salkin was appropriate in her $elf-$erving assessment of the toilet as offering an inexpensive and accessible high quality legal training," right?!?! Just while you think law faculty professors have tapped their mighty innovation-sticks dry, absolutely requiring a protracted refractory interval earlier than one other nice thrust of intellectual power can sprout, they pleasantly shock, making one wonder if it's capsule enhanced because it just doesn't appear truthful. Whether you understand the effect of your feelings or not, your detrimental feelings are attracting more damaging in your life, so it is essential that you just perceive the role that your emotions play within the law of attraction, and find out how to change these emotions. Soon afterwards, Frans Timmermans was appointed First Vice-President of the European Commission in charge of, inter alia, the Rule of Law.
Interestingly enough, nevertheless, younger men who have been unwell-behaved (particularly sexually, however in different issues as well) had been usually married youthful than may be traditional (even when it meant the young couple had to live in his parents' residence) so as to curtail his shameful actions. Yes, that Los Angeles, the one with the museum-quality collection of regulation schools rivaling solely that found in Boston, NYC, DC, and Chicago, indisputably the epitome of mass legal education west of the Mississippi and east of Tokyo and it is nonetheless a great place for brand new legal professionals. I suppose your criticism has extra to do with the Overton Park line of case regulation and the APA itself. As regular readers have come to study, my precise message, best captured in my ACC guidebook , is targeted on deepening relationships between regulation departments and legislation corporations.
Reminder: Students matriculating on the College of Law beginning in Fall 2015 (present 2Ls in the class of 2018 or later) are required to complete a clinic or externship to graduate. Statistics show that lower than two-thirds of law college graduates in Texas from 2015 have full-time jobs as legal professionals 10 months after graduating. Don't go to any law school in Texas or for that matter any neighboring state (possible exception for some states in Mexico; I do not know circumstances there).


text: A&A Pool & Spa Cleaning has been serving Sherman Oaks CA and the greater San Fernando Valley for years. We understand that heat waves can hit at anytime and that's why our swimming pool technicians are here to guarantee that you have a sparkling pool that you can use year round. We offer a variety of services, including swimming pool cleaning, swimming pool maintenance, weekly pool cleaning and spa maintenance. We believe that swimming pool maintenance requires a lot of knowledge and hard work, but also a lot of care. So why not trust us, the Sherman Oaks CA swimming pool experts?


text:
SBI SO Result 2016 is announced by the State Bank of India. SBI SO 2016 Result is being declared in two stages - Exam, and Interviews. Candidates who have appeared for SBI Specialist Cadre Officers Recruitment exam, can check the exam result from the link provided below on this page and know whether … [Read more...]
SBI SO 2016 – Recruitment of Specialist Cadre Officers
SBI SO 2016 Recruitment - State Bank of India (SBI) conducted SBI SO 2016 recruitment exam for Indian citizens for the recruitment of Specialist Cadre Officers in State Bank Group. Details of SBI SO 2016 recruitment such application form, educational qualification, admit card & result date, … [Read more...]


text:
One stop destination for central heating services in Wirral
Date posted:
Click here to reply via
What better than a warranty of keeping your house warm throughout the year? Nothing? Well, if that's what you also think then you should definitely give a call to C J Charles at 01516449251 or at 07811113023 as they promise the best services.
C J Charles has a solution to a number of central heating system's issues, from boiler installations, repairs, replacements to their servicing and not just that, but to make the heating systems work efficiently we also provide you with power flushing. Apart from this, we also specialize in underfloor heating, so, if are searching for Central Heating Services in Wirral look at the services provided by us by clicking here - http://www.cjcharles.co.uk/central_heating.html


text:
Partnering Science for Impact
Joslin Solutions
Joslin brings an integrated approach to diabetes solutions by combining state-of-the-art research, novel clinical care and patient-centered education in nutrition and exercise.
Joslin solution areas for diabetes management and control are based on its research, innovations in the clinic, and tools to reach the physician and patient community.
- New targets for diabetes in glycemic control and insulin resistance
- New drug targets for treatments in diabetes complications
- Targets in obesity management and diabetes control
- Beta cell regeneration methods and targets for beta cell stimulation
- Glucose sensors and novel approaches for insulin pump therapy
- Markers, tools, and software-based approaches in nutrition and exercise
News | Joslin Research
- Medalist Study Underlines Importance of Blood Glucose Control in Older Adults with Type 1 Diabetes
- Lloyd Paul Aiello, M.D., Ph.D., Receives the 2017 JDRF Excellence in Clinical Research Award for Type 1 Diabetes Research
- Joslin Diabetes Center Creates Center for Cell-Based Therapy for Diabetes (CCTD)
- Connecting the dots between insulin resistance, unhealthy blood vessels and cancer
- Forging New Defenses against Diabetic Kidney Disease
Page last updated: September 19, 2017


text:
220 W Lexington Blvd, Glendale WI 53217-5016 Sold for $169,500 on 05/06/2016
Low Maintenance Brick Ranch!!
Features of 220 W Lexington Blvd Glendale WI 53217-5016
- Single Family Home
- Status: Sold
- 5 Total Rooms
- 3 Bedrooms
- 1 Full Bathrooms
- Est. Square Footage: 1,275
- Garage Size: 1.5
- Garage Type: Attached
- Est. Year Built: 1955
- School District: Glendale-River Hills
- County: Milwaukee
- Postal Municipality: Glendale
- Listing Company: Shorewest - South Metro
- Price/SqFt: $141
Property Description for 220 W Lexington Blvd Glendale WI 53217-5016
220 W Lexington Blvd Glendale WI 53217-5016 - Location, Location, Location on quite street but only minutes away from shopping, entertainment, dinning and freeway. Beautiful hardwood floors throughout, eat-in kitchen, large combo living and dining room allows openness and space great for openness and space. Tons of natural light makes home sparkle. Basement waiting for you to add your finishing touch. Backyard with patio great for summer BBQ.
Room Dimensions for 220 W Lexington Blvd Glendale WI 53217-5016
- Master BR: 13.0 x 11.0
- BR 2: 11.0 x 10.0
- BR 3: 10.0 x 10.0
- Kitchen: 11.0 x 10.0
- Living Rm: 18.0 x 13.0
- Dining Rm: 9.0 x 7.0
- Full Baths: 1
Other rooms
- Block, Full Storage Shed
Interior Features
- Heating/Cooling: Natural Gas
- Water Waste: Municipal Sewer, Municipal Water
- Appliances Included: Dishwasher, Dryer, Microwave, Oven/Range, Refrigerator, Washer
Building and Construction
- 1 Story
- Exterior: Patio Ranch
Land Features
- Waterfront/Access: N
|MLS Number
|New Status
|Previous Status
|Activity Date
|New List Price
|Previous List Price
|Sold Price
|May 9 2016 8:44AM
|Apr 1 2016 10:12AM
Estimated Monthly Payment
Make An Appointment
Katie Murphy
Listing Agent • 414.690.8123
Get More Info
Receive more detailed information on this propertyAsk a Question
Community Homes Near 220 W Lexington Blvd
Buyer understands that the information which is contained on Data pages is obtained from a number of different sources and which has not been independently verified or confirmed by the various real estate brokers and agents who have been and are involved in this transaction. If any particular measurement or data element is important or material to buyer, Buyer assumes all responsibility and liability to research, verify and confirm said data element and measurement. REALTOR *MLS* Equal Housing Opportunity


text:
The 'terrible threes' may result in a few tantrums and tears, but it's a brilliant age where your child will really start to develop their personality and grow in confidence and independence.
The concealed sensor inside her hand makes her face and heart light up in a lovely soft glow.
As far as toys for children age 3-years are concerned, Kinetic Sand is a safe bet.
Oh translates into one of the most gorgeous soft toys we have ever seen.
Each doggie has a different number of baby puppies in her tummy!
This awesome jungle set has working features including trap doors and a working vine lift.
Featuring the tree that appears in most episodes, the playset has plenty of features.
It takes the original concept of marble runs and track and balls to a whole new level.
Doctor Who fans can own their ride-in Dalek that they can actually get inside and operate!
Puzzle Globe is a terrific toy for children and is especially suited for kids aged 3-years old.
These wooden role play vehicles are perfect for all junior budding engineers and builders.


text: Dakine builds its gloves, like these Crossfire Gloves, with Pacific Northwest winters in mind! Designed to keep you warm and dry and fit for every function. Details: Synthetic suede with silicone grip palm, nylon/poly with DWR treatment shell, 150g tricot lining, slip on stretch to fit cuff closure, touch screen compatible, two year limited warranty. Material: Man Made Suede, Nylon.


text:
The Federal Executive Council, FEC, on Wednesday observed a minute silence in honour of Nigeria's one-time Permanent Representative to the United Nations and First Republic minister, Maitama Sule.
Speaking shortly before observing the moment of silence in Abuja, Acting President Yemi Osinbajo described the late elder statesman as a father of the nation and one of the great contributors to the growth and stability of the country.
"He will be sadly missed by all of us who regarded him as a mentor and as a father of the nation and one of the great contributors to the growth and stability of the nation.
"Yes, he was buried yesterday. I'm sure many of us followed the proceedings.
"The Federal Government was represented by delegation led by the Chief of Staff to the President, Abba Kyari, and several members of the Federal Executive Council."
NAN recalls that President Muhammadu Buhari's letter of condolence was handed over to Governor Abdullahi Ganduje of Kano State while acting President Osinbajo expressed condolences over the demise of the elder statesman via a statement on Monday.
Maitama Sule, the Danmasanin Kano and a distinguished diplomat, died in the early hours of Monday in a hospital in Cairo, Egypt.
His remains were buried in Kano on Tuesday.


text:
Registered participants will receive specific joining instructions two weeks before the conference. In the meantime, we hope you find the following, more general, ECRM Practical Information helpful.
Conference Location
Department of Business Studies
University of Roma TRE
via Silvio D'Amico
77-00145, Rome
Website http://www.uniroma3.it/
Conference Location
University Information
The 17th ECRM Conference is organized in collaboration with the Department of Business Studies, Roma TRE University, Rome, Italy. Roma TRE University is a public research university, the second largest in Rome, with enrollment of over 35,000 students and about 1,400 academic and professional staff. The university offers undergraduate degrees in a wide range of fields as well as Master and Doctoral degrees.
Local Information
Rome, the capital city of Italy, has for almost 3,000 years been an important city, globally influencing art, architecture and culture. The city is home to ancient ruins such as the Forum and the Colosseum which are powerful reminders of the former Roman Empire. Home to the Vatican City, headquarters of the Roman Catholic Church, Rome has the beautiful St. Peter's Basilica and the Vatican Museums, which house masterpieces such as Michelangelo's Sistine Chapel frescoes. The city offers fantastic shopping and foodie venues as well, to keep visitors entertained.
About Dubliin
Dublin, and indeed Ireland, has come a long way in the last 100 years. Our music, art and literature have travelled the world and made an impact on millions of hearts and minds. In fact, in 2010 Dublin was designated as a UNESCO City of Literature in recognition of its cultural profile and international standing as a city of literary excellence. Today, the city is a multi-cultural, creative hub and as we reflect, we also celebrate the evolution of one of the world's greatest cities.
Dublin has a whole host of interesting cultural activities available that won't trouble your wallet. The Irish Museum of Modern Art (IMMA) on the grounds of the 17th century Royal Hospital Kilmainham contains magnificent sculptures, ancient chestnut trees and inviting avenues, while the National Gallery of Ireland near Trinity College is a great spot to appreciate fine art. You can also stop by the 'Dead Zoo', aka the Natural History Museum and marvel at the animal specimens on display – including a dodo skeleton – or visit the Irish Jewish Museum at the Walworth Road Synagogue. Remember, all are completely free!
Getting to ECRM
The main airport Leonardo da Vinci (Fiumicino) is located about 26 km from the city centre. The centre of city is easily reached:
From and to Fiumicino airport by train Leonardo Express
At the airport's railway station the Shuttle-Train Leonardo Express direct service (without calling at other stations) takes just 32 minutes to get to Termini Station (intersecting with the Metro Lines A & B) .
Conference Accommodation
Conference dinner
Conference Dinner
Key locations on Google Maps
Key locations for the 17th European Conference on Research Methodology for Business and Management Studies ECRM 2018 in Rome are shown on this google map:


text:
¿Contraseña olvidada?
NFL rookie minicamps are hardly a barometer for future success. Players are learning the basics, such as the names of coaches, where to find the locker and how to execute a play call. But http://www.authentictampabayrays.com/Wade-Boggs-Jersey a few NFL-transferrable traits should stand out. And one thing -- OK, two things -- usually on display with Pittsburgh Steelers first-round pick T.J. Watt: His hands.A highlight video from the Steelers' Twitter feed shows Watt violently smacking a tackling dummy. Yes, it's a quick clip, but it brings home what the Steelers liked about Watt during Joey Bosa Youth Jersey the pre-draft process -- his ability to win with his upper-body. In late April, both coach Mike Tomlin and general manager Kevin Colbert praised Watt's hand usage as advanced for a player who started one full season at outside linebacker at Wisconsin. "He beats people with is upper body just as much as his lower body, and usually you don't see that type of hand usage and that type of know-how http://www.officialnikechargershop.com/joey-bosa-jersey-for-sale-c-11.html in a one-year player," Colbert said. Wisconsin coach Paul Chryst said Watt showcased his hands early in his collegiate career. Despite Watt's position change from tight end and a knee injury that cost him the 2014 season, Chryst quickly noticed Watt the pass-rusher could beat tackles in one-on-one settings with several different hand maneuvers. "He had some natural stuff to him where you knew he would be good," said Chryst of Watt, who finished last year with 15.5 tackles for loss. "We felt pretty good about his (first) fall camp as a pass-rusher. He will work, and he's smart enough to know how to get better, smart enough to listen to caching and watch what other great pros do. He'll put the time in." BEREA, Ohio -- Cody Kessler will begin the Cleveland Browns' offseason practices as the starting quarterback.Though coach Hue Jackson said the job is open, he said Kessler earned the chance to open organized team activities as the starter ahead of Brock Osweiler, Kevin Hogan and second-round draft pick DeShone Kizer. "This thing's Adidas Authentic Dalton Prout Womens Jersey open. It really is," Jackson said Saturday after the team's second rookie camp practice. "But Cody's done a great job. That's the reason I brought his name up first. He's really improved. He's worked his tail off, and he deserves the right and the opportunity to walk in this building and walk out there first. "And they've got to take it from him." Jackson would not commit to Osweiler as the backup, saying Hogan's year with the team has him ahead. Kizer will also get his share of reps. When the Browns acquired Osweiler from Houston in March, they did so to obtain Houston's second-round draft choice in 2018. Osweiler was not in the long-term plans, but he now http://www.authenticcolumbusbluejackets.com/authentic-dalton-prout-jersey figures in the mix. "He's competing," Jackson said. "He's here. And I said from the beginning, if a guy is in our locker room we're going to treat him like any of our other players." Osweiler's 21 starts are more than double the total for the other three combined. "I think in this league we all know you can't have enough good quarterbacks, enough guys to train at the position," Jackson said. "You never know how it's going to unfold, and things do happen, but he's competing. "He's done a good job. He's been great in the room with the guys. He's been a good person in the building. We're going to continue to allow him to do that and see what he has to show for us and kind of go from there." Jackson said the breakdown on who gets first-team reps for veteran minicamp in June will be settled at that point. For now, Kessler gets the first chance. As for figuring out reps for four quarterbacks for an open position, Jackson shrugged. "I'll find a way," he said. "I've been through this before, a few times."
¿Quieres participar en esta sección?
Si ya eres miembro, entra / loguéate.
Si aún no estás registrado en lospimentoneros.com:
Resultados del Real Murcia CF
Calendario del Real Murcia CF
Segunda B
Copa del Rey
¿Por qué eres del Real Murcia CF?
Los partidos memorables
Ídolos de la afición
Listado de aficionados del Real Murcia CF
Plantilla del Real Murcia CF
Ficha del Real Murcia
Historia del Real Murcia
Palmarés del Real Murcia
Equipación del Real Murcia
Estadio del Real Murcia
Escudo del Real Murcia
Himnos del Real Murcia
Presidentes del Real Murcia
¿Qué puedo hacer en Lospimentoneros.com?
Regístrate en lospimentoneros.com
Contactar con lospimentoneros.com
Recomienda lospimentoneros.com
Staff de lospimentoneros.com


text:
Encontrar uma cópia na biblioteca
|Western stories
Historical fiction
|Pessoa Denominada:
|Benjamin Capps; James Auchiah; Will Henry; Dorothy M Johnson; Elmer Kelton; C L Sonnichsen; Ernest B Speck; Anna Lee Waldo
|Tipo de Documento
|Material de arquivo
|Todos os Autores / Contribuintes:
Benjamin Capps; Will Henry; Dorothy M Johnson; Elmer Kelton; C L Sonnichsen; Ernest B Speck; James Auchiah
|Número OCLC:
|16 boxes (6.5 linear ft.)
Capps' papers from his college days through 1993. Includes diaries relating his experiences as a school teacher, machinist, and writer, 1946-1970; correspondence with colleagues and members of the Western Literature Association, 1968-1993; classwork, unpublished stories and books, 1946-1987; and research materials, manuscripts, and galleys for his publications from 1949-1993. Also included are files related to his plagiarism suit against Anna Lee Waldo and her publisher, Avon Books, for words and ideas used in the novel, SACAJAWEA, 1981-1983. Research materials include Indian history, especially the Kiowas in the late 1800s; typescripts by Kiowa historian, James Auchiah; and post returns and maps of some West Texas and Indian Territory forts 1865-1871. Correspondents include Will Henry, Dorothy M. Johnson, Elmer Kelton, C.L. Sonnichsen, and Ernest Speck.
Ítens Similares
- Capps, Benjamin, -- 1922-2001 -- Archives.
- Authors, American -- Texas -- Archives.
- Authors, American -- 20th century -- Archives.
- American literature -- 20th century -- Manuscripts.
- Auchiah, James -- Manuscripts.
- Henry, Will, -- 1912-1991 -- Correspondence.
- Johnson, Dorothy M. -- Correspondence.
- Kelton, Elmer -- Correspondence.
- Sonnichsen, C. L. -- (Charles Leland), -- 1901-1991 -- Correspondence.
- Speck, Ernest B. -- Correspondence.
- Waldo, Anna Lee.
- Fortification -- Texas -- Sources.
- Kiowa Indians -- History -- Sources.