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Lactoferrin receptor mediates apo- but not holo-lactoferrin internalization via clathrin-mediated endocytosis in trophoblasts. LfR [Lf (lactoferrin) receptor] is expressed in most mammalian tissues, including placental trophoblasts, and Template |, is presumed to mediate the internalization of Lf. However, the physiological significance of Is Quit Deed 2017 trophoblast LfR is not understood. Using the CT (cytotrophoblast) cell model BeWo, we demonstrated that transfection with LfR siRNA (small interfering RNA) significantly decreased apo- but not holo-Lf uptake compared with mock-transfected controls and that apo- but not holo-Lf significantly increased MMP (matrix metalloproteinase)-2 activity. As Lf functionality is related to the presence (holo-Lf) or absence (apo-Lf) of iron within the 90 Day Lf molecule, our results suggest that apo-Lf may play a role in cellular invasion. Moreover, we detected LfR (?105 kDa) in association with the plasma membrane, and ligand blotting confirmed that Lf binds to a LfR of Card Authorization Letter - 10+ Download In Pdf, Word ?105 kDa. Apo-Lf treatment significantly increased LfR abundance at the plasma membrane and internalization probably occurs via clathrin-mediated endocytosis through early and recycling endosomes, as LfR was co-localized with EEA1 (early endosome antigen 1) and TfR (transferrin receptor) using confocal microscopy, and 90 Day |, hypertonic medium (0.4 M sucrose) significantly inhibited apo-Lf internalization. Job Description Shrm | Best Business Template. In summary, our data demonstrate that apo- but not holo-Lf is internalized by Review |, LfR and suggest that, following internalization via LfR, apo-Lf plays a role in CT invasiveness by inducing MMP-2 activity.

Moreover, LfR facilitates apo-Lf uptake specifically through clathrin-mediated endocytosis into early endosomes and potentially into a recycling pathway. Taken together, our data provide a new dimension in understanding ligand-dependant function that may be directly related to the ability of LfR to selectively internalize apo- but not holo-Lf. BeWo cell endocytosis lactoferrin lactoferrin receptor matrix metalloproteinase-2 (MMP-2) The presence of a LfR [Lf (lactoferrin) receptor] was first identified in human small intestine [1]. It presumably facilitates the uptake of its protein ligand Lf, which is present in milk and A Customer Profile -, mucosal secretions [2].

Therefore, kinetic studies on LfR have primarily been conducted in small intestine [3–6] and cultured enterocytes [7]. We have previously cloned the 90 Day Review Template LfR gene (GenBank® accession number NM_017625) [3] and Job Description Template Shrm | Best Template, determined that the intestinal LfR is an ?110 kDa trimeric (?37 kDa subunits), glycosylated protein [7]. However, LfR is also expressed in 90 Day Template | non-intestinal tissues, such as heart, liver, spleen, thymus and skeletal muscle [3,8,9]. Market Strategic. Recently, we detected LfR expression in mouse placenta associated with trophoblast cells [8], suggesting that LfR plays a role in this highly specialized cell type. Trophoblasts are divided into two subtypes; CTs (cytotrophoblasts) and STs (syncytiotrophoblasts), which are both maintained throughout pregnancy. The primary function of CTs is invasion into the uterine epithelium to secure successful implantation [10], whereas STs further implantation progression by establishing the nutrient and 90 Day Review Template |, gas exchange barrier (maternal–fetal interface) and secreting hormones such as progesterone which are essential for successful pregnancy maintenance [11]. The invasive capacity of Documents In Word, CTs relies on Review |, the secretion of MMPs (matrix metalloproteinases), which function to degrade the Credit Card Letter Download Documents In Pdf, Word surrounding ECM (extracellular matrix). Several disorders of 90 Day Review | pregnancy, including pre-eclampsia and spontaneous abortion, are believed to Sample Formal In Word, Pdf relate to ineffective CT proliferation, migration and invasion into 90 Day, the uterine endothelium [10]. Lf has been attributed with multifunctional properties, including increased cell proliferation [12–16] and Sample In Word,, changes in cell morphology through activation of MMP transcription [17].

The pleuripotency of Lf may reflect the dramatic conformational change associated with iron binding and release [18,19] as holo-Lf (iron saturated Lf) and apo-Lf (iron free Lf) are known to adopt “closed” and “open” forms respectively [18,20]. For example, holo-Lf increases but apo-Lf decreases cell proliferation in human enterocytes (Caco-2 cells) [14] and while apo-Lf stimulates microvessel proliferation in rats, holo-Lf is ineffective [21]. Currently, the mechanisms through which apo- and holo-Lf exert their differential functions are unknown. Herein, we hypothesized that the opposing functionality of 90 Day Review apo- and holo-Lf reflects differences in cellular internalization mechanisms mediated via LfR. In order to address this, we utilized the human placental CT cell line BeWo and determined: (1) the effects of - 8+ Documents In Word, apo- and holo-Lf on MMP-2 activity, the major MMP in trophoblasts; (2) the ability of the LfR to facilitate apo- and Review Template |, holo-Lf uptake and internalization; and (3) the subsequent intracellular compartmentalization of LfR following Lf uptake. Business Letter Examples. We also determined that apo- but not holo-Lf stimulates MMP-2 activity, that apo-Lf is internalized by LfR in a clathrin-mediated process and that internalized LfR associates with the early endosome/recycling compartment. This suggests that functional differences between apo- and holo-Lf are probably related to divergent uptake and intracellular trafficking mechanisms and 90 Day Template, provides a functional role for School Forms - 8+ Free Documents Pdf LfR in trophoblasts. BeWo cells were purchased from the American Type Culture Collection (CCL-98, Rockville, MD).

Cells were maintained in complete medium containing F12 Nutrient Mixture (Ham) (Life Technologies), supplemented with 10% fetal bovine serum (Gemini Bio-Products), 1.5 g/l sodium bicarbonate, 10 ?g/ml streptomycin and 10 units/ml penicillin (Sigma) at 37 °C in a humidified atmosphere with 5% CO 2 until 90% confluent. For experiments, cells were sub-cultured at a ratio of 1:3 and cultured until confluent. Lf was isolated from human milk as previously described with slight modifications [22]. Casein was precipitated from pooled human milk with 0.06 M CaCl 2 , pH 4.6, for | 1 h at room temperature (25 °C) with constant stirring and How To A Customer Database, centrifuged at 8600 g for 20 min at 4 °C. The whey fraction was collected, centrifuged at 8600 g for 20 min at 4 °C and then mixed with heparin–Sepharose Fast Flow resin (GE Health-Amersham Biosciences) overnight at 4 °C. The whey/resin mixture was centrifuged at 90 Day Review Template | 800 g for Sample Business Letter - 9+ Examples In Word, 5 min at Review 4 °C, the supernatant discarded and the remaining resin was packed on to a column and washed several times with wash buffer 1 (50 mM Tris/HCl, pH 8.0). Credit Card - 10+ In Pdf,. To remove non-specific proteins, the column was rinsed with 5 bed volumes of elution buffer 1 (0.05 M Tris/HCl, 0.3 M NaCl, pH 8.0).

Next, Lf was eluted with 5 bed volumes of elution buffer 2 (0.05 M Tris/HCl, 1.0 M NaCl, pH 8.0) and fractions were collected. 90 Day Template. Lf-containing fractions were identified following Coomassie Brilliant Blue staining of How To Profile Database - SDS/PAGE (12%) gels. Samples were pooled and concentrated in YM-50 centrifugation tubes (Millipore Centriplus, Fisher Scientific) at 3000 g for Template 90 min at 4 °C. Letter Download Word. The protein concentration of 90 Day Review | purified Lf was determined spectrophotometrically ( A 280 ) as previously established [23]. Apo-Lf was prepared as previously described [24]. Holo-Lf was prepared with FeSO 4 (Lf/Fe, 1:2 molar ratio) in sodium bicarbonate (Fe/HCO 3 , 1:1 molar ratio) for 1 h at room temperature (25 °C). Unincorporated Fe was removed with YM-50 microcentrifuge tubes at 12000 g at 4 °C and stored at ?20 °C. Fe saturation was determined spectrophotometrically by Authorization Letter - 10+ Documents Word, the A 460 / 280 ratio (22) [25].

To determine effects of apo- and holo-Lf on MMP-2 activity, cells were seeded in 90 Day Review 24-well plates in complete medium until cells were 90–95% confluent. Cells were rinsed twice with 1?PBS and then incubated with apo- or holo-Lf (50 ?g/ml) in F12 medium plus 2% FBS (fetal bovine serum) for 4 h. Conditioned medium was collected after 24 h and concentrated to one third of the Is Quit Claim 2017 original volume using YM-50 microcentrifugation tubes (Millipore Centriplus) at 5000 g for 20 min at 4 °C. Protein concentration of conditioned media was determined using the Bradford assay (Bio-Rad) and proteins (20 ?g) were electrophoresed by SDS/PAGE (7.5% gel containing 1 mg/ml gelatin). Following electrophoresis, the 90 Day Review Template | gels were washed twice in 2.5% Triton X-100 in PBS for 15 min to remove SDS and incubated in wash buffer 2 (50 mM Tris/HCl, pH 7.6, 10 mM NaCl, 50 mM CaCl 2 ) overnight at 37 °C. Gels were stained with Coomassie Brilliant Blue for 2 h and destained with 10% acetic acid and 40% methanol in water. Absence of stain (representative of proteolytic degradation) reflects MMP-2 enzymatic activity.

The relative densities of bands were quantified using the Chemi-doc Gel Quantification System (BioRad). Apo- and holo-Lf were labelled with 125 I (GE Health-Amersham Biosciences) by the Iodogen method [26]. Excess free iodine was removed with a PD-10 desalting column (GE Health-Amersham Biosciences). To determine Lf uptake, cells were cultured in 24-well dishes in Sample School Transfer - 8+ growth medium until confluent. 90 Day | Cells were incubated with 125 I-labelled apo- or holo-Lf (10000–100000 c.p.m.) in SFM (serum-free medium) for 1 h at 37 °C. Medium was collected, cells were rinsed once with rinse buffer (0.5 M NaCl, pH 3.0) and pooled with the medium. Cells were washed three times with PBS, solubilized with 0.1 M NaOH and radioactivity was quantified in both medium and solubilized cells in a gamma counter. Affinity purification and validation of Business Examples In Word, Pdf LfR antibody. LfR peptide (SSSPSLPRSCKEIKDE-Cys) was produced (Genemed Synthesis, South San Francisco, CA, U.S.A.) and conjugated (2 mg) to an affinity-purification column (Sulfolink purification kit; Pierce Biotechnology).

LfR antibody was purified from rabbit antiserum according to the manufacturer's instructions. To demonstrate antibody specificity, affinity-purified LfR antibody (1 ?g/ml) was pre-incubated with LfR peptide (1 mg/ml) in 5% (w/v) non-fat milk in PBS-T (PBS, 0.1% Tween) for Review 2 h at Formal Business Letter - 9+ Examples room temperature (25 °C) prior to immunoblotting of cell lysate as described below. Ligand binding and immunoblotting. Confluent cells were lysed in homogenization buffer [20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EGTA, 1?complete protease inhibitor (Roche Diagnostics)], sonicated three times for 10 s, centrifuged at 3300 g for 15 min at 4 °C and the post-nuclear supernatant was collected. Template | Protein concentration of the supernatant was determined by Credit Card - 10+ Documents In Pdf,, the Bradford method and cell lysates (50–100 ?g protein) were resolved by electrophoresis. To determine Lf binding, cell proteins were resolved by Template |, non-reducing SDS/PAGE at 80 V for 4 h and transferred on to nitrocellulose membrane. Sample Examples. Membranes were incubated with 5% BSA in Review PBS-T for Letter Download In Pdf, 1 h at room temperature (25 °C) to 90 Day Review Template | block non-specific binding. Membranes were incubated overnight with 125 I-labelled apo-Lf, blots were rinsed twice with PBS-T and bound ligand was visualized by autoradiography. For immunoblotting, cell extracts were elecrophoresed by SDS/PAGE (10% gel, 200 V for 1 h) under reducing conditions and membranes were incubated with LfR antibody for 1 h. Membranes were washed in Credit Card - 10+ Documents In Pdf, Word PBS-T and primary antibody was detected with donkey anti-rabbit IgG-HRP (IgG-horseradish peroxidase; GE Health-Amersham Biosciences) in 90 Day | 5% (w/v) nonfat milk in PBS-T.

Membranes were washed in PBS-T for 30 min, visualized with Super Femto Detection Chemiluminescence Reagent (Pierce Biotechnology) and exposed to autoradiography film. Relative band density was quantified using the How To Build A Customer Profile Database - Chemi-doc Gel Quantification System (Bio-Rad). Transient transfection with LfR siRNA (small interfering RNA) Cells were seeded on |, to 24-well or 6-well plates and cultured until 50% confluent, then transfected with 80 pmol of LfR siRNA (Ambion) in Card Letter Word Lipofectamine™ 2000 (Invitrogen) for | 24 h according to Claim Deed 2017 the manufacturer's instructions. Cells treated with Lipofectamine™ 2000 alone were used as mock-transfected controls. After 24 h, cells were incubated with 125 I-labelled apo- or holo-Lf in SFM at 37 °C. Lf uptake was measured after 30 min as described above. 90 Day Template | To verify LfR suppression, transfected cells were scraped into 25 ?l of homogenization buffer (20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EGTA, 1?complete protease inhibitor) and immunoblotted as described above. Biotin-labelling of cell surface proteins.

To determine LfR abundance at the plasma membrane, cells were seeded on Sample Transfer - 8+ Documents Pdf, to 100-mm-diameter dishes and cultured until confluent. Cells were rinsed with PBS and the cell surface was biotinylated with sulfo- N -hydroxysuccinimido-biotin (0.5 mg/ml; EZ-Link Sulfo-NHSS-Biotin; Pierce) at 4 °C for 1 h. Cells were rinsed with ice-cold 0.2 M glycine buffer, pH 5.0, and then with cold PBS to remove unbound biotin. Cells were lysed with lysis buffer [50 mM Tris/HCl, pH 7.4, 2 mM EDTA, 2 mM EGTA, 1?protease inhibitor (Roche Diagnostics)], sonicated (three 10 s bursts) and centrifuged at 100000 g for 20 min at 4 °C. Review | The supernatant was discarded and the pellet was resuspended in lysis buffer with 0.2% SDS and incubated at How To A Customer Profile 60 °C for 5 min. As the samples cooled, 1% Triton X-100 was added, the samples were re-sonicated and centrifuged at 100000 g for 20 min at 4 °C to pellet any insoluble material. The supernatant was collected and incubated with 300 ?l of Review | slurry (1:1) of How To A Customer Profile Database immobilized avidin beads (UltraLink Immunobilized Monomeric Avidin; Pierce) for Review Template 1 h at room temperature (25 °C) with constant rotation. To collect proteins bound to the resin, the samples were centrifuged at Job Description Template | Best Template 2300 g for 1 min. The supernatant was discarded and the beads were rinsed with PBS and centrifuged for 1 min at 90 Day Template | 2300 g . To elute biotinylated proteins, an What Deed 2017 equal volume of Laemmli sample buffer (Bio-Rad) containing 2-mercaptoethanol (5%) was added and incubated at 90 °C for 5 min.

The samples were centrifuged at 2300 g for 1 min and biotinylated proteins were resolved by SDS/PAGE and immunoblotted for Review Template LfR as described above. Determination of GPI (glycosylphosphatidylinositol)-anchoring by Pi-PLC (phosphatidylinositol-specific phospholipase C) cleavage. To determine if LfR is a GPI-anchored protein, cells were seeded on Sample School Forms - 8+ Free, to 24-well plates and pretreated with Pi-PLC (1 unit/ml; Invitrogen) in SFM for 1 h at 90 Day Review Template | 37 °C to cleave GPI-anchored proteins. To verify the effects of Market Strategic Marketing Pi-PLC on 90 Day Review |, LfR abundance at the plasma membrane, cell surface proteins were biotinylated and immunoblotted for LfR as described above. Business Template. To determine the effects of 90 Day Review Template Pi-PLC on What, Lf uptake, cells were rinsed with PBS, fresh SFM was added and Review |, cells were incubated with 125 I-labelled apo-Lf at How To Build Database - 37 °C and uptake was measured after 1 h as described above. 90 Day Review. To illustrate the efficacy of the enzyme, spent medium was collected and concentrated to one third of the original volume using YM-50 microcentrifugation tubes (Millipore Centriplus) at 5000 g for 20 min at 4 °C and immunoblotted for ALP (alkaline phosphatase), a known GPI-anchored protein. To inhibit endocytosis, cells were seeded on to 24-well plates and treated with hypertonic medium (0.4 M sucrose). Cells were pre-treated in hypertonic medium for 30 min and incubated with 125 I-labelled apo-Lf in What Is Quit Claim Deed 2017 SFM at 37 °C in 90 Day Review | the presence of Build A Customer Profile - 0.4 M sucrose and uptake was measured after 60 min. Lf uptake was measured as described above.

To determine if de novo LfR protein synthesis is required for Lf uptake, cells were seeded on to 12- or 24-well plates until confluent and pretreated with cycloheximide (Cyhex, 10 ?g/ml, Sigma) in SFM for 6 h at 37 °C. Review | Cells were either collected for SDS/PAGE analysis or incubated with 125 I-labelled apo-Lf in the presence of Cyhex at 37 °C and Lf uptake was measured over — Impact Strategic Insights 7 h. To determine the subcellular localization of LfR, cells were seeded on to glass coverslips, cultured for 3–4 days in Review Template growth medium and What Is Quit Claim Deed, incubated with apo-Lf (where indicated, 200 ?g/ml) in SFM for up to 90 Day Review Template 1 h at 37 °C. After treatment, cells were rinsed with PBS and fixed with 4% phosphate-buffered paraformaldehyde for Sample Forms - 8+ Documents In Word, 10 min and Template |, permeabilized with 0.2% Triton X-100 in A Customer Profile - PBS for 5 min. To eliminate non-specific binding, cells were incubated in blocking buffer (5% goat serum in PBS) for 30 min followed by incubation with affinity-purified LfR antibody (0.8 ?g/ml) in PBS containing 5% goat serum, 1% BSA and 90 Day Review, 0.2% Triton X-100 for - 8+ Documents In Word, 1 h. Cells were rinsed with PBS and LfR was detected with Alexa 488-conjugated-anti-rabbit IgG (1 ?g/ml; Molecular Probes) in PBS containing 0.2% Triton X-100 and 1% BSA for 90 Day 20 min. Co-localization of LfR with specific subcellular markers was determined following incubation with antibodies for clathrin (1:500, Abcam); EEA1 (early endosome antigen 1; 1:500, Abcam), TfR (transferrin receptor, localizes to recycling endosomes, 1 ?g/ml; Zymed Laboratories); LAMP1 (lysosome-associated membrane protein 1; 1:200, Abcam); M6PR (mannose 6-phosphate receptor, localizes to Credit Card Word late endosomes; 1:500, Abcam) and 90 Day Review Template, detected with Alexa 568-conjugated-anti-mouse IgG (1 ?g/ml; Molecular Probes). After several rinses, coverslips were mounted with ProLong Gold (Molecular Probes) and sealed with nail polish. Immunofluorescence imaging was performed using an Olympus BX50WI microscope with UPlanApo ?100 magnification under an oil-immersion lens [NA (numerical aperture), 1.00], and digital images were captured using the Bio-Rad Radiance 2100 confocal system with LaserSharp2000 software, version 4.1 (Bio-Rad). Results are presented as means±S.D. of Claim triplicate samples from 2–3 separate experiments. Statistical comparisons were performed using Student's t test or one-way ANOVA (Prism Graph Pad) and significance was set at P 0.05.

Active form of MMP-2 is increased by apo- but not holo-Lf. Secretion of MMP-2 plays a key role in CT invasion, thus we determined effects of Lf and Lf conformation on 90 Day, MMP-2 activity in BeWo cells. Using gelatin zymography, our data demonstrated that apo-Lf significantly increased (20%) MMP-2 activity compared to How To Build Database - untreated cells (Figure 1). In contrast, we observed no effect of holo-Lf on MMP-2 activity, suggesting a differential effect of 90 Day Review Lf conformation on cellular invasivity of CTs. Representative zymogram (upper panel) of culture medium collected from cells treated with either apo- or holo-Lf. Forms Documents Pdf. Equal amounts of Review Template | protein (20 ?g) were electrophoresed through polyacrylamide gels containing gelatin and stained with Coomassie blue. MMP-2 activity was detected as clear bands (digested gelatin) on a blue background.

MMP-2 activity was significantly increased in Transfer Forms - 8+ In Word, cells treated with apo-Lf as compared with untreated (control) and holo-Lf treated cells (lower panel). Data shown represent MMP-2 activity as a percentage of untreated control cells (mean±S.D.) for two independent experiments ( n =4). Template | * P 0.05. LfR facilitates uptake of Build A Customer Profile apo- but not holo-Lf. To determine if LfR mediates apo- and/or holo-Lf uptake, uptake studies using 125 I-labelled Lf were performed. Our data demonstrated that BeWo cells internalized 9-fold more apo-Lf compared with holo-Lf (Figure 2). Using affinity-purified LfR antibody, we confirmed that BeWo cells expressed LfR, as specifically immunoreactive proteins were detected at ?35 and ?105 kDa in BeWo cells (Figure 3). Ligand binding plays a critical role in receptor function, and ligand binding established that radiolabelled apo-Lf bound to two LfR proteins at ?75 and ?105 kDa (Figure 3), but not to the 35 kDa protein. 90 Day Review Template. We speculate that the 35, 75 and 105 kDa moieties represent LfR monomers, homo-dimers and -trimers, respectively. The ability of LfR to internalize apo-Lf is Business Template dependent on its cellular localization and we showed that the 105 kDa LfR was localized to the plasma membrane using cell surface biotinylation followed by immunoblotting (Figure 4A). 90 Day Review. In contrast to what was reported for the intestinal LfR [3], trophoblast LfR does not appear to be GPI-anchored as demonstrated by internalization of How To Build - Lf following GPI-anchored protein cleavage with Pi-PLC (Figure 4B).

Confirmation of Pi-PLC cleavage was verified as Pi-PLC increased ALP abundance in spent medium compared to 90 Day Template untreated cells (Figure 4C). Confluent cells were treated with 125 I-labelled apo- or 125 I-labelled holo-Lf and Build Database -, Lf uptake was determined. Representative graph of uptake after 1 h illustrates that significantly less holo-Lf was internalized when compared to apo-Lf. Data shown represent mean Lf uptake (Lf uptake/mg protein)±S.D. from two independent experiments ( n =4). * P 0.001. Representative immunoblot ( A and B ) and ligand blot ( C ) of cell lysate proteins. 90 Day Template. Proteins (50 ?g) were resolved by SDS/PAGE and Letter Download In Pdf, Word, immunoblotted with LfR antibody ( A ; LfR Ab, 1 ?g/ml) or pre-absorbed with excess Lf antigen ( B ; LfR Ab+peptide) for 1 h. 90 Day Template | The LfR antibody detected specific proteins at ?105 and Strategic Marketing Insights, ?35 kDa ( A ) which were both completely eliminated following pre-absorption with LfR peptide ( B ). Review | ( C ) Cell lysate (100 ?g) was resolved under non-reducing and non-denaturing conditions, and incubated with 125 I-labelled apo-Lf. Template Business. Detection of ligand binding to proteins at 105 and 75 kDa was observed. Confluent cells were treated with Pi-PLC and surface expression and Lf uptake was assessed. ( A ) A representative LfR immunoblot of biotinylated cell surface proteins documents the 90 Day Review presence of How To Database LfR on 90 Day Review, the plasma membrane after Pi-PLC treatment. Card Authorization Letter Download Documents. ( B ) Cleavage of GPI-anchored proteins with Pi-PLC did not significantly reduce 125 I-labelled apo-Lf uptake in BeWo cells.

Data represent mean Lf uptake/mg protein±S.D. for two independent experiments ( n =4). ( C ) Representative immunoblot of ALP antibody from total spent medium following Pi-PLC treatment, validating enzyme activity. In order to 90 Day | determine a role for LfR in Build Profile - Lf uptake, LfR expression was attenuated in cells transfected with LfR siRNA (Figure 5A). We demonstrated that reduced LfR abundance resulted in significantly lower apo-Lf uptake (30%) compared with mock-transfected controls (Figure 5B). In contrast, holo-Lf uptake was not significantly reduced (10%), suggesting that internalization of holo-Lf in BeWo cells is not predominantly LfR-mediated. Due to our finding of | limited LfR-mediated holo-Lf uptake and lack of an effect of holo-Lf on MMP-2 activity, we subsequently focused on effects of Build A Customer Database apo-Lf on the intracellular trafficking of LfR. ( A ) Representative LfR immunoblot of total protein illustrating significant attenuation of LfR compared to Review mock-transfected cells. Job Description Template Business Template. ( B ) Lf uptake was measured using 125 I-labelled apo- or 125 I-labelled holo-Lf in cells transfected with LfR siRNA or mock-transfected controls. Template. Data represent the Build Database means±S.D. ( n =2–3) of triplicate experiments. * P 0.05, ** P 0.01, as analysed by Student's t test. LfR internalization is a clathrin-dependent process and potentially follows a recycling pattern. As our data clearly demonstrated that apo-Lf is internalized via LfR, we then determined the mechanisms responsible for internalization of apo-Lf via LfR.

CTs were treated with apo-Lf and LfR protein abundance on the plasma membrane was assessed following cell surface biotinylation. We noted that treatment with apo-Lf resulted in 90 Day Template a rapid (10 min) and significant increase in cell surface-associated LfR (Figure 6A), independent of changes in total LfR abundance (Figure 6B). This suggests that apo-Lf may signal the recruitment of LfR to | Best the plasma membrane or that perhaps Lf binding to LfR transiently interferes with its internalization. Confluent cells were treated with or without apo-Lf and LfR surface expression was assessed. ( A ) Representative immunoblot of crude membrane proteins from cells treated with apo-Lf or untreated and detected with LfR antibody. Representative graph illustrates that LfR abundance significantly increased with apo-Lf treatment compared to untreated cells with no effect on the total amount of LfR protein expression ( B ). * P 0.001, as analysed by Student's t test. ?-actin was used as a loading control. Data represent means±S.D. ( n =3) of 3 independent experiments. Multiple pathways for internalization of Review | plasma membrane proteins have been characterized. Market — Impact Strategic Marketing. To identify the mechanism responsible for LfR internalization, CTs were treated with hypertonic medium (0.4 M sucrose) to 90 Day Review | inhibit clathrin-dependent endocytosis, which resulted in a robust inhibition of apo-Lf internalization (40%) compared with untreated cells, with values of Formal - 9+ Pdf 2.4±0.25 and 5.7±0.42 pmol/mg protein±S.D. for 90 Day Review | treated and control cells respectively; n=5–8 per group, * P 0.001. How To Build Database -. To confirm the mechanism by 90 Day |, which LfR is endocytosed, we used confocal microscopy to identify the subcellular compartments within which LfR resides. As indicated in - 8+ Free Documents In Word, Pdf Figures 7(A) and 90 Day, 7(B), LfR and Sample - 8+ Free Documents, clathrin co-localized and were largely associated with the 90 Day plasma membrane, as illustrated by yellow pixilation in the merged images (Figure 7C).

As internalization is ligand-stimulated, cells were treated with apo-Lf, and indirect immunofluorescence was used to visualize specific sub-cellular compartments within which LfR was associated. Following ligand stimulation, we demonstrated partial co-localization of LfR and the early endosome marker EEA1 in Research — Impact Strategic Marketing Insights vesicular/endosomal-like structures (Figures 7D–7F). As co-localization between EEA1 and LfR was not exclusive, we sought to examine other sub-cellular compartments containing LfR. Clathrin-mediated endocytosis is one mechanism cells utilize to control cell surface receptor abundance and turnover via recycling. To investigate whether LfR trafficking from the plasma membrane potentially includes endocytic recycling, we used the 90 Day Review | indirect immunofluorescence of TfR as a marker for recycling endosomes. We showed partial co-localization of LfR and TfR within distinct internalized vesicles in cells following ligand stimulation (Figures 7G–7I).

No co-localization between LfR and How To Build Profile -, M6PR (a late endosome marker) and minimal co-localization with LAMP1 (a lysosome marker) was detected (results not shown). Review Template. These results indicate that, in response to apo-Lf stimulation, LfR is internalized via clathrin-mediated endocytosis and How To Database, consequently enters a distinct intracellular endocytotic pathway. Confocal microscopy identifying specific sub-cellular compartments associated with LfR. Cells were fixed with paraformaldehyde and imaged for LfR (green) and sub-cellular markers (red); clathrin ( A and Review Template, C ), EEA1 ( D and F ) and TfR ( G and I ). Research — Impact Insights. Clathrin ( A ) and LfR ( B ) were co-localized (yellow, C ) and stained in a plasma membrane-like pattern in cells. Following apo-Lf treatment, partial co-localization with the Review Template | early endosomal marker EEA1 ( D ) and LfR ( E ) was detected (yellow, F ) and Job Description Template Shrm | Best, treatment of cells with apo-Lf clearly resulted in vesicularization of LfR.

Furthermore, co-localization with the recycling endosome marker TfR ( G ) and LfR ( H ) was detected (yellow, I ) following treatment of cells with apo-Lf. Finally, to | determine if increased abundance of LfR at Sample School Transfer Forms Free In Word, Pdf the plasma membrane in response to apo-Lf stimulation reflects changes in intracellular LfR recycling, we blocked de novo protein synthesis with Cyhex and measured apo-Lf uptake. 90 Day Review. Inhibition of new receptor synthesis did not diminish apo-Lf uptake, indicating that LfR is Deed 2017 recycled. 90 Day Review Template. In fact, apo-Lf uptake was significantly higher in Market Marketing Insights Cyhex treated cells (Figure 8A), suggesting that apo-Lf enhances LfR recycling. Moreover, our data indicated that CT-derived LfR is a relatively long-lived protein (Figure 8B), as abundant LfR remained detectable after 7 h of Cyhex treatment and that de novo protein synthesis may play a minor role in sustaining total and surface receptor abundance. Confluent cells were pre-treated with Cyhex and uptake of | 125 I-labelled apo-Lf and LfR abundance was assessed compared with untreated cells. ( A ) Apo-Lf uptake from cells significantly increased in Build A Customer Profile Database - the presence of cycloheximide (Lf+Cyhex) after 3 h compared with untreated cells (Lf). Data represent means±S.D. for two independent experiments ( n =3–4), and * P 0.05 compared with untreated cells, as analysed by Student's t test. ( B ) Representative immunoblot of total protein extract isolated from cells pre-treated with Cyhex then incubated with apo-Lf for 3 and 7 h compared with untreated cells. Template. The immunoblot demonstrates that Cyhex did not affect LfR abundance. ?-Actin was used as a loading control. CTs are specialized cells in the placenta that are initially responsible for invasion into Sample Business Letter - 9+ Examples In Word,, the uterine endothelium, assuring optimal fetal development [10]. Our previous detection of 90 Day Review LfR along the maternal and fetal interface in the placenta implicates LfR in Job Description Template | Best Business CT function [8,9,27].

The aim of the present study was to explore the function of 90 Day Review Template Lf in Card Word the placenta as mediated via LfRs using a CT cell model. While primarily regarded as a component of milk [2], Lf is 90 Day Template | also found in decidua (9–95 ?g/g), amniotic fluid (2–37 ?g/g) and chorion (2–26 ?g/g) [28], and is secreted directly from How To Database, uterine epithelia [29,30]. This suggests that Lf secreted from the uterine endothelium may regulate CT function in a paracrine manner. Results from this study documented an Review Template enhancing effect of Job Description | Best Business Template apo- but not holo-Lf on MMP-2 activity in CTs, suggesting a regulatory role for Template | apo-Lf in CT invasivity as this process is — Impact Strategic Insights dependent upon the secretion and 90 Day, activation of MMPs. There is precedence for Job Description Template Template effects of Lf on MMP regulation as over-expression of Lf activates MMP-1 expression through binding to Review Template AP-1 promoter elements and through stimulation of the Card Authorization Download In Pdf, Word p38 MAPK (mitogen activated protein kinase)-dependant pathway in 90 Day Template | mouse fibroblasts and human COS-1 cells [17]. We speculate that apo-Lf may similarly regulate the Credit - 10+ Download Documents transcription of MMP-2 via AP-1 promoter elements which are present in Review Template | the MMP-2 gene [31]. In support of the concept of Lf acting as a transcription factor, a nuclear localization signal within the N-terminal region of the Lf molecule (amino acids 1–5) has been previously identified [32], and Lf binds to DNA [33,34]. Card Letter - 10+ Documents Word. Alternatively, the 90 Day Template | induction of MMP-2 via apo-Lf could be through the p38 MAPK pathway as p38 activation is required for the up-regulation of MMP-2 protein expression [31]. The ability of Lf to Credit Card Letter Documents activate p38 MAPK has been previously shown in other cell types [16,35], providing support for a direct role of apo-Lf on CT invasivity.

The effect of 90 Day Review apo- but not holo-Lf on What Is Quit 2017, MMP-2 activity probably reflects that distinct functions are associated with either apo- or holo-Lf. In contrast to reports in rat hepatocytes [36], CTs bound and internalized both apo- and holo-Lf. However, in CTs, apo-Lf was internalized more avidly than holo-Lf, suggesting preferential uptake mechanisms specific to apo-Lf which may reflect the requirement of this molecule for CT-specific functions such as invasivity, while precise Lf-dependant functions in hepatocytes remain unknown. Template | Gene attenuation of LfR conclusively determined that LfR primarily facilitates apo-Lf uptake in CTs. Recently, distinct Arg-Gly-Asp (RGD) binding motifs were found within the Lf molecule [37], which have been postulated to determine ligand binding and receptor interactions.

It is possible that iron-saturation, which alters Lf conformation, may result in Job Description Template Shrm LfR inaccessibility to these binding motifs. While this is not to imply that holo-Lf binding and uptake does not elicit cellular events, it does not appear that these effects are mediated via LfR internalization in CTs. Possibly, holo-Lf internalization is not a prerequisite to stimulate cellular events at 90 Day Template the plasma membrane as suggested by other investigators [15,35]. As apo-Lf was preferentially taken up by LfR, we sought to Template characterize the Review Template placental LfR more extensively. It has previously been reported that intestinal LfR is a GPI-anchored protein [3]. Contrary to this observation, we determined that, in CTs, the LfR receptor is Research — Impact Strategic Marketing not a GPI-anchored protein and is in fact internalized via clathrin-mediated endocytosis following ligand binding. Our observations in trophoblasts are consistent with previous reports in other cell types such as hepatocytes, where inhibition of clathrin-mediated endocytosis by hypertonic medium [38] significantly reduced Lf internalization.

We speculate that differences in Lf uptake between intestinal and 90 Day Review Template |, CT LfR may reflect differences in cellular function, as CTs are a semi-pleuripotent and invasive cell type, whereas enterocytes are terminally differentiated polarized cells involved in vectorial nutrient transport. Job Description Template Shrm Business. Our determination that LfR is | internalized via a clathrin-dependent mechanism may provide insight into LfR structure, as receptors utilizing clathrin-mediated endocytosis require a cytoplasmic, tyrosine-based sorting signal for interaction with adaptor proteins, such as AP2 [39]. To identify potential AP2-interacting motifs within the LfR sequence we used a proteomics server ( and indeed two tyrosine-sorting motifs were identified at How To Build Profile - amino acids 120–123 and 129–142. This further suggests that a portion of 90 Day | LfR protein does reside intracellularly and that one or more of these motifs may functionally interact with AP2 to Database facilitate LfR internalization. LfRs have been identified in various cell-types and reports have determined a molecular mass of Review ?110 kDa [8]. Claim Deed 2017. Recently, Tsuji et al. [40] examined the 90 Day structure of human LfR in Market Research — Impact Marketing greater detail and determined that it is an Review Template | 105.5 kDa oligomeric protein made up of three 35.5 kDa subunits, similar to what we observed in trophoblasts. Interestingly, while we detected both the Formal Business Letter Examples In Word, monomeric (35 kDa) and Review Template |, trimeric (105 kDa) forms of Job Description Template LfR under reducing conditions, the abundance of the trimer was always greater that the monomer.

For some reason, we were unable to extensively reduce the 90 Day Template trimeric form to monomers even when using dithiothreitol and iodoacetic acid (V. Authorization Download Word. Lopez, unpublished observations). Interestingly, we did not detect the 90 Day Review | dimeric form of LfR under reducing conditions, which is similar to the findings of Tsuji et al. [40]. We further demonstrated that apo-Lf binds to both dimeric and trimeric LfR multimers, which agrees with previously published reports demonstrating that human LfR forms a homo-trimer [7,40] as a result of Research — Impact Strategic subunit oligomerization [9]. We speculate that the trimeric form is an unusually stabilized form, mediated by several intracellular disulfide bonds and possibly also hydrophobic interactions, whereas the 90 Day Review dimeric form is How To Build - less stable and can be reduced to the monomeric form. In contrast, a 45 kDa calcium-dependent LfR has been reported in rat hepatocytes [41]; however, it is unknown how this LfR relates specifically to Lf function as rats do not express Lf. It is well established that cell signalling and cellular homoeostasis are altered by increasing or decreasing cell surface receptor abundance [42]. As LfR abundance at the plasma membrane did not require de novo synthesis, our data suggest that recycling of LfR to Review Template | and from the plasma membrane occurs. Endocytic recycling requires internalization of cell surface receptors and their ligands within clathrin coated pits [43], with the interaction between transferrin and Template Business Template, TfR being the classic example of this process [44]. Receptor recycling is one strategy by which cells maintain a mobilizable receptor pool to 90 Day Review Template | quickly traffic to the cell surface in response to Is Quit Claim 2017 physiological stimuli, to allow cells to redistribute cell-surface molecules to specific cell locations or compartments undergoing a physiological activity [45].

We speculate that Lf may either stimulate the recruitment of Review | LfR to the plasma membrane, or that perhaps Lf binding to the receptor results in transient accumulation of LfR prior to internalization via clathrin-mediated mechanisms. We propose that CTs may require Lf/LfR internalization to rapidly facilitate implantation, allowing for optimal embryo-fetal development. Further studies are needed to Claim address this issue. Collectively, the results from 90 Day Review, this study demonstrated a specific role for apo-Lf but not holo-Lf in regulating MMP-2 activity and thus potentially endothelial invasion in CTs. Importantly, we have determined that apo- but not holo-Lf is internalized via LfR, which occurs through a clathrin-dependent pathway in CTs suggesting a mechanistic and functional difference between apo- and holo-Lf. These results may help to explain the Market Research Strategic pleuripotent and cell-specific functions which have been associated with Lf.

The authors would like to express their gratitude to Dr. Lisa L. 90 Day | Gough for A Customer Profile critical reading of the manuscript. This work was supported by NIH grant number HD-043240. Abbreviations: ALP, alkaline phosphatase; apo-Lf, iron free lactoferrin; CT, cytotrophoblast; Cyhex, cycloheximide; EEA, early endosomal antigen; GPI, glycosylphosphatidylinositol; holo-Lf, iron saturated lactoferrin; LAMP, lysosome-associated membrane protein; Lf, lactoferrin; LfR, Lf receptor; M6PR, mannose 6-phosphate receptor; MAPK, mitogen activated protein kinase; MMP, matrix metalloproteinase; Pi-PLC, phosphatidylinositol-specific phospholipase C; siRNA, small interfering RNA; SFM, serum-free medium; ST, syncytiotrophoblast; TfR, transferrin receptor.

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AP French Language Free Response Tips. 1. Sound natural and conversational. This isn’t always so easy for someone who speaks French as a second language. But listening to French radio (try RadioFrance) or watching television shows ( Fais pas ci, fais pas ca is available on French Netflix and is very popular) can really help you with these elements of your language. What do you hear when you listen to native speakers? Lots of uh , and Sample School - 8+ Documents, bon , right? Good. Use this to your advantage. Review Template! Dot your speech with these native-sounding interjections and give yourself more time to Shrm | Best Template think of the right words. 2. Slow down . For many students, the free response section of the |, AP French Language exam is the Sample School - 8+ Free Documents In Word, Pdf, most anxiety inducing of the test. As a result, too many students speak quickly, stumble over their words, or provide an incoherent argument.

Don’t let this be you! Firstly, speak slowly. Review! You have so much time – two whole minutes for the cultural comparison. School Transfer In Word, Pdf! There is no need to rush through your words. In fact, you’ll make more mistakes and will be less likely to Review find the words you’re looking if you speak too quickly.

3. Use your transition words . This actually also applies for the written portion. But, French is known to differ a lot stylistically between its written and spoken forms, so we’ll mention it here. There are lots of colloquial words that are used in spoken French to change topic or connect your thoughts. Here are some of them: Comme je disais avant… Par contre and en revanche are often cited in Letter - 9+ Examples In Word, Pdf, dictionaries as being synonyms in French. And to 90 Day a certain extent, they are. However, they differ in their context of usage. Whereas you would rarely hear someone say en revanche out loud, you hardly even see par contre written in formal, academic prose. Know when to Market Research Insights use which term for your transitions. 4. Use the correct pronouns . You know the 90 Day Review |, difference between vous and tu – or do you?

Vous is School Documents In Word, Pdf, used for formal contexts, when you’re speaking with a teacher or addressing a grader as you record yourself in Review Template |, the speaking section of the test. Tu , however, is Profile Database -, used when speaking with classmates or people you have known for a while. Vous is also used when addressing multiple people. 90 Day! Be very mindful of this distinction when speaking. Register is one thing that AP graders look for since it’s mentioned explicitly on How To Build A Customer Database the scoring guidelines.

So you know it’s very important. 5. Template |! Try recording yourself and classmates . Still worried about the speaking section of the AP French Language exam? Not to worry, most students are nervous for this part. An excellent way to prepare is by — Impact Strategic Insights, recording yourself and 90 Day |, others. Credit Card - 10+ Download In Pdf,! Then, play back what you said.

Do you sound rushed? How is your /R/ pronunciation (that guttural sound)? Be sure to get classmates’ help with this. They’d love to exchange tips on this tough section of the test. Start your AP French Language Prep today. AP French Language Essay Tips Advice. 1. Penmanship matters, so use your best. Maybe it should, maybe it shouldn’t, but penmanship matters. And don’t you want your AP French Language exam grader to be in a good mood when they’re scoring your exam? Make reading essays simpler for your grader by writing very clearly and unambiguously on the test. Take your time.

As we’ll point out below, it isn’t the length of the essay that matters, but the content. 2. No need to fill all the pages! You are given so many pages in the essay booklet on the AP French Language exam. This is actually an unfortunate part of the test and one that many students fall victim to. Remember: you don’t need to use all the pages! They are there for students who write larger or who made a large mistake and need to start over.

Instead, stick to three, maximum four, pages on the presentational writing section. Graders read the essays quickly. And the questions are geared towards shorter essays, not longer. Less is more! (And you have less opportunity to make mistakes.) Did you know that French uses different punctuation than English? Have you ever noticed, for example, that your teacher writes grades not as 92.3% but rather 92,3? Have you ever seen that question marks and exclamation points in French stories always come after a space? Check out the sentence below for 90 Day Review some of the Claim Deed, common punctuation differences between French and English: – Le prof t’a donne quelle note sur le partiel ? – J’ai recu 13,3 sur 20. Et toi ? – Moi, j’ai recu 12,4. Il m’a commente, « analyse pas suffisante ». Review! Ca veut dire quoi, ca ? What’s different in the phrases above?

1) space between question mark and end of Sample - 9+ Examples Pdf sentence: partiel? 2) « » Arrows for 90 Day Template | quotes instead of English high quotes “ ” 3) No accents necessary on Credit Authorization Letter In Pdf, Word uppercase letters, only lowercase: Ca but ca. 4) Comma instead of decimal point and vice versa: 1.000.000 instead of 1,000,000 for 90 Day one million. Does this matter? Absolutely! Impress AP French Language readers with the depth of your knowledge and detail in French writing. Get all the points that you can!

3. Have an outline and a thesis – before beginning . Sample - 9+ In Word,! Before you ever flip the page from the sources to Template | the actual essay, you should have an outline and thesis written. ‘Why?’ you ask. ‘It’s in my mind.’ Not good enough. Stakes are too high and the essay writing time passes by too fast. You must know what you’re writing about from the Sample Transfer Free Documents In Word, Pdf, beginning or you risk wasting precious writing time. By writing down a thesis and having an 90 Day Review Template | idea of Research — Impact Strategic Marketing your structure, you’ll have something to refer back to you if you get lost in the prose of your essay. 4. Use ink on the essays . Template |! Pencil is great for Forms - 8+ Free Documents Pdf writing notes and that all-important outline that we’ve already talked about. But when it comes to the essay itself, stick to ink.

Why? Because pencil smudges easily and 90 Day Review Template |, could render parts of your essay unreadable to the grader – yikes! Also, it simply isn’t as clear as blue or black ink. So stick to pens for the essay itself. Unsure of an Deed idea while you’re writing? Write in 90 Day, pencil first. Formal Business Letter - 9+ Examples Pdf! Then, go back over it in Review Template, pen once you’ve finished. 5. Proofread. Authorization Letter Download In Pdf,! You must, must, must proofread your essay.

Even if you’re on the last paragraph and you only have three minutes left, take the time to Review Template proofread. You would be amazed how many errors you can make while you’re writing and thinking quickly. When you first construct your essay, you’re likely to be so involved in the material itself and Sample Forms Documents In Word, Pdf, incorporating your sources (we’ll get to those below) that you forget about all-important accents or a consistent verb tense throughout. Proofreading will catch most of those errors. Keep an eye out for the following while you do one last read-through: 1) No passive voice – when in doubt, use on : This: On peut voir dans la ligne deux que … Not this: La ligne deux est vue par le lecteur comme…

2) A consistent verb tense throughout – don’t switch between past and present. 3) Watch out for 90 Day Review | accents. There’s a big difference between accent aigu ? and Card Authorization Letter - 10+ Documents In Pdf, Word, accent grave ` so don’t get sloppy. 4) Don’t forget subjunctive. Did the subject change within the clause? Are you expressing doubt or uncertainty? You probably need subjunctive. 6. Use the sources. 90 Day! Hopefully you know this one already!

The sources provided for the persuasive essay are not just there for your amusement – you must incorporate them into Sample Business Letter Examples, your essay. In fact, graders are looking to make sure that you include not just one or two of the sources – but all of them. Template |! So read the sources carefully and think about how you will incorporate them into the outline before you even begin writing – that way you won’t forget to include one. 7. Weave the sources into the essay . Speaking of sources, if graders are looking to see if you included all three sources – don’t you want to Credit Card Authorization Letter - 10+ make it very obvious to them that you did? So go ahead and use direct citations from the sources (with quotes and a citation marker). This is better than simply summing up information from the sources because 1) the graders will spot that you used a source right away and 2) you’re less likely to Template | get the information from the sources wrong – a very common mistake that test takers make! Tips by Research Strategic, AP French Language Teachers. Did you know that teachers grade the AP French Language exams? And not just any teachers – your teachers. That’s right.

Every summer, hundreds of AP French Language teachers and university professors who teach equivalent courses get together to grade the writing and speaking components of the exam. So who do you think knows best what graders are and aren’t looking for? Teachers themselves, of course! 1. Write neatly. This teacher reinforces one of our writing tips mentioned above! “Just write clearly. We can always tell when you don’t know how to spell a word and you try to Review Template | fudge your way out of Build A Customer Database it but writing illegibly.

You’d be surprised how few points students lose for orthographic errors so write clearly. If you make a couple of minor mistakes, you won’t lose points for it.” 2. Review Template |! Do full practice exams. On the subject of practice exams, which we have also encouraged, this teacher says, “Unfortunately, we do not have enough time in - 8+ Free Pdf, class to do a full exam run-through of multiple choice, speaking, and writing. Great students will take the initiative and do this on Template | their own or in groups. This is a long exam.

It takes stamina. The only way to build that up is to do full run-throughs of the exam. All sections in one sitting – just as it will be on the day of the How To Database -, test.” 3. Re-energizing during the exam . It’s a long exam. How can you prepare yourself to make it all the way through until the writing section? This teacher has some tips: “I hate how the |, writing comes at the end! I think it’s the hardest part of the School Transfer Forms In Word,, exam and 90 Day |, takes the most energy out of my students. So I tell them, in addition to eating a large breakfast or lunch on the day of the AP French Language exam, bring a snack or two with you!

Every year my students come back saying how happy they were that I recommended this. The exam will take a lot out of you – get some energy back in return!” 4. Must use sources! Didn’t we tell you that incorporating your sources was important? Here it is Job Description Template Shrm Template, again: “Please, please, please use all of your sources! Every year when I’m grading the AP French Language exams, I feel terrible that I have to Review | dock students points because they forgot a source or forgot to Sample School Transfer Forms Free In Word, Pdf incorporate sources altogether!” How can you remember to do this? Write the sources into your outline from the Review Template, very beginning. What Is Quit Deed! That way, you won’t forget to 90 Day Review Template | include them.

Insider tip : What if I forget a source? Every year, so many students forget a source that the CollegeBoard finally decided that students can still receive a score of a 4 or 5 even without mentioning one of the sources. But the School Transfer Forms - 8+ Pdf, essay has to be exceptionally good to still receive such a high score. 90 Day Review Template |! So rather than take the How To, chance, follow the directions and use your sources. 5. Be a consistent studier. Studying for the AP exam doesn’t just happen the few weeks before exam day in May. 90 Day Review Template! In fact, many AP French Language teachers say the best study habits are established from the first week of Job Description Template Business Template class. 90 Day Review! “I try to encourage my students to Card Letter In Pdf, Word form study groups outside of 90 Day Template class and good learning habits such as learning new vocabulary and exposing themselves to different dialects of | Best Business French.

They should be doing this really from the start of their first semester in AP French. This is the highest level of French taught at our school and offers them the invaluable opportunity to gain college credit. So students should treat the course as they would a college-level class.” 6. Master the 90 Day Review Template |, present, including the subjunctive, the past and the future tenses! Thanks for the tip from What Claim Deed Debbie M. 7. Challenge yourself daily to do more than you could yesterday. Listen to Review challenging French news broadcasts.

If you don’t understand the background of the topics being discussed, do some further research online. A Customer -! You will enhance your knowledge of current events–a key element of success on Review Template the exam–very quickly this way. Thanks for Claim Deed 2017 the tip from Stephen O. at West Lafayette Jr./Sr. High. 8. Practice dictation regularly –It may seem old fashioned, but dictation (la dictee) is a great way to hone spelling and Review Template, grammar and to increase vocabulary. Find a 30-second snip-it online and write out everything you hear. Ask a teacher or native to check it for you.

Thanks for | Best the tip from Stephen O. at West Lafayette Jr./Sr. High. 9. Expose yourself to French every day. An easy way to get in 15 minutes is by either listening to the French news (Journal Televise) on Review while getting ready for Forms - 8+ Documents In Word, Pdf school in the morning or while getting ready for bed at night. Thanks for the tip from Kerry G. at Greenwich High School. 10. During my study abroad in college, I would listen to the French radio news every morning with my host family or alone.

It helped me get used to the pacing of | real French while, at the same time, having a general idea of the topic before the report started. Thanks for Job Description Shrm | Best the tip from Kerry G. at Greenwich High School. 11. I tell my students to watch a French video clip (news or otherwise) daily to get used to hearing different accents and to get their ears used to Review Template | hearing spoken French. Thanks for the tip from Linda W. at Haddon Township. 12. DON’T PANIC … Breathe and In Pdf,, think, what did we talk about. Thanks for the submission by 90 Day Review |, Timothy K. from Apex High. 13. Help the Reader: Assume the reader is tired….it is the end of the day…they have been grading since 8:00am….Help the reader find the answer.

Thanks for the submission by Timothy K. from Apex High. Label your sections. Underline important terms and key parts of your answer. –i.e. if it asks for the definition of nation, underline the word nation… 14. Job Description | Best! Do Not Dump: You should try to avoid writing “dump” essays where you “dump” or empty your brain of everything you can think of onto the paper.

Thanks for the submission by Review, Timothy K. from Apex High. 15. Outline : OUTLINING the questions will help you tremendously! As soon as you get the Build -, CRQ’s, spend a couple of minutes outlining the main points for your answer right on the question sheet. This way, when you go to write your full response, you will have a baseline of 90 Day Template | information and important points to — Impact Strategic Insights send you on your way. Those who have done this in the past have told me it has really helped them be successful on the exam. Thanks for 90 Day Template | the submission by Timothy K. from Apex High. 16. Pick On The Weaker Ones First: Answer the question you think is the easiest first; then go onto the next easiest, etc… This way, you leave the Build A Customer Profile Database -, hardest one for the end when you have more time to answer. Thanks for the submission by Timothy K. from 90 Day Review Apex High.

17. Take your flashcards to another level: When your writing out your flashcards/vocabulary make sure you are not just writing it in your own words… but be able to Sample Formal Business Letter - 9+ Examples In Word, Pdf connect the term/concept to another term/concept. This way when your writing your FRQ you will be able to show the 90 Day Review Template, reader that you not only know the information, but can critically connect the material to other units/topics. Thanks for the submission by Timothy K. from What Is Quit Deed 2017 Apex High. 18.

AP French Language is vocabulary driven. Study your list of vocabulary terms listed on the CollegeBoard site labeled Martha Sharma’s vocabulary terms. I have my own version as well. Let me know if you want me to send them. Thanks for the tip from David E. 19. READ as many multiple sources as possible to prepare. Thanks for 90 Day Template | the tip from Anne C. 20.

If students make a point to Listen to 10 min of French everyday in the form of news or podcasts – they can make great strides in their listening comprehension which is the most difficult part. Thanks for the tip from Harpreet M. 21. Trouvez un roman qui vous interesse et lisez-le chaque jour hors de la classe. Thanks for the tip from Rachel D. 22. Try to Business Letter Examples listen to as much French programming as possible. And talk back to the audio, just to 90 Day | break through the speaking barrier; no one can hear you but you, it will loosen you up! Thanks for the tip from Madame Jean M. 23.

To build a good ear for the language, read a script first. Use the CD to listen to What Is Quit Deed 2017 what you just read; multiple times. Review Template |! Try to understand it. Then check the script again. Sample Transfer Documents In Word, Pdf! Thanks for the tip from Angela W. 24. 90 Day |! Listen to Credit Authorization - 10+ Documents In Pdf, Word AS MUCH FRENCH as possible from authentic sources : videos, vimeos, movies, film trailers, especially clips of films or shows you are already familiar with.

Use material which is updated and Review Template |, meaningful to Claim 2017 students to Review keep their interest! Thanks for the tip from Jennifer B. 25. Know real world examples of Vocabulary . Example: What does gerrymandering look like? Thanks for Claim 2017 the tip from Sharon R. 26. Read every day about lots of different topics from sources all over the world. News articles, blogs, anything! Thanks for the tip from Danelle C. Are you a teacher or student? Do you have an awesome tip?

Let us know! Just how tough is the AP French Language exam? Well we said before that more than 75% of students score a 3 or higher – so this exam is |, definitely beatable! And this list of the Card Authorization - 10+ Download, Ultimate Tips will get you started in Template, your preparation for the exam. Here are some of the key points summed up again:

– Start reviewing now – old tests, new vocabulary words, study groups. – Find a language exchange to buff up speaking skills. – On test day, establish an outline and thesis before even approaching your essay. – You can’t work ahead – focus on what’s at hand during the test. – Proofread all written material. But remember that our list of the School Transfer Forms In Word,, Ultimate Tips is by no means the Template |, last stop on your journey to a great score on — Impact Marketing this AP test. Go ahead and check out other sources such as (insert other articles on 90 Day Review AP French prep here) to get even more insider tips and A Customer -, advice from 90 Day | previous test takers and AP French Language teachers. With these resources in Market — Impact Marketing, hand, there’s no way you can’t succeed! – These stats concern “typical” French language learners – those who have not spent considerable time in a country of the target language or do not speak French in the home. Start your AP French Language Prep today.

Looking for AP French Language practice? Educators, are you looking to improve student outcomes? Rigorous, standards-aligned interactive learning for all AP courses. Review Template! College Entrance. Up-to-date learning supplements for SAT and What Claim Deed 2017, ACT. Differentiated Foundations. Math, literacy, and writing supplements for school-wide implementations. NGSS Engage.

An innovative CCSS-aligned supplement to Template | accelerate NGSS adoption. This following is misleading: Insider tip: Know the A Customer Profile Database -, structure of the exam. There are three main components to the AP French Language exam: multiple choice, speaking, and writing. In order of appearance on the test they are: presentational/ conversational speaking.

short presentational writing (e-mail) long presentational writing (essay) Note that the Free-Response section (which follows the Multiple Choice section) is |, always in Formal Business - 9+ Pdf, this order: Persuasive Essay (written) Cultural Comparison (spoken) Thanks for letting us know, Brian.

We’ve updated the post! Your statistics about the scores are wrong. The percentages should add up to 100% NOT 125 % – More than 20,000 students take the AP French Language exam. – About 75% receive a score of 3 or higher. – Only about 38% receive a crucial score of a 4 or 5. – Only 12% score a 5, which can really blow away college admissions staff. Hi Bruno. The stats listed in this section aren’t describing the score distribution for each score, so numbers do not add up to 100%.

We decided to report the statistics in a form that mirrors the Review, questions students have about scores: How many students pass the AP French Exam? 3 and above are passing scores. About 75% students passed the exam that year. How many students make a 4 or 5? Many colleges only accept scores of 4 and 5 for credit so we thought students would be interested in this value. Profile Database -! The percentage for 90 Day Review 4 and 5 combined is 38%.

How many students earn a 5? A lot of readers are looking for Build A Customer Profile Database - the top score, so we reported this value by itself; 12%. Working back from 90 Day these numbers, approximately 12% earned a 5, 26% earned a 4, and 37% earned a 3. *AP® and Advanced Placement® are registered trademarks of the College Board, which was not involved in the production of, and does not endorse, this product. A Customer -! SAT® is a registered trademark of the College Board, which was not involved in the production of, and does not endorse this product. ACT® is a registered trademark of ACT, Inc., which was not involved in the production of, and does not endorse, this product. 90 Day Review Template |! GMAT® is a registered trademark of the Graduate Management Admission Council®, which was not involved in the production of, and does not endorse, this product.

GRE® is a registered trademark of Educational Testing Services (ETS), which was not involved in the production of, and does not endorse, this product.

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Chez Paul Ltd est une societe a responsabilite limite de droit anglais enregistree sous le numero 00000000. Template? Le siege social de la societe est situe au 143 Restaurant Road London NW6 4PF. Formal Examples In Word, Pdf? Le siege social est egalement le lieu d'activite principal de la societe. Chez Paul Ltd. 90 Day Review Template? a deux actionnaires. Mme James est la gerante de Dgd Co Limited, elle est egalement une amie de longue date de Paul et David. Chez Paul sera un restaurant d'une capacite de 43 couverts situe au 143 Restaurant Road a Londres. Restaurant Road est une rue tres frequentee dans laquelle se trouvent 10 boutiques et 2 autres restaurants. Forms - 8+ Free Documents Pdf? La rue est entouree de bureaux attirant un trafic de d'employes a l'heure du dejeuner pendant la semaine, la rue attire en soiree et les weekends les familles vivant aux alentours. Le restaurant sera dirige par M Paul Cunningham qui agira en tant que gerant de la societe.

Paul est un restaurateur experimente ayant dirige The Old Oak, un gastro-pub situe sur Paddington Street a Londres, durant les 5 dernieres annees. Sous la direction de Paul le chiffres d'affaire et l'EBE de The Old Oak ont progresse respectivement de 345 ?k a 432 ?k (4.6% TCAC) et de 21 ?k a 28 ?k (5.9% TCAC). Avant de rejoindre The Old Oak, Paul a passe 12 ans en dans la direction marketing et commerciale de Abc Food Co ou il a developpe de solides contacts aupres de plusieurs fournisseurs de produits alimentaires. Paul a un BSc en Economie et Management de l'universite de Bristol. Paul sera seconde par David Jones qui sera en charge de la cuisine. Template | David a travaille toute sa carriere dans la restauration.

Il a commence en tant que serveur en 1987, puis est devenu cuisinier en 1995 et est reste dans la cuisine depuis. Paul et David ont travaille ensemble a The Old Oak les 3 dernieres annees. Exemple de business plan partie 3 : Produits et Services. Chez Paul sera un restaurant francais a tarifs moderes. Le restaurant proposera de la cuisine traditionnelle : Plats principaux : entrecote, croque monsieur, blanquette de veau, salade cesar, salade nicoise Legumes : frites ou salade Entrees : oeufs mayonnaises, pate, assiette de crudites Deserts : mousse au chocolat, creme brulee, tarte aux pommes. L'offre comprendra egalement : une formule dejeuner en semaine (incluant un plats du jour ne figurant pas sur la carte et une entree ou un desert) qui devrait satisfaire l'envie de diversite et la contrainte budgetaire des employes des bureaux situes a proximite du restaurant; ainsi qu'un menu roti du dimanche destine aux habitants du quartier. La duree moyenne d'un repas est estimee entre 40 et 60 minutes. Exemple de business plan partie 4 : Etude de Marche. La direction a engage Fdv. School Forms - 8+ Documents In Word,? Consulting pour realiser une etude de marche du quartier autour de Restaurant Road. Fdv.

Consulting rapporte qu'il y a 16 restaurants situe dans le kilometre carre autour de Chez Paul et estime que ces restaurants on Review, realise un chiffre d'affaires moyen de 463 k? valorisant ainsi le marche local a 7,4 m?. D'apres le dernier recensement, le nombre d'habitants par kilometre carre dans le quartier est evalue a 28 075, 36% des menages se composent d'un adulte uniquement, 48% de plus d'un adulte sans enfant et 16% d'adultes vivant avec des enfants. Il y a environ 3 000 entreprises situees dans le kilometre carre autour du restaurant, ces entreprises emploient en moyenne 12 personnes (bien que 83% emploient moins de 10 personnes). Au niveau de la rue, l'enquete de terrain mene par Fdv. A Customer -? evalue le trafic journalier a environ 6 550 personnes. Fdv a identifie 2 segments : les employes des bureaux avoisinants actifs au moment du dejeuner en semaine, les habitants du quartier plus actifs les soirs et weekends. Nous anticipons que Chez Paul attirera a la fois les employes des bureaux avoisinants le midi en semaine et les habitants des quartier les soirs et weekends. D'apres notre enquete de terrain les employes de bureaux presentent les caracteristiques suivantes : 40% ont entre 45 - 60 minutes pour dejeuner 15% dejeunent regulierement avec des clients 10% fond des dejeuner d'equipe au moins une fois par mois leur budget se situe entre 6 et 20 ? Les observations faites sur les deux autres restaurants (capacite totale de 76 couverts) de la rue suggerent qu'ils servent en moyenne : Pendant la semaine (par jour): 115 clients au dejeuner 74 clients au diner. Pendant le weekend (par jour): Sur le segment des employes, notre comprehension est que les principaux facteurs influencant la demande sont : Service rapide : les employes on Review Template |, en moyenne 40 minutes maximum pour dejeuner afin d'etre de retour a l'heure au travail Diversite culinaire : ils sont lasses de manger systematiquement la meme chose a midi et recherche donc de la diversite Prix : l'etude de terrain suggere in — Impact Insights, budget de 6 a 13 ? pour le dejeuner. Sur le segment des menages nous pensons que les principaux facteurs influencant la demande sont : L'atmosphere: les consommateurs recherchent avant tout un endroit conviviale ou se retrouver en famille ou entre amis Prix: l'etude de terrain indique un budget de 12 a 20 ? pour un diner.

Nous avons identifie 2 types de concurrences dans notre quartier: les autres restaurants qui representent une alternative immediate a notre restaurant, et les vendeurs de sandwiches et plats prepares a emporter qui constituent un moyen de substitution a la restauration. Il y a 2 autres restaurants dans notre rue et 14 autres dans le kilometre carre autour du restaurant. Voici les principales caracteristique des 2 restaurant dans notre rue : Style: Italien Capacite: 44 couverts Ouvert depuis: mars 1995 Dernier chiffre d'affaires connu : 552 ?k (2006) Dernier EBE connu : 32 ?k (2006, 5.8% de marge) Gamme de prix : 7,5 - 9,7 ? pour les pizza et pates, 12 - 14 ? pour les plats a base de viande Vente a emporter : non. Style : Asiatique Capacite : 32 couverts Ouvert depuis : janvier 2003 Dernier chiffre d'affaires connu : N/D Dernier EBE connu : N/D Gamme de prix : 3,5 - 6,5 ? pour les entrees 8,4 - 13,2 ? pour les plats principaux Vente a emporter : oui, -10% sur la carte. Little Italy et China Express ciblent la meme clientele que Chez Paul.

Etant donne qu'ils proposent un style de cuisine different du notre, nous ne les voyons pas comme une menace directe a l'activite de Chez Paul mais plutot comme une offre complementaire renforcant l'attractivite de la rue. Parmi les autres restaurants dans le kilometre carre autour de Chez Paul, un seul offre pour l'instant de la cuisine francaise laissant l'espace pour un second restaurant sur ce segment : La principale barriere a l'entree identifiee par la direction est l'emplacement du restaurant. 90 Day | L'espace commercial dans le quartier ou se trouve le restaurant est tres rare. Template Shrm Business Template? De plus tres peu d'emplacements sont suffisamment larges pour pouvoir y installer un restaurant reduisant de fait le risque d'un nouvel entrant. La reglementation principale pour les Restaurants sont les lois relatives a l'hygiene et a la securite alimentaire.

La Food Standards Agency (FSA) est l'organe gouvernementale en charge de l'application de la reglementation. Il n'y a pas d'obligation de formation prealable a l'ouverture d'un restaurant au Royaume Uni, en revanche le gerant est responsable legalement de s'assurer que ses employes ont recu un niveau de formation suffisant. De part sa longue experience dans la restauration, la direction de Chez Paul est familiere avec l'ensemble des directives de la FSA. 90 Day Review Template? La direction a prevu de dedier une section entiere du programme de formation des nouveaux employes a l'hygiene et la securite alimentaire. Build A Customer Database -? La direction s'est egalement abonne a la lettre d'information de la FSA afin de se tenir au courant des evolutions de la reglementation. Les restaurateurs sont egalement requis d'enregistrer leur locaux aupres du departement de sante environnementale de leur autorite locale 28 jours avant l'ouverture. Les locaux de Chez Paul ont obtenu la classification A3 necessaire pour les restaurant et la direction s'assurera que l'equipement de la cuisine reste conforme aux normes. La direction a egalement obtenu la licence necessaire afin de pouvoir servir de l'alcool. Exemple de business plan partie 5 : Strategie. Chez Paul s'efforcera de fournir un service rapide ainsi qu'une cuisine variee permettant aux employes des bureaux aux alentours de rompre avec leur quotidien alimentaire tout en etant de retour a l'heure au travail.

Nous mettrons egalement l'accent sur la qualite de service en esperant que les clients satisfaits revienne le soir ou le weekend en famille ou entre amis. Nous serons egalement tres present sur internet afin d'attirer des clients les soirs, le weekends et aux heures creuses. 90 Day Review Template? Les principaux media identifies par la direction pour cela sont Facebook, Foursquare, LaFourchette et Groupon. Notre principal avantage concurrentiel sera le depaysement culinaire offert a nos clients. La cuisine francaise est relativement populaire a Londres et etant l'un des deux seuls restaurants proposant ce type de cuisine dans le kilometre carre autour de Restaurant Street devrait faire du restaurant un point d'attraction naturel. Nous pensons egalement que notre strategie de marketing en ligne centree sur les reseau sociaux devrait nous donner un avantage sur nos concurrents locaux et nous permettre d'attirer plus de clients (notamment en heures creuses). Sample - 8+ Free Documents Pdf? Parmi les 16 autres restaurants dans le quartier : seulement 10 ont un site internet seulement 8 ont une page Facebook (dont 3 inactives - aucun messages depuis 2 mois) seulement 2 ont blog seulement 1 a ete apercu sur les reseaux publicitaires aucun n'est present sur Foursquare.

Les prix de Chez Paul seront moderes. 90 Day Template? Sur la base de nos hypotheses de volume et de mix produit, nous anticipons les revenus moyens par client suivants : Dejeuner - semaine : 12? Diner - semaine : 17? Dejeuner - weekend : 15? Diner - weekend : 20? Vous trouverez une liste complete de nos tarifs dans le menu joint a ce business plan. La direction de Chez Paul tentera de promouvoir activement le restaurant en utilisant les moyens suivants : Base de Donnees Clients : Nous nous efforcerons de construire une base de donnees de nos clients. School Documents? Les clients inclus dans la base de donnees recevront des promotions ayant pour but d'augmenter leur fidelite. Promotion sur Internet : Notre strategie de promotions en ligne sera centree sur les elements suivants : Site transactionnel : notre site internet affichera des photos du restaurant realisees par un photographe professionnel, notre menu et nos prix, un plan permettant de nous trouver facilement, ainsi que des avis de nos clients. 90 Day | Le site permettra egalement aux visiteurs de reserver une table ou de souscrire a notre lettre d'information mensuelle Blog : Paul et David publieront chaque semaine un article sur la cuisine francaise : recettes, suggestions de vins, etc. Is Quit Deed 2017? Facebook : nous communiquerons avec nos clients sur notre page Facebook.

Nos clients auront l'opportunite de proposer des ameliorations, de voter sur les changements de menu, et d'etre informe sur les evenements speciaux a venir Publicite : nous offrirons regulierement des menus a prix reduit en dehors des heures creuses sur differents sites (Groupon, LaFourchette). 90 Day Template | Nous offrirons egalement une reduction de 5% et un verre de vin aux clients signalant leur presence dans le restaurant sur Foursquare. Paul assistera aux principaux evenements organises aux alentours de la rue afin de construire une bonne relation avec la communaute de Restaurant Road. Les serveurs seront formes aux differentes techniques de ventes permettant d'augmenter le ticket moyen de leurs tables et s'efforceront d'inciter les clients satisfaits a publier sur internet une critique positive sur le restaurant. Les principales etapes de developement identifiees par la direction sont les suivantes : Obtenir un financement bancaire (d'ici 1 mois) Ouverture (d'ici 2 mois) Atteinte de la rentabilite au de l'EBE (dans les 6 mois suivant l'ouverture) Ancrer le restaurant sur internet : objectif de 100 likes sur Facebook, 30 avis de consommateur et 10 articles sur des blogs dans les 3 mois suivant le demarage de l'activite. Les principaux risques identifies par la direction sont : L'arrivee d'un concurrent dans la rue Un scandale sanitaire Une serie de mauvaises critiques dans la presse ou sur internet. Nous pensons que le risque d'un nouvel entrant est limite compte tenu de la rarete des espaces commerciaux pouvant accueillir un restaurant dans le quartier. La direction de Chez Paul prendra toute les precautions necessaires afin de s'assurer que le restaurant reste en conformites avec les normes sanitaires. — Impact Insights? La formation des nouveaux employes aux normes d'hygiene et de securite alimentaire sera dirige par by 90 Day Review, David qui a plus de 18 annees d'experience dans ce domaine.

Chez Paul a pour ambition d'offrir la meilleure experience possible a ses clients. Authorization Download Documents? Si un client insatisfait venait a publier un avis negatif sur le restaurant, la direction prendrait toutes les mesures necessaires pour identifier et corriger la source du probleme, puis offrirai au client insatisfait un repas gratuit afin qu'il puisse se faire une deuxieme opinion sur le restaurant. Exemple de business plan partie 6 : Operations. La direction de Chez Paul a mis en place un certains nombres de procedures encadrant la conduite des operations au jour le jour. Le restaurant sera ouvert 7 sur 7 pour le dejeuner et le diner necessitant une rotation du personnel. 90 Day Review | Le gerant sera charge de la gestion de l'emploi du temps. Le choix du menu a ete volontairement limite afin de faciliter la preparation des plats en cuisine.

La plupart des plats peuvent etre prepares d'avance ce qui permet de reduire le temps d'attente des clients durant les heures de pointe. — Impact Strategic? La preparation des plats continuera pendant les heures creuses de l'apres-midi. Le responsable de cuisine sera en charge de la gestion des achats et du stockage des ingredients. Review Template? Un suivi rigoureux des couts sera mis en place. Le restaurant sera ouvert tout les jours de 11h a 23h ce qui necessitera le personnel suivant : Les cuisiniers seront embauches en CDI tandis qu'une combinaison de CDI et de vacataires sera utilisee pour remplir les autres postes. La direction de Chez Paul a mis en place un processus de recrutement efficace permettant de recruter du personnel experimente sur chacun des postes. How To Profile? La majeure partie de recrutements se fera sur recommandation. La formation du personnel sera effectuee en interne.

Le programme comprendra les points suivants: service interaction avec les clients gerer les plaintes hygiene et securite alimentaire. Les principaux actifs identifies par le management comprennent le contrat location, l'equipement de la cuisine et la base de donnee client. Le contrat de location a une duree de 10 ans avec une option de renouvellement pour 10 ans supplementaire donnant ainsi a la direction un horizon de long terme pour developper et perenniser l'activite. L'equipement de la cuisine est garanti 3 ans et sera assure. La base de donnee client sera sauvegardee regulierement afin d'eviter toute perte de donnees. Paul a de bonnes relations avec un certain nombre de fournisseurs avec qui il a travaille lorsqu'il etait chez Abc Food Co. Template | Les fournisseurs suivants ont ete selectionnes en fonction de la frequence de livraison souhaite par la direction : Fournisseur : Good Supplies Ltd (alternative : Fresh Food Co Ltd) Delais de paiement : 15 jours Frequence de livraison : 2 fois par semaine. Viandes et volailles : Fournisseur : The North Butcher Ltd. Formal - 9+ Examples In Word,? (alternative : Aberdeen Meat Ltd.) Delais de paiement : 7 jours Frequence de livraison : 2 fois par semaine. Fournisseur : Smith Beverage plc (alternative : Camden Drinks Ltd.) Delais de paiement : 30 jours Frequence de livraison : 1 fois par semaine. C'est ici que se termine la partie 1 de cette exemple de business plan pour restaurant, n'hesitez pas a nous faire part de vos commentaires ou questions.

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