Measuring Cytotoxicity in HepG2 Cells

Measuring Cytotoxicity in HepG2 Cells Measuring cytotoxicity in HepG2 cells treated with cisplatin or 5-fluorouracil, via flow cytometry as a measure of effectiveness of the drugs at treating cancer Introduction Cytotoxicity is the quality of being toxic to cells, resulting in cell death (Rang et al. 2012). In the majority of therapeutics cytotoxicity is not a desirable characteristic. However, in the treatment of cancer many chemotherapy drugs, through cytotoxicity are designed to selectively induce apoptosis, a form of cell death (Rang et al. 2012). There are three main forms of cell death, these are: apoptosis, autophagy and necrosis. Apoptosis is the physiological process by which cells are destroyed in a controlled manner (Elmore 2007). There are four basic steps involved; the first is cell shrinkage, blebs begin to form and condensation of chromatin occurs. Next, membrane blebbing occurs and enzymes begin the process of nuclear fragmentation. In the late stages of apoptosis, the cell is broken down into apoptotic bodies which contain the cells components and nuclear fragments. The final stage is the phagocytosis of apoptotic bodies (Ulukaya et al. 2011). One of the main ways in which phagocytes recognise apoptotic cells is the presentation of phosphatidylserine (PS). Ordinarily, this phospholipid is found on the inner leaflet of the membrane bilayer, during apoptosis it translocates to the outer leaflet and binds to a PS receptor present on the phagocyte (Wu et al. 2006). In autophagy, targeted components such as organelles are isolated in an autophagosome; a form of vesicle with a lipid bilayer membrane. These autophagosomes are degraded by lysosomes (Kroemer, G et al. 2009). Necrosis is often thought of as uncontrolled cell death. However, there is an increasing amount of evidence that suggests it is regulated by transduction pathways and other mechanisms (Kroemer, G et al. 2009). It is caused by factors such as trauma, infection and toxins. It is characterised by the swelling of cells which causes the cell membrane to rupture, resulting in the release of intracellular components. These intracellular components can trigger a local inflammatory response. In contrast to apoptosis, the phagocytic stage does not occur in necrosis (Golstein, Pierre and Kroemer, Guido 2007). As a result, dead cells and their intracellular components remain at the site of necrosis, this build-up of debris may lead to conditions such as gangrene. Due to its detrimental effects, it’s preferential that necrosis is not produced by therapeutics. Cisplatin and 5-fluorouracil are chemotherapy agents, which both cause apoptosis by interfering with DNA via its replication and repair. Cisplatin crosslinks with purine bases in DNA which interferes with DNA repair mechanisms, this means that DNA damage is not fixed triggering apoptosis (Dasari and Bernard Tchounwou 2014). 5-flurouracil is an antimetabolite, it is metabolised into 5-fluorodeoxyuridine monophosphate (5-FdUMP) which is then incorporated into both DNA and RNA and also inhibits thymidilate synthase (Parker and Stivers 2011). HepG2 cells are derived from the hepatocellular carcinoma cells of a 15 year old, American, Caucasian, male and are epithelial in nature. They are commonly used as an in vitro model of human hepatocellular carcinoma (Costantini et al. 2013). Flow cytometry is a powerful tool that allows for the rapid differentiation and characterisation of particles including cells. A flow cytometer is made up of three main components: fluidics, optics and electronics. When a sample is injected into a flow cytometer, the fluidics system is responsible for producing a stream of single particles, this relies on the principle of laminar flow. This allows the particles to be interrogated by the laser beam individually. When a particle passes through the laser beam it scatters light. The amount of forward scatter is proportional to the size of the cell. The side scatter is proportional to the complexity of the cell. If a fluorophore is present, it can be excited by a specific wavelength of light. It will then undergo a Stokes shift and a different wavelength of light will be emitted which can be measured by the electronics of the flow cytometer (Macey 2007, Maxwell and Hannon-Fletcher 2009). The fluorophore can be conjugated with proteins suc h as annexin V which have a known high affinity to specific molecules present on the cell surface. Annexin V is a 35.7kDa, calcium sensitive, phospholipid binding protein. It has a high affinity to phosphatidylserine. The binding of fluorophore labelled annexin V is used as a common measure of apoptotic cells by flow cytometry (van Genderen et al. 2008). Aims To measure the effectiveness of two anti-cancer drugs by measuring the level of apoptosis in a population of HepG2 cells after being treated with cisplatin (15 µg/mL) or 5-fluorouracil (25 µg/mL) for 48 hours and compare those to a population of untreated cells via flow cytometry. Objectives To count and fix a population of HepG2 cells before staining them with fluorescently labelled Annexin V in preparation for analysis using a flow cytometer. To measure the level of fluorescence in 5000 treated and untreated cells using a flow cytometer as well as measuring the minimal and maximal fluorescence in a negative and positive control. Based on the fact the fluorescence is directly proportional to apoptosis determine which treatment is significantly better and provide evidence from the literature as to why this is the case. Methods Please refer to laboratory schedule (Hatch 2014). Following the collection of data descriptive statistics and a one-way ANOVA test were carried out the results of which can be found in the appendix. Results The negative control was used to identify if there was any background fluorescence. In table 1 (found in the appendix) the mean fluorescence for the negative control was 144. Therefore, fluorescence that is significantly higher than this level can be attributed to the excitation of the fluorophore (Alexa 488). Figure 1 Cytotoxicity of HepG2 cells measured as fluorescence using flow cytometry. The bars are means of triplicates, the standard error has been used for the error bars. *statistically significant compared to control (blue for negative, red for positive), p In figure 1, a greater level of fluorescence was seen for the samples treated with cisplatin and 5-fluorouracil compared to that of the untreated sample. Cisplatin produced the greatest level of fluorescence of the two treated samples. Discussion The data used was a class set, some of the data provided was produced by those running the laboratory session as some samples did not contain a pellet which was needed to run the flow cytometry analysis. The five sets of data were compared with one another, it was found that the differences were statistically significant (p As mentioned in results, cisplatin showed a greater mean fluorescence compared to 5-fluorouracil, Results were 726 and 540 respectively. Seeing as fluorescence is directly proportional to phosphotidylserine expression, which in turn is a direct measure of apoptosis. It can be assumed that cisplatin produced the highest level of apoptosis and is most cytotoxic to HepG2 cells. Although a higher concentration of the drugs were used (150 µg/mL) this result has also been seen in the literature (Brenes et al. 2007). Although it was not stained with annexin V, the negative control showed a mean fluorescence of 144; this is due to auto fluorescence of the cells. The fluorophore which was conjugated with annexin V was Alexa 488. It fluoresces at a wavelength of 488nm, this is within the green part of the spectrum. It is well documented that molecules found within all cells, including hepatocytes also fluoresce at a green wavelength, the most notable of these are flavins which fluoresce at around 560nm (Croce, Anna Cleta et al. 2007, Croce, Anna C et al. 2014). FAD (flavin adenine dinucleotide) is an example of a flavin and is a redox cofactor used in metabolism. HepG2 cells are a liver carcinoma cell line. Due to their cancerous nature there would be a high level of metabolism within these cells and hence, a high level of FAD. The low level of fluorescence seen in the untreated sample is expected as there should be no apoptosis induced by cytotoxicity. Therefore, it is not surprising that the difference between the fluorescence of the untreated sample and the negative control and the treated samples is less statistically significant. In this experiment, only one stain was used in this experiment (Annexin V), this allows for the identification of cells that are cytotoxic. To evaluate the true effectiveness of a chemotherapy drug, the cell viability and necrosis is also needed. Necrosis and late stage apoptosis can be measured using propidium iodide (PI). PI binds to nucleic acids and is impermeable to intact membranes, it is often used in combination with annexin V as it fluoresces red and there is little overlap between the wavelengths of red and green light. Therefore, compensation is not needed after flow cytometry has been carried out. It has been observed in the literature that the cytotoxicity of 5-fluorouracil is dependent on both dose and exposure time (Okamura et al. 2008). We only looked at one dose and exposure time for both drugs. It has also been observed that the use of cisplatin in combination with 5-fluorouracil or other chemotherapy drugs has a synergistic and additive effect in hepatoma cell lines (Kogure et al. 2004), both of these could be investigated further is the experiment were carried out again. Conclusion In conclusion, cisplatin was shown to produce a higher level of apoptosis in HepG2 cells than 5-fluorouracil. However, to fully evaluate the effectiveness of an anti-cancer agent, measures of viability and necrosis are also needed. The differences between the two drugs may be due to the different mechanisms by which the drugs work. The introduction of 5-dUMP into DNA and RNA is a more subtle mechanism compared to the cross-linking of DNA via cis-platin. This cross-linking may be detected by cells more readily resulting in higher levels of apoptosis. As HepG2 cells were used, these results may only be the case for hepatocellular carcinomas. References Brenes, O., Arce, F., Gà ¤tjens-Boniche, O., and Dà ­az, C. (2007) â€Å"Characterization of Cell Death Events Induced by Anti-Neoplastic Drugs Cisplatin, Paclitaxel and 5-Fluorouracil on Human Hepatoma Cell Lines: Possible Mechanisms of Cell Resistance.† Biomedicine pharmacotherapy = Biomà ©decine pharmacothà ©rapie 61 (6), 347–55 Costantini, S., Di Bernardo, G., Cammarota, M., Castello, G., and Colonna, G. (2013) â€Å"Gene Expression Signature of Human HepG2 Cell Line.† Gene 518 (2), 335–45 Croce, A.C., Ferrigno, A., Santin, G., Piccolini, V.M., Bottiroli, G., and Vairetti, M. (2014) â€Å"Autofluorescence of Liver Tissue and Bile: Organ Functionality Monitoring during Ischemia and Reoxygenation.† Lasers in surgery and medicine 46 (5), 412–21 Croce, A.C., De Simone, U., Vairetti, M., Ferrigno, A., and Bottiroli, G. (2007) â€Å"Autofluorescence Properties of Rat Liver under Hypermetabolic Conditions.† Photochemical photobiological sciencesà ¢Ã¢â€š ¬Ã‚ ¯: Official journal of the European Photochemistry Association and the European Society for Photobiology 6 (11), 1202–9 Dasari, S. and Bernard Tchounwou, P. (2014) â€Å"Cisplatin in Cancer Therapy: Molecular Mechanisms of Action.† European journal of pharmacology 740, 364–378 Elmore, S. (2007) â€Å"Apoptosis: A Review of Programmed Cell Death.† Toxicologic pathology 35 (4), 495–516 Van Genderen, H.O., Kenis, H., Hofstra, L., Narula, J., and Reutelingsperger, C.P.M. (2008) â€Å"Extracellular Annexin A5: Functions of Phosphatidylserine-Binding and Two-Dimensional Crystallization.† Biochimica et biophysica acta 1783 (6), 953–63 Golstein, P. and Kroemer, G. (2007) â€Å"Cell Death by Necrosis: Towards a Molecular Definition.† Trends in biochemical sciences 32 (1), 37–43 Hatch, E. (2014) â€Å"M19BMS Laboratory Schedule†. Coventry University Kogure, T., Ueno, Y., Iwasaki, T., and Shimosegawa, T. (2004) â€Å"The Efficacy of the Combination Therapy of 5-Fluorouracil, Cisplatin and Leucovorin for Hepatocellular Carcinoma and Its Predictable Factors.† Cancer chemotherapy and pharmacology 53 (4), 296–304 Kroemer, G., Galluzzi, L., Vandenabeele, P., Abrams, J., Alnemri, E.S., Baehrecke, E.H., Blagosklonny, M. V, El-Deiry, W.S., Golstein, P., Green, D.R., Hengartner, M., Knight, R.A., Kumar, S., Lipton, S.A., Malorni, W., Nuà ±ez, G., Peter, M.E., Tschopp, J., Yuan, J., Piacentini, M., Zhivotovsky, B., and Melino, G. (2009) â€Å"Classification of Cell Death: Recommendations of the Nomenclature Committee on Cell Death 2009.† Cell death and differentiation 16 (1), 3–11 Macey, M.G. (2007) Flow Cytometryà ¢Ã¢â€š ¬Ã‚ ¯: Principles and Applications. ed. by Macey, M.G. Humana Press, Inc Maxwell, P. and Hannon-Fletcher, M. (2009) â€Å"Advanced Techniques in Diagnostic Cellular Pathology†. in Advanced Techniques in Diagnostic Cellular Pathology. Hoboken: Wiley, 69–97 Okamura, M., Shimada, J., and Sakagami, H. (2008) â€Å"Comparative Analysis of Cell Death Induction by Cisplatin and 5-FU in Human Oral Squamous and Hepatocellular Carcinoma Cell Lines†. Anticancer research 260, 253–259 Parker, J.B. and Stivers, J.T. (2011) â€Å"Dynamics of Uracil and 5-Fluorouracil in DNA.† Biochemistry 50 (5), 612–7 Rang, H., Dale, M., Ritter, M., Flower, R., and Henderson, G.. (2012) â€Å"Anticancer Drugs†. in Rang and Dale’s Pharmacology. 7th edn. Elesevier, 676–677 Ulukaya, E., Acilan, C., and Yilmaz, Y. (2011) â€Å"Apoptosis: Why and How Does It Occur in Biology?†. Cell biochemistry and function 29 (6), 468–80 Wu, Y., Tibrewal, N., and Birge, R.B. (2006) â€Å"Phosphatidylserine Recognition by Phagocytes: A View to a Kill.† Trends in cell biology 16 (4), 189–97 Appendix Table 1: Table showing mean fluorescence for each sample where 1.00 represents the negative control, 2.00 represents the positive control, 3.00 the untreated sample, 4.00 the sample treated with cisplatin, 5.00 with 5-fluorouracil N Mean Std. Deviation Std. Error 95% Confidence Interval for Mean Minimum Maximum Lower Bound Upper Bound 1.00 3 144.3333 11.93035 6.88799 114.6967 173.9700 136.00 158.00 2.00 3 1866.6667 32.65476 18.85323 1785.5477 1947.7856 1836.00 1901.00 3.00 3 214.3333 10.59874 6.11919 188.0046 240.6621 203.00 224.00 4.00 3 864.0000 20.22375 11.67619 813.7614 914.2386 849.00 887.00 5.00 3 540.6667 51.93586 29.98518 411.6508 669.6825 484.00 586.00 Total 15 726.0000 647.67772 167.22967 367.3280 1084.6720 136.00 1901.00 Table 2 Descriptive statistics where 1.00 is the negative control, 2.00 is the positive control, 3.00 is the untreated sample, 4.00 is the sample treated with cisplatin and 5.00 is the sample treated with 5-fluorouracil. N Mean Std. Deviation Std. Error 95% Confidence Interval for Mean Minimum Maximum Lower Bound Upper Bound 1.00 3 144.3333 11.93035 6.88799 114.6967 173.9700 136.00 158.00 2.00 3 1866.6667 32.65476 18.85323 1785.5477 1947.7856 1836.00 1901.00 3.00 3 214.3333 10.59874 6.11919 188.0046 240.6621 203.00 224.00 4.00 3 864.0000 20.22375 11.67619 813.7614 914.2386 849.00 887.00 5.00 3 540.6667 51.93586 29.98518 411.6508 669.6825 484.00 586.00 Total 15 726.0000 647.67772 167.22967 367.3280 1084.6720 136.00 1901.00 Table 3 ANOVA analysis carried out on the data Sum of Squares df Mean Square F Sig. Between Groups 5863955.333 4 1465988.833 1655.612 .000 Within Groups 8854.667 10 885.467 Total 5872810.000 14 Table 4 Ad-hoc test Tukeys range tests and LSD showing statistical significance between groups. All differences between groups are statistically significant (p Multiple Comparisons Dependent Variable: Fluorescence (I) Treatment (J) Treatment Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval Lower Bound Upper Bound Tukey HSD 1.00 2.00 -1722.33333* 24.29632 .000 -1802.2945 -1642.3722 3.00 -70.00000 24.29632 .094 -149.9612 9.9612 4.00 -719.66667* 24.29632 .000 -799.6278 -639.7055 5.00 -396.33333* 24.29632 .000 -476.2945 -316.3722 2.00 1.00 1722.33333* 24.29632 .000 1642.3722 1802.2945 3.00 1652.33333* 24.29632 .000 1572.3722 1732.2945 4.00 1002.66667* 24.29632 .000 922.7055 1082.6278 5.00 1326.00000* 24.29632 .000 1246.0388 1405.9612 3.00 1.00 70.00000 24.29632 .094 -9.9612 149.9612 2.00 -1652.33333* 24.29632 .000 -1732.2945 -1572.3722 4.00 -649.66667* 24.29632 .000 -729.6278 -569.7055 5.00 -326.33333* 24.29632 .000 -406.2945 -246.3722 4.00 1.00 719.66667* 24.29632 .000 639.7055 799.6278 2.00 -1002.66667* 24.29632 .000 -1082.6278 -922.7055 3.00 649.66667* 24.29632 .000 569.7055 729.6278 5.00 323.33333* 24.29632 .000 243.3722 403.2945 5.00 1.00 396.33333* 24.29632 .000 316.3722 476.2945 2.00 -1326.00000* 24.29632 .000 -1405.9612 -1246.0388 3.00 326.33333* 24.29632 .000 246.3722 406.2945 4.00 -323.33333* 24.29632 .000 -403.2945 -243.3722 LSD 1.00 2.00 -1722.33333* 24.29632 .000 -1776.4689 -1668.1978 3.00 -70.00000* 24.29632 .016 -124.1356 -15.8644 4.00 -719.66667* 24.29632 .000 -773.8022 -665.5311 5.00 -396.33333* 24.29632 .000 -450.4689 -342.1978 2.00 1.00 1722.33333* 24.29632 .000 1668.1978 1776.4689 3.00 1652.33333* 24.29632 .000 1598.1978 1706.4689 4.00 1002.66667* 24.29632 .000 948.5311 1056.8022 5.00 1326.00000* 24.29632 .000 1271.8644 1380.1356 3.00 1.00 70.00000* 24.29632 .016 15.8644 124.1356 2.00 -1652.33333* 24.29632 .000 -1706.4689 -1598.1978 4.00 -649.66667* 24.29632 .000 -703.8022 -595.5311 5.00 -326.33333* 24.29632 .000 -380.4689 -272.1978 4.00 1.00 719.66667* 24.29632 .000 665.5311 773.8022 2.00 -1002.66667* 24.29632 .000 -1056.8022 -948.5311 3.00 649.66667* 24.29632 .000 595.5311 703.8022 5.00 323.33333* 24.29632 .000 269.1978 377.4689 5.00 1.00 396.33333* 24.29632 .000 342.1978 450.4689 2.00 -1326.00000* 24.29632 .000 -1380.1356 -1271.8644 3.00 326.33333* 24.29632 .000 272.1978 380.4689 4.00 -323.33333* 24.29632 .000 -377.4689 -269.1978 *. The mean difference is significant at the 0.05 level.

Objections Of The Croquette And Hoop Skirt :: essays research papers

While reading selections from The Tatler and The Spectator, two subjects that I found most interesting were that of the coquette and her place in society, and also that of fashion, in particular, the hoop skirt. The Webster’s Dictionary defines a coquette as "a woman who flirts." One of the reasons why ‘she’ appears so frequently in these essays is because these men sense a danger in them. The croquette is a figure of self-sufficiency and 18th century women were not supposed to be self-sufficient. Women of the 18th century were supposed to be ‘sugar and spice and everything nice’ and, above all, dependent on men. When a croquette comes into the picture, however, the men are the ones who can’t handle themselves. The croquette is a flirtatious woman who knows very well the powers of her sex and uses them to her advantage. Just by looking and acting a certain way around men, she learns how to ‘control’ them to a certain degree. Although the men realize what she is doing, they are powerless to stop it. It is also apparent to these men that women are able to be deceitful, and that the coquette may not be the beginning or the end of th is deceitfulness. And they find this to be very dangerous to the ideas of knowing your respective places in their society. Addison and Steele seem to think that the croquette is not only a lady who tends to flirt with every man she comes into contact with, but any young lady who outwardly makes every attempt to find a suitable marriage partner. In No. 110, of The Tatler (478-482), Addison speaks of a certain young woman being accused of catching the lust of several young men and breaking their hearts. He explains how she seems very innocent when proposed with the accusation and that this is how she obtains that she is not in the wrong. The way he words this essay makes me think that he believes that it is criminal for a woman to use the "Motion of her Eyes and Turn of her Countenance"(p 479) to capture the eyes of a possible suitor. Because a woman is able to use her ‘special powers’ in such a way is deceitful and therefore may lead to other wrongful doings which, in turn, disturbs the way Addison believes the opposite sexes serve opposite sides of society.

Pre 1914 Poetry William Blake Essay

These poems ‘Holy Thursday (experience)’ and Holy Thursday (innocence) are set on Ascension Day in a service in St. Paul’s church. This was a special occasion for the orphans who came from London Charity Schools. The ‘Holy Thursday (innocence)’ poem can be interpreted in two different ways. The impression we get at first is that the orphans are treated well and they lead happy lives but after reading ‘Holy Thursday (experience)’ you start to realise that there is a negative way of understanding the same poem. This view shows the orphans to be mistreated and very unhappy. The phrase ‘their innocent faces clean’ suggests children that are being well looked after rather than being abandoned and roaming the streets of London. There is a suggestion that the children have companions, are well behaved and have a sense of order by the line ‘the children walking two by two’ This is further added to by the phrase ‘In red, blue and green’ which implies that they were dressed in bright, smart uniforms rather than rags. The children have angelic guardians to nurture and protect them, as implied by the lines ‘Grey-headed beadles walked before’ who have ‘wands’ are described ‘as white as snow’ which makes us feel that these are enchanted guardians who are pure and magical. Another phrase that adds to this is the sentence ‘Wise guardians to the poor’. There is further reference to the good work that the guardians are doing when William Blake uses the term ‘Multitudes of lambs’ implying the guardians are shepherding and guiding innocent creatures. The idea of lambs conjures up the image of animals all grouped together making sure that they are all safe. The orphans are referred to as flowers in the second paragraph, implying delicate, natural and beautiful. Flowers signify peace implying that the children are good-natured. ‘Seated in companies they sit’ like good well-behaved pupils in a school, to say their nature is calm and peaceful rather than loud and rowdy. Their god-fearing nature is implied by the words ‘raising their innocent hands’ probably referring to prayer as they are hopeful and eager. In the last paragraph William Blake is saying the children enjoy going to church, praying and singing hymns as †like a mighty wind they raise to heaven the voice of song.Overall the poem has a lively rhythm with pace to give it a beat and fluidity. Now I am going to analyse ‘Holy Thursday (experience)’ poem. In the first stanza Blake describes England as a country which is ‘rich and fruitful’. This would appear to be his own experience of life in England but this statement can be interpreted in different ways. Blake could have meant that England is rich in that there is fruit and food but it is poor because of the amount of orphans. He uses ‘holy’ to infer that England is a Christian Country and asks why babies should be reduced to misery and fed and looked after by people who don’t care for them ‘Cold and usurious hand?’. In the second stanza he asks three rhetorical questions. We know ‘the trembling cry’ isn’t a song and that whatever is crying is probably alone and maybe crying out for help. ‘Can it be a song of joy?’ Perhaps it could be a song of joy for the favoured few who live in the rich and fruitful land but for the many poor children roaming the streets of London it isn’t. ‘It is a land of poverty’. In the previous poem ‘Holy Thursday (innocence)’ he says that the children ‘raise to heaven the voice of song’. He obviously believes that songs can lift a spirit and in ‘Holy Thursday (experience)’ it hurts him, that there are no songs of joy going heavenwards form children who are so pure. Normally to see how rich a country a country is you measure the amount of wealth the country but here Blake is measuring the happiness by asking if their singing which is usually a sign of happiness from children. The third stanza describes their happiness in terms of the climate. Their lives are like a place where the; ‘sun does never shine. And their fields are bleak and bare.’ In the third line he contrasts their journey through life with that of Christ’s crown of thorns. The image that this reflects is of a painful way through life. And the next line is echoed in a later work by C.S Lewis who uses the term eternal winter to mean a place, like Siberia, that is unbearably sad and where happiness does not exist. This metaphor makes us aware that there is never any joy of warmth in their lives and that emotionally they are completely bereft and emotionally starved of love. In the last paragraph he again refers to the environment and the weather to describe a situation where everything would be all right and ‘Babe cam never hunger there’. This completely fails to show the real reason why those children are poor. Rain and sunshine won’t get them out of the grinding poverty that they are in. It is simply used as a metaphor to change the children’s situation from eternal winter to dry warm summer in which they would appear to be happy. Throughout the poem there is a lack of colour and description so it is difficult to conjure up any image other than of a grey bleak landscape, where grey people and grey children exist in a society that doesn’t value them. In ‘Holy Thursday (innocence)’ he uses descriptive words such as ‘clean’, ‘two by two’, ‘red’,’ blue’, ‘green’ and ‘as white as snow’ to conjure up a picture of London that is quite different. Blake also appears to be attacking the church in other poems for its splendour and wealth but also its lack of humanity and awareness of the ‘multitudes of lambs’ which could be led to the slaughter and misery of poverty. Reading the ‘Holy Thursday (experience)’ makes you reconsider the poem ‘Holy Thursday (innocence) and its approach. In a negative this is my interpretation.In the first stanza it is implied that the thousands of orphans are being made to scrub their faces clean so much that it hurts. This cleanliness of the children is only a faà ¯Ã‚ ¿Ã‚ ½ade to give a good impression when the phrase ‘their innocent faces clean’ appears. This implies that the children are disciplined and regimented. This makes a good impression on the carers. This is also show ‘in red and blue and green’ because it shows that they are being made to wear a uniform. Being forced to wear uniforms means that the orphans also lose their individuality. ‘Grey-headed beadles walked before’ could show that these ‘carers’ are bad people who order the children around and make them walk ‘two and two’ like in the military. This also implies that these bad people are egotistical because they only look after themselves and they might only be looking after the children for extra money. These military officers have canes to beat the children with as it says ‘with wands as white as snow.’ This idea of the children being part of a military force is backed up by the quote ‘seated in companies they sit.’ Because the army is sectioned off into companies, they stand in a certain order and they are very obedient. ‘These flowers of London town’ implies that the children are innocent and pure but like flowers they will eventually die. Flowers are also vulnerable and easily ruined. The comparison between the groups of children and the ‘multitudes of lambs’ implies that the orphans like the lamps, group together like pure innocent creatures. The image of the lamb also stands for the idea of vulnerability and sacrifice. Like the lambs the orphans are forced to do what the carers tell them to do, and may face an early death as victims of a cruel world. ‘Thousands of little boys and girls’ suggests that there are any poor orphans who are homeless. This shows that there is a large scale of poverty. The orphans plead for help by ‘raising their innocent hands.’ ‘Like a mighty wind†¦voice of song’ implies that the wind is like a destructive hurricane ready to sweep their lives away. Ironically the ‘wise guardians of the poor’ are there to look after the orphans for the money and are not concerned about the orphans at all. The rhythm of the poem in this negative view is a like a strict military march.

Perception Of Public Health Professionals - 834 Words

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Health care television shows have portrayed nurses as handmaidens to doctors, ‘naughty’, battle-axes’, sex objects, self-sacrificial angelsRead MoreA Hard Day s Night1008 Words   |  5 Pagesas the most honest job sixteen times in the last seventeen years. Saad also reports that nurses have no other serious competition to outrank them. Despite being America’s most trustworthy profession, the public does not view nurses with the same level of respect or distinction as other professionals. Historically a female profession, nurses have been known to be caring and kind and work solely under the doctor’s orders. Over the years, media has negatively influenced the way the people perceive nursesRead MoreAnnotated Bibliography : Dependa bility And Transferability966 Words   |  4 Pagesanalyzed the data using a â€Å"wholistic, selective and detailed line-by-line approach† by Van Manen. The themes that emerged were matched with verbatim passages; it was categorized into two perspectives from the consumers of the health care (patient and family) and the providers’ of health care. Descriptors of emerging themes were further enumerated and correlated between two perspectives. It is auditable in a sense that it can be subscribed to and followed by other researchers by providing rationales of theRead MoreThe Changing Context of Nursing Essay examples1635 Words   |  7 PagesThe image of nursing is dynamic, changing from the late 1800’s to present, the images are made from individual’s perceptions by what is seen in the media, how education is taught in nursing and historical figures (Daly, Speedy, Jackson, 2014). Historical figures mainly include Florence Nightingale and her, self-sacri fice and the nurses who transformed education (Fee Garofalo, 2010, p. 1591; Allen, 2010, p. 35). Education has moved forward by the enhancing strategies of teaching student nursesRead MoreWhy Smoking Is A Public Health Problem901 Words   |  4 Pagestargeting college students by opening across the street from main campus, to them this is just business, while to others this is an important public health concern. Why it is a public health problem? Hookah smoking is a public health problem that is growing rapidly nationwide, especially among young adults and college students.1 Hookah consumers have a perception that it is less harmful than smoking cigarettes, while evidence show that hookah smoke contains many of the same harmful toxins as cigaretteRead MoreNursing Professional Identity Essay1693 Words   |  7 Pagesdevelopment of nursing on contemporary representations of the nursing profession. It examines the professional identity of nursing and further discusses the contribution of modern nursing, social, cultural and political factors that influences the professional identity of a nurse. The essay also looks into the professional regulations and the role of the nurse’s and midwifery Council (NMC) in the protection of the public. Finally, this essay will discuss nursing education, the media, stereotype, and their impactsRead MoreNursing Image and Media1449 Words   |  6 PagesNursing the Silent Profession and Med ia’s influence The nursing image has become a major issue in the society as people have different perception about nursing. Some believe that nurses do their duties out of kindness. This has influenced the nursing image as most people do not see nursing as a good profession. Only few people in the society see nursing as an important profession and consider the qualification of the nurses (Younge amp; Niekerk, 2004). This has led to shortage of nurses in theRead MoreThe Issues Surrounding The Idea Of Immunization Essay1576 Words   |  7 Pagesinjections will not increase immunity levels and the possibilities of negative reactions are too great of risk. This conflict of perceptions is related to the way people perceive cognitive limitations, the psychometric paradigm and the axiomatic paradigm; these include factors such as dread, control and voluntariness. A great influence on these perceptions is health professionals’ communication strategies, who target social media, advertisements and re ports. The concept of risk means different thingsRead MoreAbbreviations. Anm Auxiliary Nurse Midwife. Chc Community1152 Words   |  5 Pages ABBREVIATIONS ANM Auxiliary Nurse Midwife CHC Community Health Center CNE Continuing Nursing Education INC Indian Nursing Council KNMC Kerala Nurses and Midwives Council MO Medical Officer PHC Primary Health Center TNAI Trained Nurses’ Association of India GNM General Nursing and Midwifery JPHN Junior Public Health Nurse NCD Non Communicable Disease JHI Junior Health Inspector GO Government Order DNO District Nursing Officer ADNS Additional Director of Nursing Services NSRead MoreEvaluating The Attitude Of Healthcare Professionals Essay1110 Words   |  5 Pagesof this study was to systematically review the attitude of healthcare professionals towards professional accreditation and to also know if there are any differences in their perception and in understanding the accreditation standard. Healthcare professionals had a doubtful attitude towards accreditation. Owners of hospitals indicated that accreditation had the potential of being used as a marketing tool. Healthcare professionals viewed accreditation programs as bureaucratic and demanding. There was