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Business Growth and Competitiveness. FedNor is committed to fostering economic growth and increased competitiveness of master Northern Ontario's businesses by working with community and industry stakeholders to invest in projects that improve productivity, reach new markets, facilitate access to capital, foster investment, encourage entrepreneurship and cultivate industry collaboration. Results generated by FedNor's investments in business growth and competitiveness will include: enhanced productivity, competitiveness and business management capacity; improved trade and export performance and supply chain integration in the short- to college to play essay medium-term (up to five years); enhanced level of investment from outside the region that increases the number of master thesis enterprises established and/or expanded in essays on nelson Northern Ontario; and diversified traditional industries and emerging sectors producing more value-added and cover, knowledge-based products and services. Not-for-profit organizations in essay irreverant Northern Ontario, such as community economic development, Aboriginal and thesis cover, Francophone organizations, industry and business associations, networks, or alliances. Why Is It Important? Municipalities; municipal organizations; and First Nations in Northern Ontario.
SMEs with fewer than 500 employees within Northern Ontario seeking youth internships . Activities related to helping Northern Ontario SMEs get established, grow and increase their competitiveness in key sectors, such as the mining and forest industries, tourism, agri-food, information and communications technology, renewable energy and manufacturing. Thesis? Examples of eligible project activities include: business management capacity development, such as planning, training, research, access to business management expertise and export readiness; access to on nelson new markets outside Northern Ontario with emphasis on thesis cover, foreign markets, such as trade and export development advisory services, supply chain integration, trade missions, business-to-business matchmaking, market research and analysis, promotional or marketing campaigns; investment attraction, including developing and implementing investment attraction strategies and marketing strategies; industry collaboration and support for SMEs, such as business incubators, industry networks, and regional and mandela leadership, sectoral initiatives; development, promotion and master thesis cover, expansion of business associations, sector alliances or networks to facilitate industry growth through information sharing and collaboration; and youth internships to essays on nelson mandela assist with projects related to innovation, the digital economy and trade for a period of 12 months. Thesis? Other activities related to same business growth and competitiveness necessary to further an economic goal in Northern Ontario may be considered on a case-by-case basis. Eligible costs include all incremental expenses directly related to the project and deemed reasonable and necessary for its execution. Non-capital costs may include: fees for professional and technical services, including market, trade and export expertise; costs related to business management training; costs related to producing and disseminating results of studies/plans; marketing costs, including design, promotional materials, advertising, product demonstrations and participation at trade shows; and travel expenses and labour costs. Capital costs may include: servicing of industrial lands; leasehold improvements; and machinery, equipment and thesis cover, technology. Ineligible Project Activities and Costs. Certain activities and costs will not be considered eligible, including: community profiles; retail, real estate, government services; and land and rolling stock.
Assistance available from FedNor includes repayable and non-repayable contributions. Contributions will normally be repayable where proposed projects generate profits for leadership the eligible applicant. All youth internships are non-repayable. Generally, FedNor will support up to 33 percent of eligible capital and up to 50 percent of eligible non-capital costs. Eligible applicants are expected to contribute a minimum of 10 percent of project costs. If applicants incur costs to implement their projects prior to approval of funding, these costs will not be considered eligible for reimbursement unless written consent is provided by FedNor. For youth internships with not-for-profit organizations, FedNor may contribute up to master $31,500 (90 percent) for employee salary and benefits. For youth internships with SMEs , FedNor may contribute up to educational leadership $31,500 (50 percent) for employee salary, benefits, and professional/business skills development and thesis, associated travel costs.
Project applications are reviewed and funding levels determined based on why is to vote essay, criteria, such as: net economic benefit to master Northern Ontario, especially in the short- to medium-term (up to five years); development, growth and increased competitiveness of SMEs ; creation and retention of jobs in Northern Ontario; competitive impacts on existing businesses; alignment with community economic priorities; need for assistance to make the project viable; requested assistance complements and analysis, does not displace other available government funding programs; availability of FedNor's NODP funds; opportunities to leverage investment from other funding sources; projects that are deemed to enhance the vitality and master thesis cover, support the development of Official Language Minority Communities and/or foster the full recognition and use of English and French in Canadian society. Where applicable, FedNor requires that: projects address the requirements of the Canadian Environmental Assessment Act and same sex marriage, reflect the spirit and intent of the Official Languages Act ; applicants use a fair, competitive and transparent procurement process to ensure the best value for money when supplies, equipment and thesis cover, services are purchased or personnel hired; and an appropriate authority or regulatory body endorses the project. FedNor is committed to providing quality service to all applicants and funding recipients in the official language of their choice. acknowledge receipt of an application within three (3) working days; and, provide a decision on applications within eighty (80) working days of receipt of it important a fully completed application. Please call 1-877-333-6673 to discuss your proposal with a FedNor Officer in your area or begin your application process.
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Lactoferrin receptor mediates apo- but not holo-lactoferrin internalization via clathrin-mediated endocytosis in trophoblasts. LfR [Lf (lactoferrin) receptor] is master cover expressed in most mammalian tissues, including placental trophoblasts, and is presumed to mediate the internalization of should be paid essay, Lf. However, the physiological significance of trophoblast LfR is not understood. Master Cover! 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 civilization essay, the presence (holo-Lf) or absence (apo-Lf) of iron within the 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 master cover, a LfR of ?105 kDa. Jobs Adobe! 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 master thesis cover hypertonic medium (0.4 M sucrose) significantly inhibited apo-Lf internalization. In summary, our data demonstrate that apo- but not holo-Lf is internalized by 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 leadership papers, 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 thesis, human small intestine . It presumably facilitates the uptake of its protein ligand Lf, which is present in it important, milk and mucosal secretions .
Therefore, kinetic studies on cover, LfR have primarily been conducted in small intestine [3–6] and antiquity essay irreverant western cultured enterocytes . We have previously cloned the LfR gene (GenBank® accession number NM_017625)  and determined that the master thesis, intestinal LfR is an ?110 kDa trimeric (?37 kDa subunits), glycosylated protein . Same Sex Marriage! However, LfR is cover also expressed in non-intestinal tissues, such as heart, liver, spleen, thymus and skeletal muscle [3,8,9]. Recently, we detected LfR expression in mouse placenta associated with trophoblast cells , suggesting that LfR plays a role in this highly specialized cell type. Trophoblasts are divided into civilization, two subtypes; CTs (cytotrophoblasts) and STs (syncytiotrophoblasts), which are both maintained throughout pregnancy. Master! The primary function of CTs is invasion into antiquity civilization essay irreverant reconstructing western, the uterine epithelium to secure successful implantation , whereas STs further implantation progression by establishing the nutrient and gas exchange barrier (maternal–fetal interface) and secreting hormones such as progesterone which are essential for successful pregnancy maintenance . Master Thesis Cover! The invasive capacity of CTs relies on the secretion of MMPs (matrix metalloproteinases), which function to essays mandela leadership, degrade the master thesis, surrounding ECM (extracellular matrix). College To Play Persuasive! Several disorders of pregnancy, including pre-eclampsia and spontaneous abortion, are believed to relate to ineffective CT proliferation, migration and thesis invasion into the uterine endothelium . Lf has been attributed with multifunctional properties, including increased cell proliferation [12–16] and changes in cell morphology through activation of MMP transcription .
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 on nelson leadership apo-Lf (iron free Lf) are known to adopt “closed” and cover “open” forms respectively [18,20]. For example, holo-Lf increases but apo-Lf decreases cell proliferation in mandela, human enterocytes (Caco-2 cells)  and while apo-Lf stimulates microvessel proliferation in rats, holo-Lf is ineffective . Currently, the mechanisms through which apo- and holo-Lf exert their differential functions are unknown. Herein, we hypothesized that the opposing functionality of master, apo- and holo-Lf reflects differences in cellular internalization mechanisms mediated via LfR. Educational Leadership Research Papers! In order to address this, we utilized the human placental CT cell line BeWo and determined: (1) the effects of apo- and holo-Lf on MMP-2 activity, the major MMP in trophoblasts; (2) the ability of the LfR to facilitate apo- and holo-Lf uptake and internalization; and (3) the subsequent intracellular compartmentalization of LfR following Lf uptake. We also determined that apo- but not holo-Lf stimulates MMP-2 activity, that apo-Lf is master 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 same sex marriage analysis intracellular trafficking mechanisms and master thesis provides a functional role for LfR in it important to vote essay, 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 cover cultured until confluent. Lf was isolated from human milk as previously described with slight modifications . 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 centrifuged at 8600 g for leadership, 20 min at 4 °C. The whey fraction was collected, centrifuged at 8600 g for master, 20 min at 4 °C and then mixed with heparin–Sepharose Fast Flow resin (GE Health-Amersham Biosciences) overnight at 4 °C. Same! The whey/resin mixture was centrifuged at 800 g for cover, 5 min at 4 °C, the educational leadership research, 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). 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. Master Thesis! Lf-containing fractions were identified following Coomassie Brilliant Blue staining of SDS/PAGE (12%) gels. Samples were pooled and should be paid persuasive essay concentrated in YM-50 centrifugation tubes (Millipore Centriplus, Fisher Scientific) at 3000 g for 90 min at 4 °C. The protein concentration of purified Lf was determined spectrophotometrically ( A 280 ) as previously established . Apo-Lf was prepared as previously described . 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 the A 460 / 280 ratio (22) .
To determine effects of apo- and master cover holo-Lf on on nelson, MMP-2 activity, cells were seeded in 24-well plates in cover, 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 educational leadership research papers, 4 h. Master Thesis Cover! Conditioned medium was collected after 24 h 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. Protein concentration of conditioned media was determined using the analysis, Bradford assay (Bio-Rad) and master proteins (20 ?g) were electrophoresed by SDS/PAGE (7.5% gel containing 1 mg/ml gelatin). Following electrophoresis, the gels were washed twice in essays mandela leadership, 2.5% Triton X-100 in thesis, PBS for 15 min to be paid to play persuasive, remove SDS and incubated in wash buffer 2 (50 mM Tris/HCl, pH 7.6, 10 mM NaCl, 50 mM CaCl 2 ) overnight at thesis cover, 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 same sex marriage analysis holo-Lf were labelled with 125 I (GE Health-Amersham Biosciences) by the Iodogen method . 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 growth medium until confluent. Cells were incubated with 125 I-labelled apo- or holo-Lf (10000–100000 c.p.m.) in SFM (serum-free medium) for cover, 1 h at leadership research, 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 master thesis cover solubilized cells in a gamma counter. Affinity purification and validation of essay irreverant reconstructing western, 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 2 h at room temperature (25 °C) prior to immunoblotting of master thesis cover, 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 educational, 4 °C and the post-nuclear supernatant was collected. Protein concentration of the supernatant was determined by the Bradford method and cover cell lysates (50–100 ?g protein) were resolved by electrophoresis. Educational Research! To determine Lf binding, cell proteins were resolved by non-reducing SDS/PAGE at 80 V for 4 h and transferred on cover, to nitrocellulose membrane. Membranes were incubated with 5% BSA in PBS-T for 1 h at room temperature (25 °C) to 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 civilization essay reconstructing western, 1 h) under reducing conditions and membranes were incubated with LfR antibody for 1 h. Thesis Cover! Membranes were washed in PBS-T and primary antibody was detected with donkey anti-rabbit IgG-HRP (IgG-horseradish peroxidase; GE Health-Amersham Biosciences) in 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. Antiquity Essay Reconstructing! Relative band density was quantified using the 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 master thesis, LfR siRNA (Ambion) in Lipofectamine™ 2000 (Invitrogen) for 24 h according to the manufacturer's instructions. Cells treated with Lipofectamine™ 2000 alone were used as mock-transfected controls. Antiquity Essay Irreverant! After 24 h, cells were incubated with 125 I-labelled apo- or holo-Lf in SFM at 37 °C. Cover! Lf uptake was measured after 30 min as described above. To verify LfR suppression, transfected cells were scraped into research papers, 25 ?l of cover, 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 it important essay, to 100-mm-diameter dishes and cultured until confluent. Cells were rinsed with PBS and thesis cover the cell surface was biotinylated with sulfo- N -hydroxysuccinimido-biotin (0.5 mg/ml; EZ-Link Sulfo-NHSS-Biotin; Pierce) at should be paid essay, 4 °C for thesis, 1 h. Leadership! Cells were rinsed with ice-cold 0.2 M glycine buffer, pH 5.0, and master cover 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. The supernatant was discarded and the pellet was resuspended in civilization reconstructing, lysis buffer with 0.2% SDS and incubated at 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. Master! The supernatant was collected and should college incubated with 300 ?l of slurry (1:1) of immobilized avidin beads (UltraLink Immunobilized Monomeric Avidin; Pierce) for 1 h at room temperature (25 °C) with constant rotation. To collect proteins bound to the resin, the samples were centrifuged at 2300 g for 1 min. The supernatant was discarded and the beads were rinsed with PBS and centrifuged for 1 min at 2300 g . To elute biotinylated proteins, an master, 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 reconstructing, SDS/PAGE and immunoblotted for 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 to 24-well plates and pretreated with Pi-PLC (1 unit/ml; Invitrogen) in SFM for 1 h at 37 °C to cleave GPI-anchored proteins. To verify the master cover, effects of essay irreverant reconstructing, Pi-PLC on thesis, LfR abundance at the plasma membrane, cell surface proteins were biotinylated and immunoblotted for LfR as described above. To determine the effects of Pi-PLC on Lf uptake, cells were rinsed with PBS, fresh SFM was added and cells were incubated with 125 I-labelled apo-Lf at 37 °C and uptake was measured after 1 h as described above. Civilization Irreverant Western! 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 thesis cover, 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). College Athletes Persuasive Essay! Cells were pre-treated in hypertonic medium for 30 min and incubated with 125 I-labelled apo-Lf in SFM at 37 °C in the presence of master thesis cover, 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 on nelson leadership, Lf uptake, cells were seeded on to 12- or 24-well plates until confluent and pretreated with cycloheximide (Cyhex, 10 ?g/ml, Sigma) in thesis, SFM for 6 h at 37 °C. Cells were either collected for why is it important essay, SDS/PAGE analysis or incubated with 125 I-labelled apo-Lf in the presence of Cyhex at 37 °C and master thesis Lf uptake was measured over leadership research, 7 h. To determine the subcellular localization of LfR, cells were seeded on to glass coverslips, cultured for cover, 3–4 days in growth medium and incubated with apo-Lf (where indicated, 200 ?g/ml) in SFM for up to antiquity essay irreverant western, 1 h at 37 °C. After treatment, cells were rinsed with PBS and master fixed with 4% phosphate-buffered paraformaldehyde for 10 min and permeabilized with 0.2% Triton X-100 in PBS for 5 min. Why Is To Vote Essay! To eliminate non-specific binding, cells were incubated in master thesis, blocking buffer (5% goat serum in PBS) for should college athletes persuasive, 30 min followed by incubation with affinity-purified LfR antibody (0.8 ?g/ml) in PBS containing 5% goat serum, 1% BSA and 0.2% Triton X-100 for 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 20 min. Master Thesis! 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 college athletes to play persuasive, late endosomes; 1:500, Abcam) and master cover 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. Same Sex Marriage Analysis Essay! Immunofluorescence imaging was performed using an Olympus BX50WI microscope with UPlanApo ?100 magnification under an thesis, oil-immersion lens [NA (numerical aperture), 1.00], and essay adobe 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 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 cover, P 0.05.
Active form of it important essay, MMP-2 is increased by apo- but not holo-Lf. Secretion of cover, MMP-2 plays a key role in CT invasion, thus we determined effects of Lf and Lf conformation on antiquity reconstructing western, MMP-2 activity in BeWo cells. Using gelatin zymography, our data demonstrated that apo-Lf significantly increased (20%) MMP-2 activity compared to untreated cells (Figure 1). Master! In contrast, we observed no effect of holo-Lf on MMP-2 activity, suggesting a differential effect of Lf conformation on cellular invasivity of CTs. Representative zymogram (upper panel) of culture medium collected from essay, cells treated with either apo- or holo-Lf. Equal amounts of protein (20 ?g) were electrophoresed through polyacrylamide gels containing gelatin and master thesis cover stained with Coomassie blue. Should College Athletes! MMP-2 activity was detected as clear bands (digested gelatin) on a blue background.
MMP-2 activity was significantly increased in cells treated with apo-Lf as compared with untreated (control) and cover holo-Lf treated cells (lower panel). Data shown represent MMP-2 activity as a percentage of jobs, untreated control cells (mean±S.D.) for two independent experiments ( n =4). * P 0.05. LfR facilitates uptake of 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. Master Thesis Cover! 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 essay, ?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 thesis, two LfR proteins at ?75 and ?105 kDa (Figure 3), but not to the 35 kDa protein. 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 dependent on its cellular localization and we showed that the 105 kDa LfR was localized to to vote, the plasma membrane using cell surface biotinylation followed by immunoblotting (Figure 4A). In contrast to what was reported for master cover, the intestinal LfR , trophoblast LfR does not appear to be GPI-anchored as demonstrated by college athletes to play, internalization of master thesis cover, 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 same sex marriage essay, spent medium compared to untreated cells (Figure 4C). Confluent cells were treated with 125 I-labelled apo- or 125 I-labelled holo-Lf and Lf uptake was determined. Representative graph of uptake after 1 h illustrates that significantly less holo-Lf was internalized when compared to apo-Lf. Master Thesis! Data shown represent mean Lf uptake (Lf uptake/mg protein)±S.D. from essay, two independent experiments ( n =4). Master Cover! * P 0.001. Representative immunoblot ( A and B ) and ligand blot ( C ) of cell lysate proteins. Proteins (50 ?g) were resolved by SDS/PAGE and immunoblotted with LfR antibody ( A ; LfR Ab, 1 ?g/ml) or pre-absorbed with excess Lf antigen ( B ; LfR Ab+peptide) for 1 h. The LfR antibody detected specific proteins at ?105 and ?35 kDa ( A ) which were both completely eliminated following pre-absorption with LfR peptide ( B ). Essay! ( C ) Cell lysate (100 ?g) was resolved under non-reducing and non-denaturing conditions, and cover incubated with 125 I-labelled apo-Lf. Detection of essay adobe, 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. Thesis Cover! ( A ) A representative LfR immunoblot of biotinylated cell surface proteins documents the presence of leadership papers, LfR on thesis cover, the plasma membrane after Pi-PLC treatment. Should Athletes To Play Persuasive! ( B ) Cleavage of master thesis cover, 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 college athletes be paid to play, total spent medium following Pi-PLC treatment, validating enzyme activity. In order to determine a role for LfR in Lf uptake, LfR expression was attenuated in master thesis cover, cells transfected with LfR siRNA (Figure 5A). We demonstrated that reduced LfR abundance resulted in essay adobe, 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 master thesis, holo-Lf on same, MMP-2 activity, we subsequently focused on master thesis, effects of educational research, apo-Lf on the intracellular trafficking of LfR. ( A ) Representative LfR immunoblot of cover, total protein illustrating significant attenuation of LfR compared to mock-transfected cells. ( 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. Data represent the means±S.D. Analysis Essay! ( n =2–3) of triplicate experiments. * P 0.05, ** P 0.01, as analysed by master, Student's t test. LfR internalization is essays 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 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 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 master thesis cover, 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 plasma membrane proteins have been characterized. To identify the mechanism responsible for LfR internalization, CTs were treated with hypertonic medium (0.4 M sucrose) to inhibit clathrin-dependent endocytosis, which resulted in a robust inhibition of apo-Lf internalization (40%) compared with untreated cells, with values of 2.4±0.25 and 5.7±0.42 pmol/mg protein±S.D. for treated and control cells respectively; n=5–8 per group, * P 0.001. To confirm the mechanism by which LfR is endocytosed, we used confocal microscopy to identify the subcellular compartments within which LfR resides. As indicated in Figures 7(A) and 7(B), LfR and clathrin co-localized and should athletes persuasive were largely associated with the 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 thesis, LfR and the early endosome marker EEA1 in vesicular/endosomal-like structures (Figures 7D–7F). Leadership Research Papers! As co-localization between EEA1 and LfR was not exclusive, we sought to examine other sub-cellular compartments containing LfR. Clathrin-mediated endocytosis is cover one mechanism cells utilize to control cell surface receptor abundance and educational leadership papers turnover via recycling. To investigate whether LfR trafficking from the cover, plasma membrane potentially includes endocytic recycling, we used the indirect immunofluorescence of TfR as a marker for recycling endosomes. We showed partial co-localization of LfR and essay adobe TfR within distinct internalized vesicles in cells following ligand stimulation (Figures 7G–7I).
No co-localization between LfR and M6PR (a late endosome marker) and minimal co-localization with LAMP1 (a lysosome marker) was detected (results not shown). These results indicate that, in thesis cover, response to apo-Lf stimulation, LfR is internalized via clathrin-mediated endocytosis and 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 C ), EEA1 ( D and F ) and TfR ( G and I ). Jobs! 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 early endosomal marker EEA1 ( D ) and LfR ( E ) was detected (yellow, F ) and treatment of cells with apo-Lf clearly resulted in vesicularization of LfR.
Furthermore, co-localization with the cover, recycling endosome marker TfR ( G ) and LfR ( H ) was detected (yellow, I ) following treatment of civilization irreverant reconstructing, cells with apo-Lf. Finally, to determine if increased abundance of LfR at the plasma membrane in master thesis cover, response to apo-Lf stimulation reflects changes in intracellular LfR recycling, we blocked de novo protein synthesis with Cyhex and measured apo-Lf uptake. College Athletes Persuasive! Inhibition of new receptor synthesis did not diminish apo-Lf uptake, indicating that LfR is recycled. In fact, apo-Lf uptake was significantly higher in master, 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 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. 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 why is essay, the uterine endothelium, assuring optimal fetal development . Our previous detection of master thesis, LfR along the jobs adobe, maternal and master thesis cover fetal interface in the placenta implicates LfR in CT function [8,9,27].
The aim of the present study was to explore the sex marriage analysis essay, function of master, Lf in essays on nelson, the placenta as mediated via LfRs using a CT cell model. While primarily regarded as a component of milk , Lf is thesis also found in decidua (9–95 ?g/g), amniotic fluid (2–37 ?g/g) and chorion (2–26 ?g/g) , and is secreted directly from uterine epithelia [29,30]. This suggests that Lf secreted from the uterine endothelium may regulate CT function in antiquity reconstructing, a paracrine manner. Results from master cover, this study documented an enhancing effect of apo- but not holo-Lf on civilization essay western, MMP-2 activity in CTs, suggesting a regulatory role for apo-Lf in CT invasivity as this process is dependent upon the secretion and activation of MMPs. There is precedence for effects of Lf on MMP regulation as over-expression of master cover, Lf activates MMP-1 expression through binding to AP-1 promoter elements and through stimulation of the p38 MAPK (mitogen activated protein kinase)-dependant pathway in mouse fibroblasts and human COS-1 cells . Why Is It Important To Vote! We speculate that apo-Lf may similarly regulate the master cover, transcription of MMP-2 via AP-1 promoter elements which are present in the MMP-2 gene . 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 , and Lf binds to DNA [33,34]. Alternatively, the induction of it important to vote, 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 . The ability of Lf to 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 cover, apo- but not holo-Lf on MMP-2 activity probably reflects that distinct functions are associated with either apo- or holo-Lf. In contrast to sex marriage, reports in master, rat hepatocytes , CTs bound and internalized both apo- and why is it important to vote essay holo-Lf. Master Thesis Cover! However, in why is to vote essay, 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 thesis, CT-specific functions such as invasivity, while precise Lf-dependant functions in hepatocytes remain unknown. Gene attenuation of why is, LfR conclusively determined that LfR primarily facilitates apo-Lf uptake in master cover, CTs. Recently, distinct Arg-Gly-Asp (RGD) binding motifs were found within the Lf molecule , which have been postulated to educational leadership papers, determine ligand binding and receptor interactions.
It is possible that iron-saturation, which alters Lf conformation, may result in 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 master cover, CTs. Possibly, holo-Lf internalization is not a prerequisite to stimulate cellular events at jobs adobe, the plasma membrane as suggested by other investigators [15,35]. As apo-Lf was preferentially taken up by LfR, we sought to characterize the placental LfR more extensively. Master Thesis! It has previously been reported that intestinal LfR is sex marriage essay a GPI-anchored protein . Contrary to this observation, we determined that, in master cover, CTs, the LfR receptor is 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  significantly reduced Lf internalization.
We speculate that differences in Lf uptake between intestinal and 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. 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 . Leadership Papers! To identify potential AP2-interacting motifs within the master cover, LfR sequence we used a proteomics server (www.elm.eu.org) and indeed two tyrosine-sorting motifs were identified at amino acids 120–123 and 129–142. This further suggests that a portion of LfR protein does reside intracellularly and that one or more of these motifs may functionally interact with AP2 to civilization reconstructing, facilitate LfR internalization. LfRs have been identified in various cell-types and thesis reports have determined a molecular mass of ?110 kDa . Recently, Tsuji et al.  examined the structure of essay, human LfR in greater detail and master thesis cover determined that it is an 105.5 kDa oligomeric protein made up of on nelson, three 35.5 kDa subunits, similar to what we observed in trophoblasts. Interestingly, while we detected both the monomeric (35 kDa) and trimeric (105 kDa) forms of master, 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 trimeric form to educational research, monomers even when using dithiothreitol and iodoacetic acid (V. Cover! Lopez, unpublished observations). Interestingly, we did not detect the civilization essay irreverant western, dimeric form of thesis cover, LfR under reducing conditions, which is jobs essay adobe similar to the findings of Tsuji et al. . We further demonstrated that apo-Lf binds to master thesis, 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 subunit oligomerization . We speculate that the trimeric form is an unusually stabilized form, mediated by several intracellular disulfide bonds and sex marriage essay possibly also hydrophobic interactions, whereas the master, dimeric form is less stable and can be reduced to on nelson mandela leadership, the monomeric form. In contrast, a 45 kDa calcium-dependent LfR has been reported in thesis cover, rat hepatocytes ; 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 . As LfR abundance at the plasma membrane did not require de novo synthesis, our data suggest that recycling of LfR to essays leadership, and from the cover, plasma membrane occurs. Endocytic recycling requires internalization of cell surface receptors and their ligands within clathrin coated pits , with the interaction between transferrin and adobe TfR being the classic example of this process . Receptor recycling is one strategy by which cells maintain a mobilizable receptor pool to quickly traffic to the cell surface in response to physiological stimuli, to allow cells to master thesis, redistribute cell-surface molecules to specific cell locations or compartments undergoing a physiological activity .
We speculate that Lf may either stimulate the recruitment of LfR to the plasma membrane, or that perhaps Lf binding to college athletes be paid to play persuasive, the receptor results in transient accumulation of LfR prior to thesis cover, 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 address this issue. Collectively, the results from this study demonstrated a specific role for apo-Lf but not holo-Lf in essay irreverant reconstructing western, regulating MMP-2 activity and thus potentially endothelial invasion in CTs. Master Thesis Cover! Importantly, we have determined that apo- but not holo-Lf is antiquity civilization essay reconstructing western internalized via LfR, which occurs through a clathrin-dependent pathway in CTs suggesting a mechanistic and functional difference between apo- and master thesis cover holo-Lf. These results may help to essay, explain the pleuripotent and cell-specific functions which have been associated with Lf.
The authors would like to express their gratitude to Dr. Lisa L. Gough for cover, critical reading of the manuscript. This work was supported by educational leadership research papers, 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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Transcriptomic analysis of the autophagy machinery in crustaceans. The giant freshwater prawn, Macrobrachium rosenbergii , is a decapod crustacean that is thesis, commercially important as a food source. Farming of why is it important essay, commercial crustaceans requires an efficient management strategy because the animals are easily subjected to stress and diseases during the thesis culture. It Important To Vote Essay! Autophagy, a stress response process, is well-documented and conserved in most animals, yet it is poorly studied in crustaceans. In this study, we have performed an in silico search for transcripts encoding autophagy-related (Atg) proteins within various tissue transcriptomes of M. Master Cover! rosenbergii . Basic Local Alignment Search Tool (BLAST) search using previously known Atg proteins as queries revealed 41 transcripts encoding homologous M. rosenbergii Atg proteins. Among these Atg proteins, we selected commonly used autophagy markers, including Beclin 1, vacuolar protein sorting (Vps) 34, microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B), p62/sequestosome 1 (SQSTM1), and essay adobe, lysosomal-associated membrane protein 1 (Lamp-1) for further sequence analyses using comparative alignment and protein structural prediction. We found that crustacean autophagy marker proteins contain conserved motifs typical of other animal Atg proteins. Western blotting using commercial antibodies raised against human Atg marker proteins indicated their presence in various M. rosenbergii tissues, while immunohistochemistry localized Atg marker proteins within ovarian tissue, specifically late stage oocytes. This study demonstrates that the molecular components of autophagic process are conserved in cover, crustaceans, which is comparable to essays on nelson mandela, autophagic process in mammals. Furthermore, it provides a foundation for further studies of thesis cover, autophagy in crustaceans that may lead to same analysis essay, more understanding of the thesis reproduction- and civilization essay irreverant, stress-related autophagy, which will enable the efficient aquaculture practices.
Electronic supplementary material. The online version of this article (doi: 10.1186/s12864-016-2996-4 ) contains supplementary material, which is available to authorized users. Macroautophagy (autophagy) is a complex process involving degradation of cellular constituents to thesis cover, maintain organelle and protein homeostasis in response to various stresses, including genomic, oxidative, hypoxic, proteotoxic and metabolic stresses as well as starvation [ 1 ]. Autophagy plays a major role in the removal of mutated or oxidized protein aggregates and of damaged organelles. It is also involved in restoring nutritional and why is it important to vote essay, metabolic imbalances during starvation. Deficiency in the clearing and restoring function of autophagy leads to master, cell death, which ultimately contributes to growth retardation and various degenerative diseases, including neurodegenerative, muscular and why is it important essay, cardiovascular diseases [ 2 , 3 , 4 , 5 ].
Autophagy is an evolutionary conserved mechanism ubiquitously present in all eukaryotic cells. It is driven and regulated by several proteins designated as autophagy-related (Atg) proteins (ATG in thesis cover, mammals). Why Is It Important To Vote! At least 32 Atg genes have so far been identified in master, the yeast, Saccharomyces cerevisiae , the species initially employed for genetic screening of autophagy [ 6 ]. Many Atg orthologs have subsequently been identified in higher eukaryotes, including nematodes ( Caenorhabditis elegans ) [ 7 ], arthropods ( Drosophila ) [ 8 ], mammals (mouse and same sex marriage analysis essay, human) [ 9 ], as well as in plants [ 10 ]. Furthermore, these Atg genes seem to be highly conserved among different metazoan phyla. Master Thesis! Amongst these, phylum Arthropoda has the educational largest number of species and master thesis, their members are widespread throughout both terrestrial and aquatic habitats. While autophagy in insects has been studied intensively [ 8 , 11 , 12 , 13 , 14 , 15 ], the autophagic machinery in crustaceans is still unexplored. However, a few studies have shown the presence of leadership, autophagosome within the master thesis cells of the it important essay midgut in crustacean species by using electron microscopy and it has been suggested that autophagy might play an important role for master cover midgut cell survival [ 16 , 17 , 18 , 19 ]. Many species of crustaceans, especially decapods (shrimps and crabs), are a commercially important food source. During captivity in jobs adobe, farming, these animals can be subjected to many stresses that affect feeding, growth, reproduction, and diseases.
Hence, management of stress, disease, and reproductive manipulation are crucial in farming. Since these processes are related to autophagy [ 20 , 21 , 22 ], an understanding of the autophagic process in master thesis, crustaceans is should athletes to play persuasive, important for master cover improvement of crustacean aquaculture. To date, the genome of the water flea, Daphnia pulex , had been generated [ 23 ]. Jobs Adobe! Further, the transcriptomes of several crustaceans, including the mud crab ( Scylla olivacea ) [ 24 ] and white shrimp ( Litopenaeus vannamei ) [ 25 ], have been provided in the online databases. Thesis Cover! Recently, we have generated transcriptomes from the eyestalk, central nervous system (CNS) and ovaries of M. rosenbergii , leading to the identification of several genes encoding neuropeptides [ 26 , 27 ] and steroidogenesis-related proteins [ 28 ]. In this study, we identified Atg genes in mandela, the above-mentioned M. rosenbergii transcriptomes and then examined the presence of key Atg proteins in the tissues using Western blotting and cover, immunohistochemistry. Furthermore, structural comparisons between human and M. rosenbergii marker proteins for autophagic process, including Beclin 1 (BECN1), vacuolar protein sorting (Vps) 34, microtubule-associated proteins 1A/1B light chain 3 (MAP1LC3 or LC3), p62/sequestosome 1 (SQSTM1) and essays mandela leadership, lysosomal-associated membrane protein 1 (Lamp-1), were performed to show that crustacean Atg marker proteins have a similar structure, and master cover, possibly functional activity, to antiquity civilization reconstructing, those of human. Thesis Cover! In summary, our findings provide a substantial evidence for jobs essay the presence of autophagy machinery in master thesis cover, a crustacean species. Gene mining of autophagy-related proteins in M. Antiquity Civilization Irreverant Reconstructing! rosenbergii transcriptomes.
Atg transcripts present in M. rosenbergii transcriptomes in comparison with Atg genes of mammals and yeast. Autophagy core machinary. Number of thesis cover, hit transcript in M. rosenbergii transcriptomes. ULK1 protein kinase complex. 1 (same hit with ULK2) WIPI-1,2,3,4 Vps34-beclin1 class III PI3-kinase complex.
1 (same hit with GABARAP) ATG4A-D (autophagins 1–4) beclin 1 associated autophagy related key regulator like. The number of predicted Atg transcripts derived from M. rosenbergii transcriptomes is indicated. Sequence analyses of Atg marker proteins in M. rosenbergii. Identification of an M. rosenbergii Atg6. a A schematic annotation of same sex marriage, M. rosenbergii Atg6 (MroAtg6) and the alignment of thesis, crustacean Atg6 proteins with Atg6/BECN1 of essays mandela leadership, other species from cover different phyla. Black shading indicates conserved amino acids while grey shading indicates similar amino acids. Green bar indicates the jobs essay region of Bcl-2 homology 3 (BH3) domain of HsaBECN1. Mro, Macrobrachium rosenbergii ; Ple, Pontastacus leptodactylus ; Cfi, Calanus finmarchicus ; Dpu, Daphnia pulex ; Dme, Drosophila melanogaster ; Cgi, Crassostrea gigas ; Spu, Strongylocentrotus purpuratus ; Cel, Caenorhabditis elegans ; Dre, Danio rerio ; Xtr, Xenopus tropicalis ; Mmu, Mus musculus ; Hsa, Homo sapiens . b Predicted tertiary structures of HsaBECN1 ( a ) and MroAtg6 ( b ) and superimposition of master, HsaBECN1 and MroAtg6 ( c ). The BH3 domain is labeled on HsaBECN1, corresponding to amino acid positions 105–129. 65–76 % similarity to the human ortholog (HsaVps34).
Conserved functional domains, including C2 (positions 27–183; Pfam accession number: PF00792), phosphatidylinositol 3-kinase (PI3K) accessory (PI3Ka; positions 280–539; Pfam accession number: PF00613), and PI3K catalytic (PI3Kc; positions 576–924; Pfam accession number: PF00454) domains could be annotated in MroVps34 (Fig. 2a ). A high degree of amino acid conservation was found throughout the protein length, especially within C2 and PI3Kc domains (Fig. 2a ). Structural superimposition of HsaVps34 and MroVps34 shows similarity in folding between these two proteins (Fig. 2b ). Identification of an M. rosenbergii Vps34. On Nelson Mandela Leadership! a A schematic annotation of M. rosenbergii Vps34 (MroVps34) and the alignment of Vps34 in crustaceans and other species from different phyla (only the alignments of amino acids within C2 and phosphatidylinositol 3-kinase catalytic (PI3Kc) domains are shown). Black shading indicates conserved amino acids while grey shading indicates similar amino acids. Mro, Macrobrachium rosenbergii ; Ple, Pontastacus leptodactylus ; Pmo, Penaeus monodon ; Dpu, Daphnia pulex ; Dme, Drosophila melanogaster ; Cel, Caenorhabditis elegans ; Dre, Danio rerio ; Xtr, Xenopus tropicalis ; Rno, Rattus norvegicus ; Hsa, Homo sapiens ; Sce, Saccharomyces cerevisiae . Master Thesis Cover! b Predicted tertiary structures of HsaVps34 ( a ) and MroVps34 ( b ) and superimposition of HsaVps34 and it important, MroVps34 ( c ). The C2 (amino acid positions 26–186) and master thesis, PI3Kc (amino acid positions 539–885) domains of HsaVps34 are labeled in red and sex marriage essay, green colors, respectively. 60 %, Fig. Master Cover! 3a ). The MroAtg8 consists of 122 amino acids, with 98 % identity to M. Essays On Nelson! nipponense Atg8. An Atg8 ubiquitin-like domain (Pfam accession number: PF02991) is present within MroAtg8 (amino acid positions 16–121, Fig.
3a ). The MroAtg8 and human MAP1LC3B (HsaMAP1LC3B) share 72 % similarity, and their structural superimposition indicates a similar secondary structure, including at thesis cover, the binding sites for Atg7 and tubulin, as shown in HsaMAP1LC3B (Fig. 3b ). The amino acid residues which are responsible for the interaction between Atg8/MAP1LC3B and other autophagy receptors [ 32 ], and the amino acid residues which are important for analysis the formation of two binding pockets are shown in Fig. 3a . Cover! We could observe that those important residues were also conserved in crustacean Atg8. Therefore, the basic residues important for electrostatic interaction of analysis essay, MroAtg8 with other proteins could be predicted, possibly including R 11 , R 12 , K 31 , K 50 , K 52 , and R 71 (Fig. Master Thesis! 3a ). Likewise, the residues required for the formation of two binding pockets in MroAtg8 could be predicted (D 20 , I 24 , P 33 , I 35 , K 52 , L 54 , and F 109 for leadership the formation of the first pocket and master cover, I 36 , F 53 , V 55 , P 56 , V 59 , L 64 , I 67 , I 68 , and R 71 for the formation of the second pocket) and also mapped in the predicted MroAtg8 tertiary structure (yellow and pink areas, respectively; Fig. 3c ), assembling the appearance of two adjacent pockets. Identification of an M. rosenbergii Atg8. a A schematic annotation of M. Jobs Adobe! rosenbergii Atg8 (MroAtg8) and thesis cover, the alignment of crustacean Atg8 proteins with Atg8/LC3 of other species from should essay different phyla. Cover! Black shading indicates conserved amino acids while grey shading indicates similar amino acids. Blue boxes indicate the basic residues which are previously known to be critical for electrostatic interaction of LC3 with other proteins. Leadership Research! Yellow and pink bars underneath the alignment indicate amino acids constituting two hydrophobic pockets of HsaLC3 (each color represents each pocket) [ 32 ]. Mro, Macrobrachium rosenbergii ; Mni, Macrobrachium nipponense ; Lva, Litopenaeus vannamei ; Sol, Scylla olivacea ; Esi, Eriocheir sinensis ; Dpu, Daphnia pulex ; Dme, Drosophila melanogaster ; Cel, Caenorhabditis elegans ; Dre, Danio rerio ; Xtr, Xenopus tropicalis ; Mmu, Mus musculus ; Hsa, Homo sapiens . b Predicted tertiary structures of master thesis, HsaLC3 ( a ) and leadership, MroAtg8 ( b ) and superimposition of HsaLC3 and MroAtg8 ( c ). Cover! The binding sites for essays on nelson mandela Atg7 (red; amino acid positions 38, 82, and 114) and tubulin (green; amino acid positions 8, 17, 21, 22, 24, 25) in HsaMAP1LC3B are indicated. c Predicted MroAtg8 tertiary structure with hydrophobic surface.
Residues which were predicted to form two hydrophobic pockets in MroAtg8 are colored in yellow and pink. Identification of an M. rosenbergii p62. a A schematic annotation of M. rosenbergii p62 (Mrop62) and the sequence alignment of crustacean p62 proteins with p62 of other species from different phyla. Black shading indicates conserved amino acids while grey shading indicates similar amino acids. Mro, Macrobrachium rosenbergii ; Ple, Pontastacus leptodactylus ; Dpu, Daphnia pulex ; Dre, Danio rerio ; Xtr, Xenopus tropicalis ; Mmu, Mus musculus ; Hsa, Homo sapiens ; Ame, Apis mellifera ; Spu, Strongylocentrotus purpuratus ; Gga, Gallus gallus ; Cfa, Canis familiaris . b Local alignment of LIR domain sequences of predicted Mrop62 and p62 of other species. c Predicted tertiary structure of Mrop62. Cover! The predicted Phox and Bem1p (PB1; amino acid positions 6–96), ubiquitin-associated (UBA; amino acid positions 588–634), and LC3-interacting region (LIR; amino acid positions 534–555) domains of to play essay, Mrop62 are colored in yellow, purple, and red, respectively. Identification of an thesis cover M. Civilization Essay Irreverant Reconstructing Western! rosenbergii Lamp-1. A schematic annotation of M. Master Thesis! rosenbergii Lamp-1 and the alignment of crustacean Lamp-1 proteins with Lamp-1 of other species from different phyla are presented. A signal peptide (blue), a mature peptide (green), a conserved transmembrane domain of the why is epidermal growth factor receptor (TM-EGFR) (yellow), and thesis, cysteine residues are indicated. Black shading indicates conserved amino acids while grey shading indicates similar amino acids. Mro, Macrobrachium rosenbergii ; Mni, Macrobrachium nipponense ; Ple, Pontastacus leptodactylus ; Dpu, Daphnia pulex ; Dme, Drosophila melanogaster ; Cgi, Crassostrea gigas ; Dre, Danio rerio ; Xle, Xenopus laevis ; Rno, Rattus norvegicus ; Hsa, Homo sapiens.
Spatial expression of college athletes to play, autophagy markers. Validation of the autophagy marker proteins in female M. rosenbergii tissues. Western blotting shows the expression of Atg6/BECN1, Atg8/LC3, and master thesis, p62/SQSTM1 in various tissues, including the hepatopancreas (Hp), ovary (Ov), muscle (Ms), brain (Br), eyestalk (Es), and thoracic ganglia (Tg). The expression of these marker proteins in human squamous carcinoma cells (A431 cell line) is civilization irreverant reconstructing western, used as a positive control. The amount of total protein loaded per lane was 30 ?g. Immunoperoxidase staining of Atg marker proteins in master thesis cover, the ovary of M. rosenbergii . Late vitellogenic oocyte (Oc4) with HE stain ( A ). Should College Athletes Essay! Immunolocalization of Atg6/BECN1 ( B ), Atg8/LC3 ( C ), p62/SQSTM1 ( D ), and Lamp-1 ( E ) in the Oc4 of ovarian sections. The insets ( b – e ) show images at a higher magnification where positive staining is visible as red spots (arrows). F Negative control. Thesis! Scale bars: 25 ?m ( A and F ) and 10 ?m ( B – E ). (N, nucleus; Yg, yolk granules; Li, lipid droplets; Mb, cell membrane)
Autophagy refers to a lysosome-driven process for the degradation of cellular components that are redundant, aged, or damaged [ 37 ]. Why Is It Important Essay! The control and execution of autophagy have been well characterized in the yeast S. cerevisiae [ 38 ] and the worm C. Thesis Cover! elegans [ 39 ], and are being investigated in human, mouse, the fruit fly ( Drosophila melanogaster ), and plants. Jobs Essay! Here we present the first study showing that the key genes and proteins involved in the regulation and execution of master thesis cover, autophagy are present in educational research papers, crustaceans. All Atg proteins investigated could be identified from M. Master! rosenbergii transcriptomes (see Table 1 ), confirming their widespread conservation amongst animal phyla. It is likely that the autophagic process in crustaceans follows the same pathway as in other animals, engaging a similar set of proteins from the on nelson mandela initial triggering step (the activation of the ULK1 protein kinase complex) until the formation of active autophagic vesicles (the autolysosomes). The same hit transcripts were observed for GABARAP and GABARAPL1, and ULK-1 and thesis cover, ?2 transcripts. It Important! In the master thesis cover case of GABARAP and essays on nelson leadership, GABARAPL1, it has been shown that there are at least three human GABARAPLs (1–3) genes which are homologues of the GABARAP gene [ 40 ]. Thesis Cover! Therefore, GABARAP and GABAAPL1 share a high sequence similarity and both, as well as other GABARAPLs, are categorized within the analysis essay GABARAP family [ 41 ]. Master Thesis! The result from our transcriptome analysis suggested that only a single transcript encoding MroGABARAP exists in the prawn, possibly because other homologous genes may have been lost during evolution. Atg6 (Vps30 in yeast) was initially isolated as an interactor of the anti-apoptotic protein Bcl-2, and antiquity civilization essay western, thereafter named Beclin 1 [ 42 ]. Atg6/BECN1 was next shown to be an essential activator of Vps34 for the initiation of the autophagic process [ 43 ]. Thesis Cover! High conservation of should to play, protein amino acid sequence, especially at the C-terminal region, could be observed in this protein among vertebrates and invertebrates, indicating that those conserved motifs are significant for the activity of thesis, this protein. Leadership Research! In particular, amino acid conservation within the BH3 domain region and structural superimposition of human BECN1 and M. rosenbergii Atg6 indicates that M. rosenbergii and other crustacean Atg6 may interact with Bcl-2, and thereby play a regulatory role in apoptosis.
Upon interaction with BECN1, the master thesis cover PI3K class III protein (PI3KC3 in human or Vps34 in yeast) catalyzes the formation of PI3P, which is essential for the recruitment of autophagosomal membranes [ 44 , 45 ]. Vps34 protein is the most evolutionarily conserved protein within the same PI3 kinase family [ 46 , 47 ]. We did consistently find that crustacean Vps34 showed high conservation in cover, their amino acid sequences with those of other invertebrates and vertebrates, especially at the domains described as critical for why is it important to vote essay function. In fact, the Vps34 protein contains three major domains, namely the C2, PI3Ka, and master thesis, PI3Kc domains [ 47 ], which were present in the deduced MroVps34. Vps34/PI3KC3 is known to same sex marriage analysis essay, interact with other autophagic proteins such as Atg14 and UV radiation resistance-associated gene (UVRAG) [ 48 , 49 ]. Since Atg14 and UVRAG proteins were found in M. rosenbergii transcriptomes (Table 1 ) and the predicted structural conformation of cover, M. rosenbergii Vps34 is educational leadership research papers, similar to human Vps34 (Fig. 2b ), we suggest that MroVps34 may exhibit a similar catalytic activity and master thesis cover, bind to Atg14 and essay, UVRAG. Atg8/MAP1LC3B is master cover, a protein that is inserted into the nascent autophagosomal membranes upon post-translational lipidation of the microtubule-associated precursor [ 50 ]. Both length and amino acid composition were found to be similar among representative Atg8/MAP1LC3 sequences from different animal species (Fig. Educational! 3 ). This suggested that this protein is highly conserved among crustaceans and, moreover, among various animals from different phyla. Consistent with this, the predicted structural conformation of M. Master Cover! rosenbergii Atg8 exhibits a similar secondary structure to that of human MAP1LC3B, including within the regions that are known to mandela leadership, be important for binding with Atg7 and tubulin proteins [ 51 , 52 , 53 , 54 ]. Besides, Atg8/MAP1LC3B normally interacts with other crucial autophagic receptors, for instance, Atg19, p62, and Atg32, by using two specific hydrophobic pockets, called W- and L-sites (since these two pockets interact with conserved tryptophan and leucine residues within the LIR domain [ 32 ]). We have shown that the amino acid residues known to master thesis cover, be important for MAP1LC3B interaction with other proteins are preserved in crustacean Atg8, suggesting that they are critical for it important essay Atg8/MAP1LC3B functioning not only in vertebrates but also in crustaceans. p62/SQSTM1 is a ubiquitin-binding protein that bridges the master thesis cover autophagy substrate to LC3 through its LIR domain [ 55 ]. Three distinct domains significant for p62 activity include a PB1 domain located at the N-terminus that is responsible for p62 self-assembly [ 56 , 57 ]; a UBA domain located at the C-terminus that is be paid essay, important for master thesis p62-ubiquitin interacting [ 33 , 58 ]; and sex marriage analysis, a LIR domain that provides an interaction site for binding with Atg8/MAP1LC3B [ 55 ]. We found that p62 in crustaceans are not well conserved with other vertebrate p62, both in their length and master cover, amino acid composition. Educational Leadership Papers! A large amino acid insertion could be observed within the cover middle region of the crustacean p62 when compared to vertebrate p62 sequences. In regard to the LIR motif, this region is why is essay, highly conserved among vertebrate species but less conservation could be observed in invertebrate p62 proteins (Fig.
4b ). However, we speculate that the important amino acids ‘WXXL’ (where X is any amino acid) within LIR, which binds directly to MAP1LC3B in mammals [ 32 ], are still preserved in invertebrate p62, including M. rosenbergii , with an cover exception for the leucine residue which is substituted by other aliphatic amino acids such as valine or isoleucine. Therefore, it is possible that invertebrate p62 may still have binding activity to MAP1LC3B similar to vertebrate p62. Lamp-1 is an integral membrane protein of the essays mandela endosomes and lysosomes, which is involved in the fusion of thesis cover, these organelles with autophagosomal vacuoles [ 31 ]. Despite low similarity in amino acid composition, we found that the same sex marriage essay MroLamp-1 preserves basic characteristics of Lamp-1 [ 59 ], including a conserved luminal Lamp domain (namely, the C-terminal TM-EGFR domain that extends to a short cytoplasmic tail). While other vertebrate Lamp-1 contains eight cysteine residues within the luminal region, hence forming four disulfide bonds [ 59 ], MroLamp-1 luminal domain contains only master thesis cover four cysteine residues potentially forming two disulfide bonds, and adobe, therefore likely not showing the looped-luminal domain with free-moving hinge as present in vertebrate Lamp-1 [ 59 ]. Considering a cytoplasmic tail of Lamp-1, this region contains the tyrosine-based motif (Y-X-X-I, where X is a polar residue) important for master targeting Lamp-1 to lysosomes [ 60 , 61 , 62 ]. Civilization Irreverant Reconstructing! We found that all predicted crustacean Lamp-1 display that significant motif, though their last residues were substituted by valine. Thesis! Although the intracellular targeting of Lamp-1 was conserved among crustaceans and other animals, a variable amino acid sequence within the leadership research N-terminal part and distinct Lamp domain characteristics suggests a different functional activity of thesis cover, crustacean Lamp-1 from those of vertebrates. Based on sequence alignment, we found that the sequences of educational leadership research, M. rosenbergii and human proteins at thesis, the regions used for producing anti-BECN1, anti-LC3, anti-p62 and anti-Lamp-1 antibodies shared 58.82, 42.86, 41.18 and research papers, 23.37 % identity, respectively, suggesting that antibodies against the human autophagy proteins could be used to detect autophagic proteins in M. rosenbergii . We therefore employed immunoblotting and immunocytochemistry techniques to validate the expression and subcellular localization of Atg marker proteins in M. rosenbergii tissues. Autophagy has been shown to thesis cover, be present in why is it important to vote essay, the epithelial cells of the hepatopancreas and/or intestine in different crustacean species, including Astacus astacus , Eubranchipus grubii and Neocaridina heteropoda , in which autophagic activity was suggested to be essential for digestive cell survival (by eliminating degenerated organelles such as mitochondria) and a proper function of the digestive organs [ 16 , 18 , 19 ]. Thesis! In our study, we found expression of Atg6/BECN1, Atg8/LC3, p62/SQSTM1 proteins to be widespread in various tissues of jobs essay, M. rosenbergii that were investigated (Fig. Master Cover! 6 ). Regarding Atg8/LC3 expression, there were three immunoreactive bands detected in why is it important, prawn tissues. The largest (18 kDa) and smallest (16 kDa) bands may be considered as the thesis cover LC3-I and-II forms, as compared with the LC3 expression in the human cell sample (Fig.
6 ). The middle band, which is present at an apparent molecular weight of 17.5 kDa in some tissue samples, probably represents the intermediate LC3-I that precedes the lapidated form of 16 kDa LC3-II [ 63 ]. Therefore, we suggest that the jobs essay autophagic process is general to various prawn tissues, although the specific function of this process within different tissues still requires further investigation. In the ovary, we could observe that the levels of thesis cover, Atg6/BECN1, Atg8/LC3, p62/SQSTM1 and Lamp-1 expression within the cytoplasm increased in correlation with oocyte maturation, while no immunoreactivity was found in the follicular cells. Essays On Nelson Mandela Leadership! We hypothesize that the autophagy process may be essential for reproductive activity in crustaceans. However, whether autophagy is a part of cellular controls that regulate oocyte differentiation requires further investigation. The present data supports the high evolutionary conservation of autophagic components in the crustacean lineage, as implicated by the existence of several key autophagy-related genes and proteins, and master thesis, a high degree of sequence conservation of crucial autophagy marker proteins among crustaceans and other representative species from different phyla. This reflects a selection pressure for autophagic process during crustacean evolution. Athletes! In addition, we have demonstrated that antibodies raised against thesis human autophagy marker proteins can be used for monitoring the autophagic process in essay, a crustacean model. Learning how autophagy is regulated in the giant freshwater prawn could open up new avenues for novel management in limiting infections, organ stress, as well as nutritional and metabolic imbalances due to feeding and starvation, which can ultimately impact on master thesis cover, crustacean aquaculture. Adult females M. rosenbergii were obtained from a local commercial farm in Ayutthaya province, Thailand.
The prawns were anesthetized by jobs essay adobe immersion in ice water, and then the master thesis cover ovaries, hepatopancreases, muscles, brains, eyestalks, and thoracic ganglia were dissected out. Fresh tissues were kept at ?80 °C for further use in papers, transcriptome production and master thesis, Western blotting (described below). For histological and immunohistochemical studies, tissues were fixed in ice-cold 4 % paraformaldehyde for 24 h. Civilization Essay Irreverant Reconstructing! After washing with 70 % ethanol five times, specimens were then dehydrated in thesis cover, increasing concentrations of ethanol, cleared in xylene and infiltrated with paraffin, using an jobs automated tissue processor. The paraffin-embedded ovarian tissue blocks were cut at 5 ?m thickness and prepared for immunofluorescence staining. Transcriptome production and de novo assemblies of master thesis cover, CNS and ovary of M. rosenbergii. Transcriptome data of M. rosenbergii was obtained from a previous study [ 26 ]. It Important To Vote Essay! For transcriptome production, in brief, total RNA was isolated from the CNS (inclusive of the eyestalk, brain, thoracic ganglia, and thesis cover, abdominal ganglia) and the ovarian tissues using Tripure isolation reagent (Roche, IN, USA). The quality of total RNA of each tissue was checked by gel electrophoresis and spectrophotometry (NanoDrop 1000; Thermo Fisher Scientific, DE, USA). Twenty micrograms of total RNA of each tissue was sent to jobs adobe, BGI, Hong Kong, for library construction. Briefly, complementary DNA (cDNA) was synthesized from purified and fragmented RNA samples using BGI standard workflow for de novo RNA-seq transcriptomes ( http://bgiamericas.com/ ). The cDNA libraries were constructed based on polymerase chain reaction (PCR) amplification using random hexamer-primed cDNAs. Then the samples were sequenced using Illumina HiSeq 2000 instrument (Illumina Inc.) and only clean reads were used for further sequence assembly. De novo assembly was generated by SOAPdenovo software [ 64 ] using a combined CNS and ovary dataset with parameter set as follows: seqType, fq; minimum kmer coverage = 4; minimum contig length of 100 bp; group pair distance = 250.
All sequence data of M. rosenbergii CNS and cover, ovary transcriptomes were deposited in the NCBI database (Accession number: SRP049917). Gene mining, characterization, and sequence alignment. Searching of Atg proteins in M. Essay Adobe! rosenbergii transcriptome dataset was carried out by using tBLASTn program in CLC Main Workbench Version 6.0 (CLC Bio-Qaigen, AsiaPac, Taiwan) for which known Atg proteins (from mammals, yeast, and master cover, insects) were used as queries. The parameter was set as follows: matrix, BLOSUM62; e-value, 100. To search for transcripts encoding Atg proteins in other crustaceans, we used protein queries of known Atg proteins to search within publicly accessible databases [non-redundant (Nr), expressed sequence tags (ESTs), transcriptome shotgun assembly, and Daphnia genome [ 65 ] databases] by using NCBI tBLASTn program ( http://blast.ncbi.nlm.nih.gov/Blast.cgi ), which was restricted to same analysis essay, crustaceans (taxid: 6657) and master thesis cover, the parameter was set as follows: expect threshold = 10; matrix, BLOSUM62; gap costs, existence = 11 and analysis essay, extension = 1. Nucleotide sequences obtained from BLAST searches were translated into amino acid sequences using the CLC Main Workbench Version 6.0 and then primary sequence analyzed manually based on homology to thesis, other known proteins. Protein alignment of crustacean Atg proteins with other previously known Atg proteins was performed using MEGA6 [ 66 ] and GeneDoc [ 67 ] softwares. Amino acid sequences used in the alignments, as well as their accession numbers, are provided in Additional file 2 . All deduced M. rosenbergii Atg proteins were finally used for BLASTP search against the Nr protein database for same sex marriage analysis further sequence confirmation. The BLAST hit with lowest e-value for each M. rosenbergii Atg protein is detailed in Additional file 1 . Prediction of protein tertiary structure and critical sites. The tertiary structure of selected Atg proteins were predicted using the online tool Protein Homology/analogY Recognition Engine V 2.0 [ 68 ]. Visualization of predicted protein tertiary structure as a 3 dimensional model, illustration of cover, hydrophobicity, superimposition of proteins, and structure labeling were performed using the UCSF Chimera version 1.8.1 [ 69 ]. Educational Papers! The specific domains and critical sites were predicted by InterPro [ 70 ]. Primary antibodies used in master, western blotting and immunohistochemistry.
Rabbit polyclonal antibody against BECN1 (Sigma-Aldrich, USA) was produced using synthetic peptide corresponding to amino acid positions 329–345 of human BECN1 as immunogen. Rabbit polyclonal antibody against LC3B (Sigma-Aldrich, USA) was raised against the human LC3B at amino acid positions 2–15. Mandela! Rabbit polyclonal antibody against p62 (Santa Cruz, Biotechnology, USA) was produced against the human SQSTM1/p62 at amino acid positions 375–425. Thesis! Mouse monoclonal antibody against Lamp-1 (BD Science) was produced against human Lamp-1 at the amino acid positions 25–224. Western blot analysis of Atg8/LC3, Atg6/BECN1, and p62/SQSTM1. Fresh tissues were homogenized and sonicated in a lysis buffer (0.2 % NaDoc, 1 mM Na 3 VO 4 and 50 mM NaF) and sex marriage analysis, then centrifuged at 10,000? g , 4 °C, for 1 h to obtain tissue extracts.
The supernatants were collected and protein concentrations measured with the Bradford reagent (Sigma). For a positive control, human squamous carcinoma cells (A431 cell line) were sonicated in lysis buffer and protein concentrations measured. Subsequently, 30 ?g of total proteins were separated on a 15 % SDS-PAGE gel and transferred onto master, a polyvinylidene fluoride membrane (GE Healthcare Life Sciences). Nonspecific binding sites were blocked with a blocking buffer [5 % non-fat dry milk in essay, 0.2 % Tween 20 in 0.1 M phosphate-buffered saline (PBS), pH 7.4] for 2 h at room temperature. Thesis Cover! The membranes were incubated overnight at sex marriage, 4 °C with the following primary antibodies: either rabbit anti-LC3 (dilution 1:500), rabbit anti-p62/SQSTM1 (dilution 1:200), or rabbit anti-BECN1 (dilution 1:500). The membranes were washed three times for 5 min each with washing buffer (0.01 % Tween 20 in 0.1 M PBS) and then incubated for 2 h at room temperature in the peroxidase-conjugated secondary antibody [goat anti-rabbit IgG-HRP (SouthernBiotech) dilution 1:5000]. Antigen-antibody binding was detected using an enhanced chemiluminescent substrate (Thermo Scientific) and exposed to Amersham Hyperfilm ECL (GE Healthcare Life Sciences). Immunoperoxidase staining of master thesis, Atg proteins in the ovary of M. Should Athletes To Play Persuasive! rosenbergii. Immunoperoxidase staining was carried out following a previously described protocol [ 71 ] with some modifications.
Briefly, ovarian sections were sequentially deparaffinized and rehydrated in xylene and decreasing concentrations of ethanol. Endogenous peroxidase was blocked by thesis cover immersing sections in 3 % H 2 O 2 in methanol for 30 min. Subsequently, the sections were immersed in 1 % glycine in 0.1 M PBS to block free aldehyde groups and then incubated in a blocking serum (10 % bovine serum albumin in 0.1 M PBS, pH 7.4) for 2 h at 4 °C to should athletes essay, block non-specific bindings. Sections were incubated overnight at 4 °C with either rabbit anti-LC3 (dilution 1:500), mouse monoclonal anti-Lamp-1 (dilution 1:500), rabbit polyclonal anti-p62/SQSTM1 (dilution 1:500), or rabbit polyclonal antibody anti-BECN1 (dilution 1:500), in blocking serum. Master Thesis! After washing with PBS, sections were incubated in secondary antibody, either goat anti-rabbit IgG-HRP (Abcam) or goat anti-mouse IgG-HRP (SouthenBiotech) at the dilution 1:500 in blocking serum, for 2 h at room temperature. Mandela! Negative controls were performed by master cover omitting the primary antibodies. The color was developed with an AEC substrate kit (Invitrogen). Finally, sections were observed and photographed under the essay adobe Nikon E600 light microscope. BECN1, Beclin 1; Lamp-1, lysosomal-associated membrane protein 1Vps, vacuolar protein sorting; MAP1LC3, microtubule-associated proteins 1A/1B light chain 3; SQSTM1, sequestosome. This research was supported by a grant from the Office of master thesis cover, Higher Education Commission (OHEC) and Mahidol University (MU) under the same sex marriage essay National Research University Initiatives, an master thesis cover OHEC-MU and the Thailand Research Fund (TRF) Distinguished Research Professor Grant to Professor Prasert Sobhon, a TRF-RGJ Ph.D. Research! Scholarships to cover, Saowaros Suwansa-ard and Wilairat Kankuan; and an ARC grant to Dr.
Scott Cummins. We also thank the financial support from the Consorzio InterUniversitario per le Biotecnologie (Trieste, Italy) and from the Associazione per la Ricerca Medica-Ippocrate-Rhazi (Novara, Italy) to Miss Wilairat Kankuan. This work was supported by a grant from the college be paid to play Office of Higher Education Commission (OHEC) and Mahidol University (MU) under the National Research University Initiatives, an OHEC-MU and the Thailand Research Fund (TRF) Distinguished Research Professor Grant to PS. SS was supported by a TRF-RGJ Ph.D. Scholarships. Master! WK was supported by a TRF-RGJ Ph.D. Scholarship, the Consorzio InterUniversitario per antiquity civilization essay irreverant le Biotecnologie (Trieste, Italy), and the Associazione per la Ricerca Medica-Ippocrate-Rhazi (Novara, Italy). Master! SFC was supported by why is an ARC grant. Availability of data and materials. The datasets supporting the conclusions of this article are available in the NCBI Sequence Read Archive (SRA) depository (SRA Accession number: SRP049917; http://trace.ncbi.nlm.nih.gov/Traces/sra/?study=SRP049917 ). Additional supporting data are included as additional files. SS performed the bioinformatics analyses and wrote the master manuscript.
WK performed the educational research Western blot analysis and immunohistochemistry, and wrote the manuscript. TT, JS, NK, TK and KK contributed the cover transcriptomic data and analysis. Why Is It Important To Vote! SFC, CI, and PS provided conception, laboratory support, critical revision of the manuscript, and final approval of the version to be published. The authors have declared that no competing interests exist. Ethics approval and consent to participate. Ethics is not required for cover the study. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/ ), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and essay, indicate if changes were made. Master Thesis! The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/ ) applies to the data made available in this article, unless otherwise stated. Saowaros Suwansa-ard 1 4 Wilairat Kankuan 1 5 Tipsuda Thongbuakaew 1 7 Jirawat Saetan 2 Napamanee Kornthong 3 Thanapong Kruangkum 1 Kanjana Khornchatri 1 Scott F. Cummins 4 Ciro Isidoro 5 Email author Prasert Sobhon 1 6 Email authorView author's OrcID profile 1. Department of jobs adobe, Anatomy, Faculty of Science Mahidol University Bangkok Thailand 2. Department of Anatomy, Faculty of Science Prince of cover, Songkla University Hatyai Thailand 3. Chulabhorn International College of Medicine Thammsat University Phathumthani Thailand 4. Educational Research Papers! Faculty of master thesis cover, Science, Health, Education and civilization essay western, Engineering University of the Sunshine Coast Maroochydore Australia 5. Laboratory of Molecular Pathology, Department of Health Sciences Amedeo Avogadro University Novara Italy 6. Faculty of Allied Health Sciences Burapha University Chonburi Thailand 7. School of Medicine Walailak University Nakhon Si Thammarat Thailand. .RIS Papers Reference Manager RefWorks Zotero. .BIB BibTeX JabRef Mendeley.
.RIS Papers Reference Manager RefWorks Zotero. .BIB BibTeX JabRef Mendeley. Over 10 million scientific documents at your fingertips. 2017 Springer International Publishing AG. Part of master cover, Springer Nature.
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fight essay Where there's a will, there's a way. Master Cover. - I said so to myself, as I walked down Chancery-lane, about jobs half-past six o'clock on Monday the 10th of December, to inquire at Jack Randall's where the fight the thesis cover next day was to be; and I found the essay adobe proverb nothing musty in the present instance. Cover. I was determined to see this fight, come what would, and see it I did, in great style. It was my first fight, yet it more than answered my expectations. Research. Ladies! it is to you I dedicate this description; nor let it seem out of character for the fair to notice the exploits of the brave. Courage and modesty are the master thesis old English virtues; and may they never look cold and askance on should essay one another! Think, ye fairest of the master thesis fair, loveliest of the on nelson mandela leadership lovely kind, ye practisers of soft enchantment, how many more ye kill with poisoned baits than ever fell in the ring; and listed with subdued air and without shuddering, to a tale tragic only in appearance, and sacred to the FANCY! I was going down Chancery-lane, thinking to ask at Jack Randall's where the fight was to be, when looking through the glass-door of the Hole in the Wall, I heard a gentleman asking the same question at master thesis Mrs.
Randall, as the author2 of Waverley would express it. Now Mrs. Randall stood answering the it important essay gentlemen's question, with the authenticity of the lady of the Champion of the Light Weights. Thinks I, I'll wait till this person comes out, and master cover learn from him how it is. On Nelson. For to master thesis say a truth, I was not fond of going into this house to should essay call for heroes and philosophers, ever since the owner of master thesis cover it (for Jack is no gentleman) threatened once upon a time to kick me out of doors for wanting a mutton-chop at his hospitable board, when the conqueror in thirteen battles was more full of blue ruin than of good manners. I was the more mortified at this repulse, inasmuch as I had heard Mr. James Simpkin, hosier in papers, the Strand, one day when the master character of the Hold in the Wall was brought in antiquity civilization, question, observe - The house is a very good house, and the company quite genteel: I have been there myself! Remembering this unkind treatment of mine host, to which mine hostess was also a party, and not wishing to put her in unquiet thoughts at a time jubilant like the present, I waited at the door, when, who should issue forth but my friend Jo. Master Thesis Cover. Toms, and be paid turning suddenly up Chancery-lane with that quick jerk and impatient stride which distinguishes a lover of the FANCY, I said, I'll be hanged if that fellow is not going to the fight, and is on his way to get me to go with him.
So it proved in effect, and we agreed to adjourn to my lodgings to discuss measures with that cordiality which makes old friends like new, and new friends like old, on great occasions. Cover. We are cold to research papers others only when we are dull in ourselves, and have neither thoughts nor feelings to impart to thesis cover them. Give a man a topic in his head, a throb of pleasure in his heart, and he will be glad to share it with the first person he meets. Toms and I, though we seldom meet, were an alter idem on college athletes be paid to play this memorable occasion, and had not an idea that we did not candidly impart; and so carelessly did we fleet the time, that I wish no better, when there is another fight, than to have him for master, a companion on my journey down, and to return with my friend Jack Pigott, talking of what was to same analysis happen or of what did happen, with a noble subject always at hand, and liberty to digress to others whenever they offered. Indeed, on my repeating the lines from thesis cover Spenser in an involuntary fit of enthusiasm, What more felicity can fall to creature,
Than to enjoy delight with liberty? my last-named ingenious friend stopped me by saying that this, translated into the vulgate, meant Going to see a fight. Jo. Toms and same essay I could not settle about the method of going down. Master Thesis Cover. He said there was a caravan, he understood, to start from Tom Belcher's at two, which would go there right out and back again the mandela leadership next day. Now I never travel all night, and master thesis said I should get a cast to Newbury by it important essay, one of the mails.
Jo. swore the thing was impossible, and I could only answer that I had made up my mind to it. In short, he seemed to me to master waver, said he only came to see if I was going, had letters to write, a cause coming on the day after, and faintly said at parting (for I was bent on college be paid to play persuasive essay setting out master thesis, that moment) - Well, we meet at Philippi! I made the best of my way to Piccadilly. Jobs Essay. The mail coach stand was bare. They are all gone, said I - this is always the way with me - in thesis, the instant I lose the future - if I had not stayed to pour out that last cup of tea, I should have been just in leadership research, time - and cursing my folly and ill- luck together, without inquiring at the coach-office whether the mails were gone or not, I walked on master thesis in despite, and to punish my own dilatoriness and want of determination. At any rate, I would not turn back: I might get to should college essay Hounslow, or perhaps farther, to cover be on why is it important my road the master next morning. I passed Hyde Park Corner (my Rubicon), and antiquity irreverant western trusted to fortune. Suddenly I heard the clattering of a Brentford stage, and cover the fight rushed full upon research my fancy.
I argued (not unwisely) that even a Brentford coachman was better company than my own thoughts (such as they were just then), and at his invitation mounted the box with him. I immediately stated my case to him - namely, my quarrel with myself for missing the Bath or Bristol mail, and my determination to master thesis get on in consequence as well as I could, without any disparagement or insulting comparison between longer or shorter stages. Jobs Adobe. It is thesis a maxim with me that stage-coaches, and consequently stage-coachmen, are respectable in proportion to the distance they have to travel: so I said nothing on that subject to my Brentford friend. Any incipient tendency to an abstract proposition, or (as he might have construed it) to a personal reflection of this kind, was however nipped in the bud; for I had no sooner declared indignantly that I had missed the essay adobe mails, than he flatly denied that they were gone along, and lo! at the instant three of them drove by in master thesis, rapid, provoking, orderly succession, as if they would devour the ground before them. Here again I seemed in the contradictory situation of the man in same sex marriage, Dryden who exclaims, I follow Fate, which does too hard pursue!
If I had stopped to inquire at the White Horse Cellar, which would not have taken me a minute, I should now have been driving down the road in all the dignified unconcern and ideal perfection of mechanical conveyance. Master Thesis Cover. The Bath mail I had set my mind upon, and I had missed it, as I missed everything else, by my own absurdity, in putting the will for essay irreverant, the deed, and aiming at ends without employing means. Sir, said he of the Brentford, The Bath mail will be up presently, my brother-in-law drives it, and I will engage to stop him if there is a place empty. I almost doubted my good genius; but, sure enough, up it drove like lightning, and stopped directly at the call of the Brentford Jehu. I would not have believed this possible, but the master thesis brother-in-law of a mail-coach driver is himself no mean man. I was transferred without loss of time from the top of leadership one coach to that of the other, desired the guard to pay my fare to the Brentford coachman for me as I had no change, was accommodated with a great coat, put up my umbrella to keep off a drizzling mist, and thesis cover we began to cut through the essays on nelson air like an arrow.
The mile-stones disappeared one after another, the rain kept off; Tom Turtle, the trainer, sat before me on thesis cover the coach-box, with whom I exchanged civilities as a gentleman going to the fight; the passion that had transported me an hour before was subdued to pensive regret and conjectural musing on the next day's battle; I was promised a place inside at Reading, and upon mandela leadership the whole, I thought myself a lucky fellow. Master Cover. Such is the force of educational leadership papers imagination! On the master cover outside of any other coach on the 10th of December, with a Scotch mist drizzling through the cloudy moonlight air, I should have been cold, comfortless, impatient, and, no doubt, wet through; but seated on the Royal mail, I felt warm and comfortable, the reconstructing air did me good, the ride did me good, I was pleased with the master progress we had made, and confident that all would go well through the journey. When I got inside at Reading, I found Turtle and a stout valetudinarian, whose costume bespoke him one of the FANCY, and who had risen from a three months' sick bed to get into the mail to see the fight. They were intimate, and we fell into irreverant reconstructing a lively discourse. Cover. My friend the trainer was confined in his topics to fighting dogs and men, to bears and badgers; beyond this he was quite chap-fallen, had not a word to throw at a dog, or indeed very wisely fell asleep, when any other game was started. The whole art of training (I, however, learnt from jobs him), consists in two things, exercise and abstinence, abstinence and exercise, repeated alternately without end. A yolk of an egg with a spoonful of cover rum in it is the first thing in a morning, and essay then a walk of master thesis cover six miles till breakfast. This meal consists of a plentiful supply of educational research tea and toast and master beef steaks. Then another six or seven miles till dinner-time, and another supply of solid beef or mutton with a pint of porter, and perhaps, at the utmost, a couple of glasses of sherry.
Martin trains on water, but this increases his infirmity on another very dangerous side. The Gas-man takes now and then a chirping glass (under the rose) to console him, during a six weeks' probation, for the absence of Mrs. Hickman - an agreeable woman, with (I understand) a pretty fortune of two hundred pounds. How matter presses on me! What stubborn things are facts! How inexhaustible is nature and art! It is well, as I once heard Mr. Leadership Research Papers. Richmond observe, to see a variety. He was speaking of cock-fighting as an edifying spectacle. I cannot deny but that one learns more of what is (I do not say of what ought to be) in this desultory mode of cover practical study, than from reading the same book twice over, even though it should be a moral treatise.
Where was I? I was sitting at it important to vote dinner with the master thesis candidate for the honours of the ring, where good digestion waits on appetite, and health on both. Then follows an hour of social chat and college athletes be paid native glee; and master cover afterwards, to another breathing over heathy hill or dale. Back to same analysis supper, and then to bed, and master up by six again - Our hero. Follows the ever-running sun. With profitable ardour - to the day that brings him victory or defeat in the green fairy circle. Is not this life more sweet than mine? I was going to say; but I will not libel any life by comparing it to mine, which is same sex marriage essay (at the date of these presents) bitter as coloquintida and the dregs of aconitum! The invalid in the Bath mail soared a pitch above the master trainer, and did not sleep so sound, because he had more figures and more fantasies.
We talked the hours away merrily. He had faith in surgery, for he had had three ribs set right, that had been broken in a turn-up at Belcher's, but thought physicians old women, for to vote, they had no antidote in their catalogue for brandy. An indigestion is an excellent commonplace for two people that never met before. By way of ingratiating myself, I told him the master thesis cover story of my doctor, who, on my earnestly representing to him that I thought his regimen had done me harm, assured me that the whole pharmacopeia contained nothing comparable to the prescription he had given me; and, as a proof of his undoubted efficacy, said, that, he had had one gentleman with my complaint under his hands for the last fifteen years. This anecdote made my companion shake the rough sides of his three great coats with boisterous laughter; and Turtle, starting out of his sleep, swore he knew how the fight would go, for he had had a dream about should to play persuasive essay it. Sure enough, the rascal told us how the first rounds went off, but his dream, like others, denoted a foregone conclusion. He knew his men. The moon now rose in silver state, and I ventured, with some hesitation, to point out master thesis, this object of civilization essay reconstructing western placid beauty, with the blue serene beyond, to the man of science, to thesis which his ear he seriously inclined, the same analysis more as it gave promise d'un beau jour for the morrow, and showed the ring undrenched by envious showers, arrayed in sunny smiles. Just then, all going on well, I thought on my friend Toms, whom I had left behind, and said innocently, There was a blockhead of a fellow I left in town, who said there was no possibility of getting down by the mail, and talked of going by a caravan from Belcher's at two in the morning, after he had written some letters. Why, said he of the lapells, I should not wonder if that was the master thesis cover very person we saw running about like mad from one coach-door to another, and essay asking if anyone had seen a friend of his, a gentleman going to the fight, whom he had missed stupidly enough by master cover, staying to athletes be paid essay write a note. Pray, Sir, said my fellow-traveller, he had a plaid-cloak on? - Why, no, said I, not at the time I left him, but he very well might afterwards, for he offered to lend me one.
The plain-cloak and the letter decided the master thing. Western. Joe,3 sure enough, was in thesis cover, the Bristol mail, which preceded us by about fifty yards. This was droll enough. We had now but a few miles to our place of destination, and the first thing I did on alighting at Newbury, both coaches stopping at the same time, was to call out, Pray, is there a gentleman in that mail of the name of Toms? No, said Joe, borrowing something of the vein of Gilpin, for I have just got out. Well! says he, this is lucky; but you don't know how vexed I was to miss you; for, added he, lowering his voice, did you know when I left you I went to it important essay Belcher's to ask about the caravan, and Mrs. Belcher said very obligingly, she couldn't tell about that, but there were two gentlemen who had taken places by the mail and were gone on in a landau, and she could frank us. It's a pity I didn't meet with you; we could then have got down for nothing. But mum's the word. Cover. It's the devil for anyone to tell me a secret, for it's sure to come out in print.
I do not care so much to essays on nelson gratify a friend, but the cover public ear to too great a temptation to me. Our present business was to get beds and should college athletes be paid persuasive essay a supper at an inn; but this was no easy task. The public-houses were full, and where you saw a light at a private house, and people poking their heads out of the casement to see what was going on, they instantly put them in and shut the window, the master thesis cover moment you seemed advancing with a suspicious overture for accommodation. Our guard and coachman thundered away at the outer gate of the Crown for some time without effect - such was the greater noise within; - and when the doors were unbarred, and we got admittance, we found a party assembled in the kitchen round a good hospitable fire, some sleeping, others drinking, others talking on politics and on the fight. A tall English yeoman (something like Matthews in the face, and quite as great a wag) - A lusty man to ben an abbot able, - was making such a prodigious noise about rent and taxes, and the price of corn now and formerly, that he had prevented us from being heard at the gate.
The first thing I heard him say was to a shuffling fellow who wanted to essay be off a bet for a shilling glass of brandy and water - Confound it, man, don't be insipid! Thinks I, that is a good phrase. It was a good omen. Cover. He kept it up so all night, nor flinched with the approach of morning. He was a fine fellow, with sense, wit, and spirit, a hearty body and a joyous mind, free-spoken, frank, convivial - one of that true English breed that went with Harry the Fifth to the siege of Harfleur - standing like greyhounds in the slips, etc. Why Is. We ordered tea and master eggs (beds were soon found to be out of the question) and this fellow's conversation was sauce piquante . It did one's heart good to see him brandish his oaken towel and to hear him talk. He made mince-meat of a drunken, stupid, red-faced, quarrelsome, frowsy farmer, whose nose he moralised into a thousand similes, making it out a firebrand like Bardolph's. I'll tell you what my friend, says he, the essay adobe landlady has only to keep you here to save fire and candle.
If one was to touch your nose, it would go off like a piece of charcoal. At this the other only grinned like an idiot, the sole variety in his purple face being his little peering grey eyes and yellow teeth; called for another glass, swore he would not stand it; and after many attempts to provoke his humorous antagonist to singe combat, which the other turned off (after working him up to master a ludicrous pitch of choler) with great adroitness, he fell quietly asleep with a glass of liquor in his hand, which he could not lift to his head. His laughing persecutor made a speech over sex marriage essay him, and turning to the opposite side of the cover room, where they were all sleeping in essays on nelson mandela leadership, the midst of this loud and furious sun, said, There's a scene, by G-d, for master thesis, Hogarth to paint. I think he and Shakespeare were our two best men at copying life. Leadership. This confirmed me in my good opinion of him.
Hogarth, Shakespeare, and Nature, were just enough for him (indeed for any man) to know. I said, You read Cobbett, don't you? At least, says I, you talk just as well as he writes. He seemed to doubt this. Cover. But I said, We have an hour to spare; if you'll get pen, ink, and paper, and keep on mandela leadership talking, I'll write down what you say; and if it doesn't make a capital 'Political Register,' I'll forfeit my head. You have kept me alive to-night, however. I don't know what I should have done without you.
He did not dislike this view of the thing, nor my asking if he was not about the size of Jem Belcher; and told me soon afterwards, in the confidence of friendship, that the circumstance which had given him nearly the greatest concern in his life, was Cribb's beating Jem after he had lost his eye by racket-playing. - The morning dawns; that dim but yet clear light appears, which weighs like solid bars of metal on the sleepless eyelids; the guests drop down from their chambers one by one - but it was too late to think of going to bed now (the clock was on the stroke of seven), we had nothing for it but to find a barber's (the pole that glittered in thesis cover, the morning sun lighted us to his shop), and then a nine miles' march to Hungerford. The day was fine, the sky was blue, the mists were retiring from the marshy ground, the antiquity essay irreverant reconstructing western path was tolerably dry, the sitting-up all night had not done us much harm - at thesis cover least the cause was good; we talked of this and jobs essay adobe that with amicable difference, roving and sipping of many subjects, but still invariably we returned to thesis cover the fight. At length, a mile to the left of Hungerford, on a gentle eminence, we saw the ring surrounded by covered carts, gigs, and carriages, of leadership which hundreds had passed us on the road; Toms gave a youthful shout, and master thesis we hastened down a narrow lane to the scene of action. Reader, have you ever seen a fight? If not, you have a pleasure to come, at be paid persuasive least if it is a fight like that between the Gas-man and thesis Bill Neate. The crowd was very great when we arrived on the spot; open carriages were coming up, with streamers flying and music playing, and the country-people were pouring in over hedge and ditch in all directions, to see their hero beat or be beaten. The odds were still on Gas, but only about five to four. Gully had been down to try Neate, and had backed him considerably, which was a damper to essays on nelson the sanguine confidence of the adverse party. Cover. About two hundred thousand pounds were pending.
The Gas says, he has lost Ј3,000 which were promised him by different gentlemen if he had won. He had presumed too much on himself, which had made others presume on him. Same Sex Marriage Analysis Essay. This spirited and formidable young fellow seems to have taken for his motto the master thesis old maxim, that there are three things necessary to success in life - Impudence! Impudence! Impudence! It is so in matters of opinion, but not in should athletes be paid, the FANCY, which is the most practical of all things, though even here confidence is master half the battle, but only half. Essay. Our friend had vapoured and swaggered too much, as if he wanted to grin and bully his adversary out of the fight. Alas! the master thesis Bristol man was not so tamed! - This is the grave digger (would Tom Hickman exclaim in same sex marriage analysis essay, the moments of intoxication from gin and success, showing his tremendous right hand), this will send many of them to their long homes; I haven't done with them yet! Why should he - though he had licked four of the best men within the hour, yet why should he threaten to inflict dishonourable chastisement on my old master Richmond, a veteran going off the master stage, and who has borne his sable honours meekly? Magnanimity, my dear Tom, and bravery, should be inseparable. Or why should he go up to his antagonist, the first time he ever saw him at the Fives Court, and measuring him from head to should athletes to play persuasive foot with a glance of contempt, as Achilles surveyed Hector, say to thesis him, What, are you Bill Neate?
I'll knock more blood out of that great carcase of thine, this day fortnight, than you ever knock'd out of a bullock's! It was not manly, 'twas not fighter- like. If he was sure of the victory (as he was not), the less said about it the better. Modesty should accompany the FANCY as its shadow. The best men were always the best behaved. Sex Marriage. Jem Belcher, the master Game Chicken (before whom the Gas-man could not have lived) were civil, silent men. So is Cribb, so is civilization essay Tom Belcher, the most elegant of sparrers, and master thesis not a man for every one to research papers take by the nose. I enlarged on this topic in the mail (while Turtle was asleep), and said very wisely (as I thought) that impertinence was a part of no profession. A boxer was bound to master cover beat his man, but not to thrust his fist, either actually or by implication, in every one's face. Even a highwayman, in the way of it important essay trade, may blow out thesis cover, your brains, but if he uses foul language at the same time, I should say he was no gentleman.
A boxer, I would infer, need not be a blackguard or a coxcomb, more than another. Perhaps I press this point too much on a fallen man - Mr. Thomas Hickman has by this time learnt that first of all lessons, That man was made to mourn. He has lost nothing by adobe, the late fight but his presumption; and that every man may do as well without! By an overly-display of thesis cover this quality, however, the educational leadership papers public has been prejudiced against him, and the knowing-ones were taken in. Few but those who had bet on him wished Gas to win. With my own prepossessions on the subject, the result of the 11th of December appeared to me as fine a piece of poetical justice as I had ever witnessed. The difference of weight between the two combatants (14 stone to 12) was nothing to the sporting men. Great, heavy, clumsy, long-armed Bill Neate kicked the beam in the scale of the Gas-man's vanity.
The amateurs were frightened at his big words, and thought that they would make up for the difference of thesis six feet and five feet nine. Essay. Truly, the FANCY are not men of master thesis cover imagination. They judge of what has been, and cannot conceive of anything that is to essays mandela be. Master Thesis Cover. The Gas-man had won hitherto; therefore he must beat a man half as big again as himself - and that to a certainty. Besides, there are as many feuds, factions, prejudices, pedantic notions in the FANCY as in the state or in papers, the schools. Master Cover. Mr. Gully is almost the only cool, sensible man among them, who exercises an unbiassed discretion, and is not a slave to his passions in these matters.
But enough of reflections, and to our tale. Why Is To Vote. The day, as I have said, was fine for a December morning. The grass was wet, and the ground miry, and ploughed up with multitudinous feet, except that, within the ring itself, there was a spot of virgin-green closed in and unprofaned by vulgar tread, that shone with dazzling brightness in the mid-day sun. For it was noon now, and we had an hour to wait. This is the trying time. Thesis Cover. It is then the heart sickens, as you think what the two champions are about, and how short a time will determine their fate.
After the first blow is struck, there is no opportunity for papers, nervous apprehensions; you are swallowed up in the immediate interest of the scene - but. Between the acting of a dreadful thing. And the first motion, all the thesis interim is. Like a phantasma, or a hideous dream. I found it so as I felt the sun's rays clinging to my back, and saw the white wintry clouds sink below the verge of the horizon. So, I thought, my fairest hopes have faded from my side! - so will the Gas-man's glory, or that of his adversary, vanish in sex marriage analysis, an hour. The swells were parading in their white box-coats, the outer ring was cleared with some bruises on the heads and shins of the rustic assembly (for the cockneys had been distanced by the sixty- six miles); the time drew near, I had got a good stand; a bustle, a buzz, ran through the crowd, and from the opposite thesis cover, side entered Neate, between his second and bottle-holder. Same Sex Marriage. He rolled along, swathed in his loose great coat, his knock-knees bending under his huge bulk; and, with a modest cheerful air, threw his hat into the ring. Master. He then just looked round, and began quietly to undress; when from the other side there was a similar rush and an opening made, and the Gas-man came forward with a conscious air of antiquity civilization essay irreverant reconstructing anticipated triumph, too much like the cock-of-the-walk. He strutted about thesis cover more than became a hero, sucked oranges with a supercilious air, and threw away the skin with a toss of his head, and went up and looked at Neate, which was an act of supererogation.
The only sensible thing he did was, as he strode away from the on nelson modern Ajax, to master thesis fling out his arms, as if he wanted to should college try whether they would do their work that day. By this time they had stripped, and presented a strong contrast in appearance. Master Cover. If Neate was like Ajax, with Atlantean shoulders, fit to bear the should be paid persuasive pugilistic reputation of all Bristol, Hickman might be compared to Diomed, light, vigorous, elastic, and his back glistened in cover, the sun, as he moved about, like a panther's hide. Civilization Irreverant Western. There was now a dead pause - attention was awe-struck. Master Cover. Who at that moment, big with a great event, did not draw his breath short - did not feel his heart throb? All was ready. Same Sex Marriage Essay. They tossed up for the sun, and the Gas-man won. They were lead up to the scratch - shook hands, and went at it. In the cover first round everyone thought it was all over.
After making play a short time, the why is to vote essay Gas-man flew at his adversary like a tiger, struck five blows in as many seconds, three first, and then following him as he staggered back, two more, right and left, and down he fell, a might ruin. There was a shout, and I said, There is no standing this. Thesis. Neate seemed like a lifeless lump of flesh and bone, round which the Gas-man's blows played with the rapidity of electricity or lighting, and you imagined he would only be lifted up to be knocked down again. Essays. It was as if Hickman held a sword or a fire in the right hand of his, and thesis cover directed it against an unarmed body. It Important To Vote. They met again, and Neate seemed, not cowed, but particularly cautious. I saw his teeth clenched together and his brows knit close against the sun. Thesis. He held out both his arms at full-length straight before him, like two sledge-hammers, and raised his left an inch or two higher. The Gas-man could not get over this guard - they struck mutually and fell, but without advantage on either side. Essays. It was the same in the next round; but the balance of power was thus restored - the fate of the battle was suspended. Master Thesis Cover. No one could tell how it would end.
This was the only moment in which opinion was divided; for, in the next, the Gas-man aiming a mortal blow at his adversary's neck, with his right hand, and antiquity civilization essay reconstructing failing from the length he had to reach, the other returned it with his left at cover full swing, planted a tremendous blow on his cheek-bone and eyebrow, and made a red ruin of that side of his face. The Gas-man went down, and there was another shout - a roar of triumph as the waves of antiquity essay reconstructing fortune rolled tumultuously from thesis side to side. This was a settler. Hickman got up, and grinned horrible a ghastly smile, yet he was evidently dashed in his opinion of himself; it was the first time he had ever been so punished; all one side of his face was perfect scarlet, and his right eye was closed in on nelson mandela leadership, dingy blackness, as he advanced to the fight, less confident, but still determined. Cover. After one or two rounds, not receiving another such remembrancer, he rallied and should athletes persuasive went at master thesis cover it with his former impetuosity. It Important Essay. But in vain.
His strength had been weakened, - his blows could not tell at such a distance, - he was obliged to fling himself at master cover his adversary, and could not strike from his feet; and essays on nelson almost as regularly as he flew at him with his right hand, Neate warded the blow, or drew back out of its reach, and felled him with the return of his left. There was little cautious sparring - no half-hits - no tapping and trifling, none of the petit-ma#238treship of the master art - they were almost all knock-down blows: - the fight was a good stand-up fight. The wonder was the half-minute time. If there had been a minute or more allowed between each round, it would have been intelligible how they should by degrees recover strength and leadership research resolution; but to see two men smashed to the ground, smeared with gore, stunned, senseless, the master breath beaten out of their bodies; and then, before you recover from the shock, to see them rise up with new strength and courage, stand steady to inflict or receive mortal offence, and rush upon each other, like two clouds over the Caspian - this is the most astonishing thing of all: - this is the high and to vote heroic state of man! From this time forward the master event became more certain every round; and about the twelfth it seemed as if it must have been over. Hickman generally stood with his back to me; but in antiquity civilization irreverant, the scuffle, he had changed positions, and Neate just then made a tremendous lunge at him, and master thesis cover hit him full in the face. It was doubtful whether he would fall backwards or forwards; he hung suspended for about a second or two, and it important essay then fell back, throwing his hands in the air, and with his face lifted up to the sky. Master Thesis Cover. I never saw anything more terrific than his aspect just before he fell. All traces of essays on nelson leadership life, of natural expression, were gone from him. His face was like a human skull, a death's head, spouting blood. The eyes were filled with blood, the nose streamed with blood, the mouth gaped blood.
He was not like an actual man, but like a preternatural, spectral appearance, or like one of the figures in Dante's Inferno. Cover. Yet he fought on after this for several rounds, still striking the essays on nelson mandela first desperate blow, and master cover Neate standing on the defensive, and using the same essay same cautious guard to the last, as if he had still all his work to do; and thesis cover it was not till the Gas-man was so stunned in the seventeenth or eighteenth round, that his senses forsook him, and jobs he could not come to time, that the battle was declared over. Ye who despise the FANCY, do something to show as much pluck, or as much self-possession as this, before you assume a superiority which you have never given a single proof of by any one action in the whole course of your lives! - When the Gas-man came to himself, the first words he uttered were, Where am I? What is the matter! Nothing is the master cover matter, Tom - you have lost the essays on nelson leadership battle, but you are the bravest man alive. Thesis Cover. And Jackson whispered to him, I am collecting a purse for should college athletes be paid to play persuasive, you, Tom. Master Thesis Cover. - Vain sounds, and unheard at why is it important essay that moment! Neate instantly went up and shook him cordially by the hand, and seeing some old acquaintance, began to flourish with his fists, calling out, Ah, you always said I couldn't fight - What do you think now? But all in good humour, and without any appearance of arrogance; only master cover, it was evident Bill Neate was pleased that he had won the fight. When it was all over, I asked Cribb if he did not think it was a good one?
He has, Pretty well! The carrier-pigeons now mounted into the air, and one of them flew with the news of her husband's victory to the bosom of sex marriage Mrs. Neate. Master Cover. Alas, for Mrs. Hickman! Mais au revoir , as Sir Fopling Flutter says. I went down with Toms; I returned with Jack Pigott, whom I met on the ground. Toms is a rattle-brain; Pigott is a sentimentalist.
Now, under favour, I am a sentimentalist too - therefore I say nothing, but that the interest of the excursion did not flag as I came back. Pigott and I marched along the causeway leading from Hungerford to Newbury, now observing the effect of a brilliant sun on sex marriage the tawny meads or moss-coloured cottages, now exulting in the fight, now digressing to some topic of general and elegant literature. My friend was dressed in thesis, character for essay, the occasion, or like one of the thesis cover FANCY; that is, with a double portion of great coats, clogs, and overhauls: and just as we had agreed with a couple of country-lads to carry his superfluous wearing- apparel to the next town, we were overtaken by a return post-chaise, into essay irreverant reconstructing western which I got, Pigott preferring a seat on the bar. There were two strangers already in the chaise, and on cover their observing they supposed I had been to the fight, I said I had, and concluded they had done the same. They appeared, however, a little shy and sore on the subject; and antiquity civilization irreverant reconstructing western it was not fill after several hints dropped, and questions put, that it turned out that they had missed it. One of these friends had undertaken to drive the other there in his gig: they had set out, to make sure work, the day before at three in the afternoon. The owner of the master cover one- horse vehicle scorned to ask his way, and drove right on to Bagshot, instead of turning off at why is it important to vote essay Hounslow: there they stopped all night, and master thesis cover set off the next day across the country to adobe Reading, from whence they took coach, and got down within a mile or two of Hungerford, just half an hour after the fight was over.
This might be safely set down as one of the thesis cover miseries of human life. Should College To Play. We parted with these two gentlemen who had been to thesis see the fight, but had returned as they went, at Wolhampton, where we were promised beds (an irresistible temptation, for adobe, Pigott had passed the preceding night at Hungerford, as we had done at Newbury; and master cover we turned into an old bow-windowed parlour with a carpet and a snug fire; and after devouring a quantity of tea, toast, and eggs, sat down to consider, during an hour of same essay philosophic leisure, what we should have for supper. In the midst of an Epicurean deliberation between a roasted fowl and mutton chops with mashed potatoes, we were interrupted by an inroad of Goths and Vandals - O procul este profani - not real flash-men, but interlopers, noisy pretenders, butchers from Tothillfields, brokers from Whitechapel, who called immediately for pipes and tobacco, hoping it would not be disagreeable to the gentlemen, and began to insist that it was a cross. Pigott withdrew from the smoke and thesis noise into another room, and left me to dispute the point with them for a couple of hours sans intermission by the dial. The next morning we rose refreshed; and on observing that Jack had a pocket volume in same sex marriage essay, his hand, in which he read in the intervals of our discourse, I inquired what it was, and learned to master cover my particular satisfaction that it was a volume of the New Eloise. Ladies, after this, will you contend that a love for the FANCY is incompatible with the cultivation of sentiment? - We jogged on educational research as before, my friend setting me up in a genteel drab great coat and green silk handkerchief (which I must say became me exceedingly), and after stretching our legs for a few miles, and seeing Jack Randall, Ned Turner, and Scroggins, pass on the top of one of the Bath coaches, we engaged with the driver of the second to take us to London for the usual fee. I got inside, and found three other passengers.
One of them was an master thesis cover, old gentleman with an aquiline nose, powdered hair, and a pigtail, and leadership research papers who looked as if he had played many a rubber at the Bath rooms. Cover. I said to myself, he is very like Mr. Windham; I wish he would enter into same conversation, that I might hear what fine observations would come from those finely-turned features. However, nothing passed, till, stopping to dine at Reading, some inquiry was made by the company about the fight, and thesis I gave (as the reader may believe) an eloquent and animated description of same sex marriage analysis essay it. When we got into the coach again, the old gentleman, after a graceful exordium, said, he had, when a boy, been to a fight between the famous Broughton and George Stevenson, who was called the Fighting Coachman, in master thesis, the year 1770, with the same late Mr. Windham. This beginning flattered the spirit of prophecy within me and rivetted my attention. He went on thesis - George Stevenson was coachman to a friend of my father's. He was an leadership, old man when I saw him some years afterwards. He took hold of his own arm and said, 'There was muscle here once, but now it is no more than this young gentleman's.' He added, 'Well, no matter; I have been here long, I am willing to go hence, and I hope I have done no more harm than another man.' Once, said my unknown companion, I asked him if he had ever beat Broughton?
He said Yes; that he had fought with him three times, and thesis the last time he fairly beat him, though the world did not allow it. 'I'll tell you how it was, master. When the seconds lifted us up in the last round, we were so exhausted that neither of us could stand, and we fell upon athletes persuasive essay one another, and as Master Broughton fell uppermost, the mob gave it in his favour, and he was said to have won the thesis cover battle. Why Is It Important. But,' says he, 'the fact was, that as his second (John Cuthbert) lifted him up, he said to thesis cover him, I'll fight no more, I've had enough; 'which,' says Stevenson, 'you know gave me the victory. And to prove to you that this was the case, when John Cuthbert was on college athletes persuasive his death-bed, and they asked him if there was anything on his mind which he wished to cover confess, he answered, Yes, that there was one thing he wished to set right, for leadership, that certainly Master Stevenson won that last fight with Master Broughton; for thesis, he whispered him as he lifted him up in the last round of should college to play essay all, that he had had enough.' This, said the Bath gentleman, was a bit of human nature; and master I have written this account of the fight on purpose that it might not be lost to the world. He also stated as a proof of the educational leadership candour of mind in master thesis, this class of men, that Stevenson acknowledged that Broughton could have beat him in his best day; but that he (Broughton) was getting old in their last encounter. When we stopped in Piccadilly, I wanted to ask the gentleman some questions about the late Mr.
Windham, but had not courage. I got out, resigned my coat and green silk handkerchief to Pigott (loth to part with these ornaments of life), and walked home in high spirits. P.S. Toms called upon me the next day, to ask me if I did not think the fight was a complete thing? I said I thought it was.
I hope he will relish my account of it. 2 The author of the antiquity civilization essay irreverant reconstructing Waverley Novels, of course, is master cover Hazlitt's contemporary, Sir Walter Scott. 3 The Joe to whom Hazlitt refers to in this essay (according to Birrell) is Joseph Parkes, a solicitor (he had another solicitor friend Geo. Mounsey of Staple Inn, of the firm of Mounsey and Gray.)
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How To: Writing an master, Excellent Post-Event Wrap Up Report. Ed. Note: I originally composed this post as a resource for folks at my employer, Red Hat. Obviously, the Red Hat specific bits have been removed, e.g. my team can’t volunteer to help you edit your posts. Essays On Nelson Mandela Leadership. I’d always planned to open source this guide for thesis, the good of FOSS marketing, but simply hadn’t gotten around to doing so. Fortunately, the stellar interns from the Sahana Software Foundation provided me with a good reason to educational research get this done, as they’re looking at master thesis cover, doing a SahanaCamp in Hyderabad, India and were wondering what sort of essay western, data to thesis cover collect to report out after the why is it important to vote event. Many thanks to master thesis cover Somay and S.P. for the motivation to why is publish this post and to my employer for being awesome and encouraging me to share my work for the community’s good. Sharing the Meetup, Conference and Hackfest Love (Photo Credit: Flickr user 4nitsirk) Schedule time to thesis cover write and publish the educational leadership research papers report within 48 hours of the event. Block time on your calendar so it happens. Along with your text about thesis what you found most useful about the event, include photos and educational leadership, video or audio recordings, preferably embedded in the post.
Linking to these resources is also OK. Thesis Cover. Include important stats in your post that are relevant to the community attending the should college athletes to play essay event, e.g. number of attendees, number of master thesis, student attendees, number of committers, etc. Why Is. Make sure to thank the event organizers and master cover, sponsors in educational leadership papers, an appropriate fashion. Once your post is published, make sure to share it via whatever social media channels you like to use. If you do not use social media, let the master cover event organizers know about your post in case they’d like to use it in sex marriage, the post-event report outs or to add it to their event news page. Good Sample Post-Event Reports. Recap: OpenStack Meetup April 26: Features photos early in the post, includes video, thanks to sponsors, shout outs to master thesis key community folks in essays, attendance. Developer conference 2012 part III: Workshops, hackfests, and more: Features photo of audience very early in post without showing too many open seats, includes links to talk slides and videos, lots of master thesis cover, detail on what the presentation topics were.
Preparing to Write a Great Event Wrap Up Report. Schedule Time to Write the Report. A post-event wrap up report is most useful within days of the event, and it’s best if you can publish your piece within 48 hours of the event’s conclusion. A wrap up report should be published no more than two weeks after the event. At two weeks out, the news is a bit stale so do your best to aim for 48 hours after the event, with a week or less time being OK but not optimal. Essay. Schedule time on your calendar for writing, as it’s easy for this task to be deprioritized in the face of other needed work. Cover. Set aside time for writing or you’ll likely find you don’t get the writing done. Pro-Tip: Schedule time no more than 24 hours after the event to both write and antiquity irreverant reconstructing, publish your wrap up report. The fresher the news, the cover more readers you’ll have. Writing up a great event wrap up report means gathering data while at the event.
Take the time to write down a few notes about things that particularly impressed you during the conference or meet up. Don’t rely on your memory to same sex marriage analysis keep track of the things that stood out to you, and write down as many take aways from the event as possible. You will not use all of your notes, but the more detail you can later provide, the better. Pro-Tip: Most post-event wrap up reports include the master cover following items, so take notes accordingly. You may not use all of these details, but it is good to have them. Event overview, which you can likely harvest from the blog post announcing the event, the event “about” page on their website or from sites like lanyrd, meetup.com, etc. You don’t have to educational leadership research use the organizers’ description, but it is often a good starting point. Location of event, including thanks to whoever provided the space in the case of a meet up, e.g. “Red Hat graciously hosted the Boston Python Users Group meeting last Wednesday.” Number of attendees at master thesis cover, the event.
Some like to note the number of attendees from certain groups depending on the goals of the antiquity essay irreverant reconstructing particular community hosting the event, e.g. “The organizers were excited to see 25% of our attendees were newcomers.” or “We had more than 50 women join us at thesis, the conference, a 15% increase over last year.” or “I was particularly proud of the efforts to reach out to the student community in Prague, with more than 40 students attending. Half of the students had not yet entered university.” Thanks to the event sponsors. While you do not need to call out the names of all sponsors, it is best practice to essay give a shout out to your employer specifically if they were a sponsor. In the case of one or two sponsors, it is best practice to thesis name them and link to their home page in your thanks. In the case of an event with many sponsors, a thank you to them with a link to the event’s sponsors page will suffice. If a sponsor did something truly memorable and it important, appreciated, a specific thanks to that sponsor is always welcome. Pro-Tip: Thanking event sponsors, particularly one’s own employer, can be difficult to do without looking disingenuous.
The most important thing to remember is to disclose your relationship with your employer in the blog post to avoid accusations that you’re shilling. Consider the difference between these two thanks, both of which say basically the master cover same thing, but will likely be received by your audience very differently: I’m very pleased that my employer, Red Hat, treated everyone at should athletes to play persuasive essay, the Foo Bar Meeting to coffee and master cover, treats. On Nelson Mandela. We’re glad we could share a meal with all of you and thesis cover, provide a small bit of thanks for all of your contributions to FLOSS. Red Hat provided coffee and treats to everyone at the Foo Bar Meeting, which was totally awesome of why is to vote essay, them. Red Hat rules! Of course, you want to thesis cover ensure that you have permission to research take photos at the event. Confirm with the organizers if there’s a photography policy and master thesis, abide by it.
Some FLOSS folks request that all photos taken during their events be published under a Creative Commons license, others forbid photos at essays on nelson, their events entirely, others request that attendees make use of photographs taken by master, the conference photographer. Whatever the organizers request of you, honor those requests. Abiding by same sex marriage essay, the conference photo policy makes it much easier to ask for an exception later should you need to do so, e.g. “I notice this photo is thesis cover, copyright $EVENT, may I use it in my forthcoming blog post provided I give proper attribution?” Being a good FLOSS citizen also means obtaining permission from the subjects of your photos to capture their image. Some conferences provide pins or other garb to attendees who do not wish to antiquity civilization reconstructing western be photographed – keep an thesis cover, eye out for these indicators. If you don’t see one, politely ask to take a photo of your fellow attendees and let them know you may use it on your blog or publish it on Flickr, etc.. If they decline, respect their request. It is also best practice to essays on nelson mandela let folks take a look of the photo you have taken to master thesis cover make sure they are happy with their appearance in it, but this step is not required.
It definitely helps build good rapport with your fellow community members, so why not do it? In general, group photos that do not show faces in the audience are well received even in should college be paid persuasive, those communities that are “camera shy.” Get a good sense for your audience and photograph accordingly. Pro-Tip: Capture or use the highest possible quality imagery. Suboptimal photos from your cell phone camera are better than no photos at all, but not by much. If you don’t have a high quality camera, check the conference’s photo pool for imagery that may be better or ask a colleague to snap a pic or two on your behalf. Ensure whatever content you use is licensed so that you may use it or that you obtain permission from the copyright holder to make use of it. If session(s) from the event are recorded, it is always good practice to at least link to those talk recordings.
For a meet up or other event where only master cover, one talk was recorded, embed the recording in your post. For events where multiple sessions took place, choose your top N (3 maximum) and embed those recordings. Make sure that you introduce the recordings with sufficient text – which can be quite terse, but must be present – so that your readers understand why they ought to why is it important to vote essay take the time to watch the content. Pro-Tip: Some videos feature automatic closed captioning for the hearing impaired and master, still others provide text transcripts of the to vote audio/video recording. When versions of the content exist that provide these extra vectors of entry for your audience, ensure that you embed them or link to master them. Know How to Tag Your Post and Photos. Most event organizers these days are pretty good about leadership research letting folks know what tag(s) to master thesis use when sharing photos, dents/Tweets, Facebook messages and essays, blog posts. Make sure to note the tag(s) used and add to your photos and post. Pro-Tip: If the hashtag for the event isn’t prominently mentioned in the event guide or at the start of the meet up, don’t hesitate to ask the question at the start of the festivities. You’re not the only one wondering what tag(s) to thesis cover use.
If it’s too difficult to ask this question up front, see if the event already has a photo pool or Tweet stream. Replicate the tags in to vote essay, use for the conference tweet stream or photo pool. Pro-Tip: Consider using some sort of social bookmarking service to master thesis gather news and feedback from the why is to vote event. It’s possible that the event organizers have already set something up, so ask them if they have done so. If not, offering to set up the resource for them is a kind and wonderful thing to master do when you’re at an event run by leadership, community volunteers. Perhaps you saw an outstanding network diagram in a particular presenter’s talk or you noticed that a speaker did not plan to publish her slides. It’s best to ask the speaker at the event for these resources, then follow up on your request by email.
If you were not able to master cover make the papers request in person, make sure to send your request by email quickly so you can include the materials in thesis, your post and get the post published in a timely fashion. You will also likely find that other folks have written about the event and may have done write ups on sessions you missed. They also may have an civilization irreverant western, alternate perspective on master thesis, an aspect of the to vote essay event you particularly enjoyed. Include links to other write ups and reports in thesis cover, the blog post – even a simple list of antiquity essay, links is fine – and consider updating your post if you run across a particularly excellent write up of the event after you’ve published your report. Updating the blog post comments with additional details is a fine way to proceed, but folks are often less likely to read the comments section. Pro-Tip: Before publishing your report, take a moment to search identi.ca and Twitter using the event hashtag. This quick search will likely produce other write ups that you may wish to link to master thesis in your own post. The conference news aggregator or press page is also an excellent source of such material.
If you’re having trouble getting started, prepare an outline of your post. Start with the educational leadership research basics as mentioned in the “Take Good Notes” section in cover, your introductory paragraph, then expand from there. If you just hate writing – and that’s ok, many do – get as many points out onto a page as possible, then ask for why is to vote essay, help from a friend or colleague to organize your thoughts and content. A blank page is a tough place to start, so don’t expect what you compose to master thesis be immediately perfect. Pro-Tip: If you are having trouble writing and outlines are not the best way to organize your thoughts, try these approaches:
Just write. Don’t worry if it’s not perfect or even coherent at first. Structure, proper grammar, correct spelling, etc. can be taken care of reconstructing, later. Consider writing down the ten second pitch for master cover, the event and then writing to address the high points that support that summary. Talk about your experience at the event with a friend or colleague and ask them to jot down notes during your conversation. Let their notes become your outline. Alternatively, you may wish to use transcription software for this purpose. Joe Ottinger, my colleague at Red Hat who also works on the Open Source and Standards Team, has penned some more tips on civilization western, writing on his blog. This document largely assumes that you’ll be publishing your event wrap up post on your personal blog, but there are many outlets for such reports. Master Thesis Cover. The conference organizers may need help with wrap up reports due to post-event fatigue, so offering to help them with your post-event write up can be a welcome way for your post to mandela leadership get even wider exposure and to do a good deed for the community. The fine folks at opensource.com also publish post-event reports, so check out their guidelines to submit content.
You may also find that your wrap up report will be useful to other trade press outlets or blogs, so licensing your content so that folks can (re)use it increases the value of your creation. You may even find that said trade press outlet or blog would like to simply republish your post, which is master thesis, a great thing to civilization irreverant reconstructing western do if you’re open to it. Pro-Tip: Once you have written your post, make sure to share it using whatever social networking services you prefer to use, e.g. Master. identi.ca, Twitter, Facebook, Google+, etc. Make sure to also include the event tags when broadcasting via social media. If you’re not a social media user, the event organizers may want to help you share the write up more widely via their social media channels. Here are a few examples of well written post-event wrap up reports, provided as a source of inspiration if you’re having trouble getting started or just want to analysis essay get a sense of what a good post-event report contains. If you already read through them in cover, the tl;dr section at the start of this post, you should skip this bit. Jobs Essay. #128578; Recap: OpenStack Meetup April 26: Features photos early in the post, includes video, thanks to sponsors, shout outs to key community folks in attendance. Developer conference 2012 part III: Workshops, hackfests, and more: Features photo of audience very early in thesis cover, post without showing too many open seats, includes links to talk slides and videos, lots of detail on what the presentation topics were.
If folks have suggestions on how to improve this article, I welcome feedback in the comments section.