Indian Removal Act Essay Example for Free

Indian Removal Act Essay Known as having adopted an Indian child as his son, Andrew Jackson was quite fond of the Indian race; however, with pressure to expand westward, he needed to transfer the Indians farther west and soon became their worst enemy. Andrew Jackson’s Indian Policy was to move the Indians westward as peacefully as possible, for the tribes that stayed in the East Coast were annihilated. Also, moving them West will help them live longer, and there is a fair exchange for the tribes moving. Another important component is the gain of Western lands and the addition of American power; this will add on to America’s size and increase America’s authority. Jackson validates his actions by saying he will pay for the land the Indians inhabit, pay for their long journey West, also known as the Trail of Tears, and support them a little while their settling in. President Jackson also said if they move west, they will enjoy sovereignty forever. Jackson made the point that with the Indians gone there will be less conflict between them and state governments, it will allow for prosperous populations and cities to flourish, and the free land will add another line of defense for America. President Jackson’s attitude toward the Indians in his speech reveals that he wants the Indians gone so America can gain land and grow stronger. He uses words like â€Å"Red Men† and â€Å"Savages† to describe them, so they seem like outcasts. He makes it sounds like the Indians are a nuisance and if they move, they will prosper as a people. Jackson says, â€Å"†¦perhaps cause them gradually†¦to cast off their savage habits and become an interesting, civilized, and Christian community.† Overall, Jackson wants the Indians gone for his own benefit. He refers to them as uncivilized, uninteresting, and having savage habits, and was proving that he is just looking for a gain for his country, not the native people who have lived there forever.

Effects of Static and Dynamic Culture Conditions

Effects of Static and Dynamic Culture Conditions Tissue engineering has been investigating the properties of scaffolds and cell culture conditions for better cell attachment, viability and proliferation. This study compares the two cell culture conditions: Static and spinner flask / dynamic cell conditions over a period of 7 days on polyglycolyic acid. The scaffolds were statistically seeded by mouse dermal 3T3 fibroblast in static culture method and on other hand seeded scaffolds were transferred to spinner flask at approx.60 rpm in dynamic culture method. Significant improvement in cell viability was not observed in both the conditions after 7 days of culturing. The cells adhesion successfully took place and expressed cytoskeleton ÃŽÂ ²-actin in both the methods but achieving maximum distribution of cells on the scaffold in dynamic method. This study reports that static culture method could produce increase in cell number approximately six times more after 7 days of culture i.e. from 1.2 x 10à ¢Ã‚ Ã‚ µ ( ±0.1610à ¢Ã‚ Ã‚ µ) cells to 6.3 x10à ¢Ã‚ Ã‚ µ ( ±110à ¢Ã‚ Ã‚ µ) cells. Surprisingly, instead of enhancing the growth of 3T3 fibroblast cells in dynamic condition, they seems to be probably undergoing cell death/loss as reported by alamar blue, hoechst DNA assays, toludine blue and western blot. Overall, static condition favoured the cell adhesion, proliferation and ÃŽÂ ²-actin expression gradually with days and produced better reproducible data compared to dynamic condition. The techniques involved in dynamic culture method needs to be more carefully investigated and improved further to draw a strong conclusion. The aim of the study is to implement the principles of fundamental techniques in tissue engineering in culture method on the three dimensional polyglycolic acid (PGA) scaffolds seeded with 3T3 fibroblast. To compare and contrast the effects on cells in spinner flask or dynamic culture condition method with the static culture condition method by observing and analysing on factors like cell adhesion, distribution, proliferation, viability and expression of cytoskeleton after culturing in the same system for 7 days using alamar blue, hoechst 33258 DNA assays, toludine blue staining and western blot analysis. Tissue engineering is a multidisciplinary field which aims in developing new approaches for functional substitutes applicable in restoration of damaged or injured tissues. These substitutes are complex constructs of living cells, bioactive molecules and three dimensional porous scaffolds, which supports cell attachment, proliferation and differentiation. Therefore, its main objective to achieve in therapy is to form a living tissue from small population of mammalian cells. For this, the ideal tissue engineering strategy so far has remained to develop tissue by seeding the specific population of cells on three-dimensional constructs which not only provides a structural support to cell mass but also can effectively influence cell attachment, growth and differentiation either by incorporation of adhesion molecules or controlled release on bioactive molecules from the scaffold. After seeding of cells onto the 3-D scaffolds construct, the cells starts proliferating which results in deposi tion of extracellular matrix components and biodegradation of scaffolds. The latter makes the porous construct of scaffold more solid 3-D. Several other factors affect the 3D tissue growth including scaffold design, seeding method and the culture condition methods. Studies have reported that high degree of cell attachment to biocompatible and biodegradable particles, while avoiding aggregate formation can be achieved using poly co-glycolic acid (PGA) scaffold of 50-100mm spherical size fabricated by electro-spinning technique. This method provides reliable, reproducible and well-characterized PGA scaffold. The surface chemistry of the scaffold helps to determine the particle size, shape, morphology and distribution. Depending on the experiments, surface modifications are performed like formation of poly l-lactide-coglycolide (PLGA) via ring opening polymerisation and fibronectin coating to scaffolds. However, it is not the part of standard protocol. Depending on the size, the required cell density for maximum attachment may differ to obtained optimal cell attachment. The seeding is usually done using the cell suspension of a particular seeding density which allows for maximum dispersion of cells and well integration into the pores of the scaffolds. But for therapeutic purposes, however, this strategy is not sufficient enough to result in an overall improvement in conditions due to severe tissue damages. This can be overcome only by achieving relatively high degree of cell attachment to the micro-particle. Several factors and parameters influence the cell adhesion like the curvature of the particles, the particle material, the electrostatic charge of the particles, the surface motif of the particles, the interaction between cell and particles, the number of cells in the tissue culture and type of cell culture method implemented. It is also important to obtain homogenous cell adhesion to the scaffolds and avoiding clumping which will lead to the formation of cell-particle aggregates. This will prevent cells from appropriate uptake of nutrient from the media and hinder their subsequent growth. The mammalian cells are usually cultured in static or bioreactors condition. Here in this study, spinner flask system is employed which is also a kind of bioreactor as it provides the 3D environment. It is a flask provided with magnetic rod which keeps rotating constantly at specified speed. The nature of growing cells requires such dynamic condition to mimic the environment similar inside the body which gives sufficient nutrient supply, waste exchange, enhances ECM and gap junction formation, and cell-cell interaction. Most importantly it also helps maintain the cells differentiated in 3D which is needed for tissue formation. This characteristic is not maintained by static culture method. Hence, many 3D culture methods have been developed such as perfusion chambers, rotary vessels and commercial perfused bioreactors with improved capacity for mass transport of nutrients and waste product. They help in formation of relatively good quality of tissue by more enhanced cell differentiati on and also maintaining in that state. The static culture method used in this study, tissue culture plastic with seeded scaffolds remains untouched in the incubator. But with static culture, alternative shaking on a shaker and resting can also be employed to provide better supply of nutrient through media. The attachment characteristic of ECM proteins such as laminin, will also depend upon the cell type used. There are particular conditions needed to be optimised with each cell type. Most of the tissue engineering experiments uses 3T3 fibroblast only to optimise the cell culture condition where there is optimum cell adhesion is obtained before using the actual stem cell of interest. This is because, 3T3 fibroblast are known to easily attach to any surfaces due to presence of the high density of integrins on their cell surface. This will not only enhance the cell attachment but will also give maximum possible interaction with the particle. Cells that have spread on the particles exhibit a clear halo of cytoplasm surrounding their nucleus after the rearrangement of their actin skeleton. The attachment and spreading of cells to a substrate surface is often seen as a basic characteristic, but is, in fact, the initial process that subsequently influences and regulates cell growth, survival, migration and differentiation. In addition, cell-to-substrate interaction, mediated by integrins, also influence cell behaviour and signalling pathways leading to modifications in upstream and/or downstream cellular activity. Thus, a desirable substrate should allow sufficient and optimal cell attachment and spreading characteristics to occur. The 3T3 fibroblast media is used in which DMEM supplemented with 10% FCS enhances the cell attachment as the serum is highly protein rich and therefore, helps in cell in adhesion by supplying the ECM-proteins as well as nourishing them. Hence, the serum conditioning step is of critical importance in maintaining cells health and attachment in the culture. Materials and Methods Scaffold preparation and serum conditioning PGA FELT Scaffolds disc of 2mm x 10mm and 45mg/cc (TE005-50-10) was provided by Smith and Nephew research group, University of Nottingham. These non-culture scaffolds were then treated in 24 well tissue culture plastics (TCP) plates with 3T3 fibroblast media containing 500ml DMEM (Sigma G7513) supplemented with 10% FCS, 2mM L-glutamine and 1% AB/AM (Sigma A5955). All the scaffolds were statistically seeded on day 1 using non-culture treated well plates to encourage the cells of mouse dermal 3T3 fibroblast to adhere to the scaffolds at seeding density of 1x 10à ¢Ã‚ Ã‚ ¶cells/ml. 3T3 fibroblast cell suspension was added in TCP plates for all test and no cells in the blanks. The plates are then incubated overnight at 37 °C, 5%COà ¢Ã¢â‚¬Å¡Ã¢â‚¬Å¡ in air. The remaining cell suspension was then again resuspended in warm media to achieve 4 x 10à ¢Ã‚ Ã‚ ¶ cells/ml cell density and was stored at -20 °C till day 7 for Hoechst analysis. 3T3 fibroblast cells were used to seed the scaffolds to observe the cell viability, cell proliferation and ÃŽÂ ²-actin expression on day 1, when the cell culture condition was maintained static and day 7, after applying the two cell culture conditions (static dynamic) and maintaining for 7 days. Static culture In static culture condition, the seeded and non-seeded (blanks) scaffolds were kept in 1ml of warm 3T3 fibroblast media per well. These five culture plates were kept in incubator and cultured for 7 days at 37 °C, 5%COà ¢Ã¢â‚¬Å¡Ã¢â‚¬Å¡ in air. Spinner flask culture Two separate spinner flask filled with 50ml warm media each was used for seeded scaffolds and non-seeded (blanks) scaffolds. These flasks were kept in incubator by loosening the side arms and setting the magnetic stirrer approximately at 60rpm and cultured for 7 days at 37 °C, 5%COà ¢Ã¢â‚¬Å¡Ã¢â‚¬Å¡ in air. After following 7 days for culture conditions, the construct was then sacrificed for alamar blue, toludine blue and Hoechst analysis. Also, in addition cytoskeleton analysis using western blot was also carried out. The assessment of two culture methods, static and dynamic was done by producing five set of readings for static condition and four set of readings for dynamic condition where the experimental analysis were conducted using three replicates for test and blanks on day1 and day7 Alamar Blue Assay Staining was done using 10% alamar blue containing 1ml alamar blue (Serotec BUF012B) and 9ml HBSS without phenol red (Sigma H1387). The stain was kept in dark at 37 °C. The scaffold was transferred from seeding and culture conditions to new 24-TCP non-cultured plate with 1ml warm alamar blue after washing three times with PBS. The plates were then incubated at 37 °C, 5%COà ¢Ã¢â‚¬Å¡Ã¢â‚¬Å¡ for 1hr. The aliquots of 3 x 100 µl of alamar blue were transferred to 3 wells of 96 microtitre well plate including the blanks to measure fluorescence using plate reader (Ex530nm/Em590nm). The excess of alamar blue solution was aspirated and washed with 1ml sterile PBS. Toludine Blue Staining Scaffold for toludine blue staining was transferred to new non-culture treated 24well TCP plate and was treated with 1ml ice cold 95% (v/v) methanol in dHà ¢Ã¢â‚¬Å¡Ã¢â‚¬Å¡O for 5 mins after washing 3 times with 1ml warm PBS. Then fixative was discarded and scaffold was allowed to air dry at RT followed by treatment with 1ml aqueous 0.1% (w/v) toludine blue (Fisher chemicals BPE107-10) for 5 mins. The scaffold was again allowed to air dry at RT. Papain Digest and Hoechst 33258 DNA Assay The aliquot of cell pellet (4 x 10à ¢Ã‚ Ã‚ ¶ cells) prepared on day1 was treated with 1ml of papain solution (1.06mg/ml, pH 6.5) (Sigma P4762) followed by overnight incubation in waterbath at 60 °C. The serial dilutions of the papain digested cell pellet using hydrolysed papain solution as diluent was prepared for 4.0, 2.0, 1.0, 0.5, 0.25, 0.125, 0.0625, 0.0312 and 0 x 10à ¢Ã‚ Ã‚ ¶ cells. In the Hoechst 33258 DNA assay, the hydrolysed papain solution was used as blank. 5 µl of each aliquots + 70  µl Hoechst dilution buffer was added in triplicates in black 96-well plate including the blank. In each well, 100  µl Hoechst 33258 working solution (Sigma S6639) was also added and fluorescence was measured using plate reader (Ex 360nm/Em 460 nm) Western Blot 100 µl cold RIPA buffer (Sigma R0278) was added to the cell pellet (4 x 10à ¢Ã‚ Ã‚ ¶ cells) and the seeded scaffolds in eppendorf from day 1 and was kept on ice for 20 mins while vortexing every 5 mins. The cells were then snap freezed by placing it on dry ice for 1 min then 1-3 min at RT. The cells are resuspended by grating and spinning for 30 mins. The supernatant was used for western blot. 10 µl of molecular weight marker and each sample were loaded onto SDS polyacrylamide gel. The electrophoresis was carried out for 90 mins at 125V. After SDS-page electrophoresis, the filter paper, nitrocellulose and sponge were soak in transfer buffer (Invitrogen NP0006) with 20% (v/v) methanol. The assembled western blot tank was run for 1 hr at 25V. The immune-detection of protein ÃŽÂ ²-actin was performed using primary antibody anti-mouse ÃŽÂ ²-actin (Sigma A2006) and secondary antibody anti-mouse horse radish peroxidises (HRP) (Invitrogen G21234). Statistical analysis All the data obtained was calculated using MS-Excel spreadsheet and statistic Independent t-test and paired t-test analysis was performed using SPSS software. Results and Discussion Morphology of 3T3 fibroblast cells The cell of mouse dermal 3T3 fibroblast was obtained from T180 flask by trypsin digest method is shown in figure1. The flask was confluent enough (80%) and morphology of the cells seems to be intact and healthy. No sign of contamination was observed prior to seeding procedure. The morphology of 3T3 fibroblast cells are of flat and spindled shape. These cells form a well-characterised and established mesh like interconnected networks. This property of fibroblast cells make them ideal for cell attachment as they show anchorage property due to presence of integrins in ECM. Hence, using this cell type achieving maximum cell adhesion onto the scaffolds becomes ideal for this experiment. Effect on Cell viability in static and dynamic conditions The alamar blue assay was performed on the static and dynamic culture condition to observe its effect on 3T3 fibroblast cell viability is shown in figure 2a and 2b. The culture method employed aims to maintain or increase the cell viability when cultured for seven days. Under static condition (Fig 2a), only 1 group out of five showed significant increase in fluorescence whereas other two groups showed more or less no change in their fluorescence produced from day 1 to day 7. Also, on contrary two groups showed significant decrease in fluorescence on day 7 (Fig 2a). Hence, variable of results were obtained between groups. On the other hand, under dynamic condition, the cell produced more fluorescence on day 7 compared to day1 expect for one group. Therefore on an average, when mean of the static absorbance reading was taken, it showed that there is significant decrease in fluorescence (fig 2b). But in dynamic method, the increase in fluorescence day 7 (Fig 2b) was not significant enou gh. The 3-D construct of PGA scaffold provides with an environment to the cells where they remain viable in culture for several weeks. Moreover, they should successfully increase the cell viability after some days. However, our study reported that the cell viability decreased tremendously in cell seeded PGA scaffolds in static cell culture condition whereas the dynamic cell culture method was able to increase the cell viability over 7 days of culture. So, when comparing the two culture methods statistically showed difference in their overall effect on the viability of 3T3 fibroblast cells where dynamic condition is more but not effective enough. So, static condition did not improve the cell viability more than dynamic culture method. Effect on Cell distribution in PGA scaffolds The three-dimensional PGA scaffolds constructs enables the fibroblastic cells to adhere and to evenly distribute throughout the porous structure. To assess the uniform 3T3 fibroblast cell distribution in two different culture conditions, toludine blue staining was carried out on day 1 and day 7 on both conditions is shown in fig 3. Toludine blue stains cell dark blue within the 3-D construct. As observed in static condition, on day 1 the cells were successfully seeded onto the scaffold but compared to day 7 the cells are not evenly distributed throughout the scaffold. Also, the scaffolds were efficiently seeded on day1 under dynamic condition as the figure 3c shows cells stained with toludine blue. Surprisingly, on day 7 (Fig 3d), the scaffolds shows no cells at all. This means, that the 3T3 fibroblast cells under dynamic condition was eventually lost or died. The spinner flask culture system might have loose the cells by day 7 due to poor adhesion or vigorous rotation. The cell seed ed on day 1 was too low or error in carryout the technique. But this was observed with all the spinner flask condition system, where the success was 2 out of 4 groups (Supplementary data 3). However, this observation is more of debate because no other factors expect the condition itself could affect cell distribution as uniform distribution was achieved in all the five static condition (supplementary data 3) which used the same scaffolds and cell type. Effect on Cell proliferation in static and dynamic conditions 3T3 fibroblast was culture over 7 days in both conditions to also observe its effect on the cell proliferation are shown in figure 4 (a, b, c d). The standard curve obtained with known cell density for both static and dynamic of all the groups (fig 4a 4b) showed increase in cell density with increase in the fluorescence. The unknown cell density of the cells from these two culture methods on day 1 and day 7 was calculated and found that 2 out 4 groups from dynamic conditions had no cells in the culture on day 7. Therefore, only other two groups were considered to evaluate the cell number on day 1 and day 7. There was significant difference in cell density over 7 days of culture in static method (n=5)(fig 4c) and on contrast there was no significant difference in cell density in dynamic method (n=2)(fig 4c). Almost all the groups showed cell density on day 1 around 1 x10à ¢Ã‚ Ã‚ µ cells/ml which was the actual cell density seeded on day 1 (supplementary data 4). This shows that se eding performed on scaffold achieved effective adhesion of all the cells present. The mean cell number from 1.2 x 10à ¢Ã‚ Ã‚ µ ( ± 0.16 x 10à ¢Ã‚ Ã‚ µ) cells on day 1 increased to 6.3 x 10à ¢Ã‚ Ã‚ µ ( ± 1 x 10à ¢Ã‚ Ã‚ µ) cells on day 7 under static culture method (fig 4d). On the other hand, dynamic culture methods showed hardly any change in cell number over 7 days of culture i.e. 2.0 x 10à ¢Ã‚ Ã‚ µ ( ± 0.92 x 10à ¢Ã‚ Ã‚ µ) cells on day 1 to 2.5 x 10à ¢Ã‚ Ã‚ µ ( ± 1.96 x 10à ¢Ã‚ Ã‚ µ) cells on day 7 (fig 4d). Previous studies have reported using other cell types that they start proliferating within 24 hrs after seeding cells on scaffolds employing dynamic culture methods. Contradicting this, our results have shown that dynamic had really poor effect on cell proliferation. Moreover, 3T3 fibroblast cells were undergoing death during seven days of culture. Whereas, static culture method shows drastic increment in the cell number and thus supporting 3T3 fibroblast cell proliferation efficiently. The scaffolds used for alama r blue assay on day 1 were used for Hoechst DNA assay with same after washing step (same for day 7 scaffolds). The washing might have been too vigorous which resulted in cell loss. It could also be possible that cells are being aspirated off from the culture which gave poor or no cell proliferation. It should be also taken into account that the success rate with dynamic culture method on cell proliferation was null out of 4 demonstrations. Expression of Cytoskeleton For the analysis of expression of cytoskeleton ÃŽÂ ²-actin on 3T3 fibroblast in two different conditions was done by western blot as shown in figure 5. The cell pellet of density 4 x10à ¢Ã‚ Ã‚ ¶ cells/ml was loaded against the cells obtained on day1 and day 7 from static and spinner flask culture method. The density of ÃŽÂ ²-actin obtained from the cell pellet was maximum. The amount of ÃŽÂ ²-actin detected on day 1 was lower than day 7 in static culture condition. It was the opposite scenario with spinner flak method where day 7 had minimum amount of ÃŽÂ ²-actin compared to day1. In some cases of spinner flask method ÃŽÂ ²-actin was not even detected on day 7 (supplementary data 5). Hence, comparatively the expression of ÃŽÂ ²-actin was higher in static culture method. Perhaps, it could be because the cell could not proliferate much as expected. Also, the culture didnt have enough cells left to express ÃŽÂ ²-actin on western blot. The formation of ECM cytoskel eton was not shown to be supported by spinner flask method. Conclusion and future work The tissue engineering scaffolds constructs have been shown more effective on cells containing serum in spinner flask/dynamic culture method rather than in static culture method. But from our data, it shows that dynamic condition only favoured cell adhesion and distribution. It was also able to produce a small increment in cell viability unlike static culture method. Contradicting the other data, cells were virtually not detected on day 7 and so is the expression of ÃŽÂ ²-actin. Not only this, all the 4 demonstration failed to show that cell growth can be effectively supported in dynamic culture method. Three seeded scaffolds were kept in spinner flask together, where there is increased chance for it to come in contact with each other. Cells may get detached from the scaffolds as it might be loosely adhere to the scaffold. The continuous rotation of magnetic rod in the flask circulates the media to provide nutrients to cells more effectively then static. Despite of this fact, the cells were either undergoing cell death or dislodged from the scaffolds or may be aspirated off from the culture. The static culture method have been effective in 3T3 fibroblast adhesion on the construct after seeding and eventually could improve tremendous cell growth by showing increase in cell proliferation over a period of seven days in culture. However, better distribution and increased ÃŽÂ ²-actin expression could only be achieved by the static culture method after 7 days as the cells proliferated more. Moreover, the success rate for this method was more compared to dynamic and produced more reliable and reproducible data. Hence, it can be concluded that static culture method supported cell growth better then the dynamic culture method. It would be interesting to investigate the technique involved in dynamic culture method more carefully to produce reliable data where it could be compared with the static method to give better understanding of the environment cells require to grow in artificial ECM-like structure and culture media. Since, within the body the cells are continuously under the force by blood flow in 3D environment, it would be useful to derive cell culture growth better in dynamic condition with enhanced technique. It is strongly recommended to carry out further research in this area to conclude spinner flask methods effect on 3T3-fibroblast cells with more reliable data. Evaluation The practical session assessed my learning in the techniques and concepts involved in tissue engineering. The demonstration on different techniques to prepare scaffolds assessed my understanding better and was helpful to apply same in this practical session by evaluating the different parameters that can be influenced by the scaffold design alone. As earlier discussed troubleshoot, implementing the technique given in protocol helped to produce the good replicates and contamination free-blanks and controls. While working in the hood with the partner, things were discussed prior to carrying out the experiment and working space was kept ready which helped in managing the use of same equipments, solution and incubation time effectively to avoid any source of contamination. Also, the exchange of results and data between several groups also led to the exchange of ideas and different cause for their results. However, the exact reason for spinner flask method to not work out is still not cle ar as all the groups got same reading where cells were present onto the scaffolds during alamar blue assay on day1 and day 7 but eventually lost when subsequent assays were done for same day. Overall, the difference between the effects of two culture method was evaluated. Acknowledgement The efforts put in by the Paula Ellis is acknowledged was carryout the change of media and taking care for the samples throughout the practical session and also during weekends. Also, Dr. Felicity Rose for giving the guidance and helping with doubt regarding the techniques and protocol. The images and data taken from all other groups are acknowledged for sharing their data used in this report. The effort of the group member is also acknowledged for managing with the working protocol load effectively. Figures Figure1. 3T3 fibroblast cells in T180 flask (10X). The image shows morphology of 3T3 fibroblast prior the trypsin digest followed by static seeding. The image was taken using Nikon (Scale bar: 80 µm) Figure 2a. Alamar blue assay for all static (n=5) and for dynamic (n=4) culture methods on day1 and day 7. The graph shows fluorescence detected  ± SD for both the culture condition. The absorbance value of non-seeded scaffold (control, Ac) was subtracted from the absorbance value obtained for seeded scaffold (As) to optimise the calculated fluorescence i.e. As-Ac. This was done for all the static and dynamic culture methods data. The statistical analysis paired t-test was at 95% significance level was done using SPSS. The calculated data is provided in the supplementary data. Figure 2b. Alamar blue assay of static and dynamic condition on day 1 and day 7. The mean of all the values on day 1 and day 7 for static (n=5) as well as dynamic (n=4) was done. The graphs shows the mean of absorbance (O.D)  ± SD. The statistical analysis was performed using paired t-test and independent t-test at 95% significance level. DAY 1 DAY 7 Figure 3. Toludine blue assay. The toludine blue staining was performed on static culture condition on day 1 (a) and on day 7 (b). Similarly for dynamic culture condition on day 1 (c) and day 7 (d) was carried out. In (a) and (b) there is darker background staining but (c) shows proper stained 3T3 fibroblast cells. No cells staining can be detected in (d) (Scale bar: 100 µm). Figure 4a. Standard curve for all static condition using Hoechst 33258 DNA assay. The standard curve was plotted using the known concentration 4.0, 2.0, 1.0, 0.5, 0.25, 0.125, 0.0625, 0.0312 and 0 x 10à ¢Ã‚ Ã‚ ¶ (blank) of 3T3 fibroblast cells against the absorbance obtained. The blank was subtracted from the test reading to standardise the graph. The graph was produced using MS-Excel, to obtain the linear regression and linear equation for each group to calculate the cell density in static culture condition. Figure 4b. Standard curve for only two dynamic condition using Hoechst 33258 DNA assay. The standard curve was plotted using the known concentration of 4.0, 2.0, 1.0, 0.5, 0.25, 0.125, 0.0625, 0.0312 and 0 x 10à ¢Ã‚ Ã‚ ¶ (blank) of 3T3 fibroblast cells against the absorbance obtained. The blank was subtracted from the test reading to standardise the graph. The graph was produced using MS-Excel, to obtain the linear regression and linear equation to calculate the cell density in dynamic culture condition on day 1 and day 7. Figure 4 c. Hoechst 33258 DNA assay was carried out on all static (n=5) and dynamic (n=2) culture condition on day 1 and day 7. The cell density was calculated using the standard curve for its own respective group. The graph shows cell density (x 10à ¢Ã‚ Ã‚ µ cells/ml)  ± SD for static and dynamic condition. The calculation was performed on excel-sheet and statistical analysis of paired t-test was done using SPSS. Figure 4 d. Hoechst 33258 DNA assay. The unknown cell density calculated from standard curve was averaged (mean) for static (n=5) and dynamic (n=2) culture methods. The graph shows cell density (x 10à ¢Ã‚ Ã‚ µ cells/ml)  ± SD for static and dynamic condition. The calculation was performed on excel-sheet and statistical analysis of paired t-test and independent t-test was done were appropriate using SPSS. Figure 5. Western blot analysis of 3T3 fibroblast cell from static and dynamic on day 1 and day 7. The expression of ÃŽÂ ²-actin in both culture methods are analysed using the rainbow marker and compared with the actual pellet of 3T3 fibroblast to cells extracted from two different culture methods on different days.

Fairytales and Folktales Essay -- Literary Analysis, Charles Perrault

Fairytales and folktales have been told around the campfire, in the living room, the class room, and before bedtime for centuries. First told orally, the â€Å"†¦ stories had to have remarkable features in order to remain memorable (Nodelman 246).† These stories were passed down from storyteller to audience until they were eventually written down and collected for consumption by the public. Due to the passing of time and fallibility the stories have changed throughout the years and slightly differ from culture to culture, however, â€Å"Stories similar to â€Å"Cinderella† can be found in historical records from as far back as the seventh century, and from a variety of places around the world (Nodelman 246).† Although the classic tales differ in various ways from their modern counterparts (such as Disney films, etc.), the characters and their journeys are still very much identifiable. For centuries, fairytales have been used for instruction; to teach children what is expected of them as they age and what terrors behold them if they do not comply with the guidelines laid out for them by their culture/society. Many of the tales were purposely frightful in order to scare children away from strangers, dark corners, and traveling off the beaten path into the dark thicket. Charles Perrault first began writing fairy tales in the late 17th century to educate his children. The morals of those tales often center on what is expected of young women; that they should remain ‘pure’ and ‘docile’. He wrote the tales in a time period when fairytales or ‘jack’ tales were looked at as instructional lessons. They were also widely told around the fire, as entertainment, for adults. Angela Carter adapted Perrault’s classic tales in the 1970’s; changing the victim... ...the end of the 1960’s – the mid 1970’s) was a revolutionary time period for women. In America, The Civil Rights Act of 1964 protected women from workplace discrimination and Roe v. Wade, 1973, guaranteed a woman’s right to choose when to be pregnant. In England, for the first time, a law was passed guaranteeing equal pay to women in Britain’s civil service (Women’s International Center 1). Carter, herself, was a self proclaimed feminist; she once said, â€Å"The Woman’s Movement has been of immense importance to me personally, and I would regard myself as a feminist writer, because I’m a feminist in everything else and one can’t compartmentalize these things in one’s life (Gamble 15).† Her writing began to be viewed, and still is viewed as feminist literature adored by college students, especially those concentration in gender related studies, and the literati alike.

Executive Summary of the Hispanic Market Essay -- Hispanic Culture Mar

Executive Summary of the Hispanic Market â€Å"Latinos are changing the way the country looks, feels, and thinks, eats, dances, and votes. From teeming immigrant meccas to small-town America, they are filling churches, building businesses, and celebrating this Latin heritage.... In America, a country that constantly redefines itself, the rise of Latinos also raises questions about race, identity, and culture – and whether the United States will ever truly be one nation.† (Larmer, pg. 50) This passage aptly describes the dawning of a new ‘enlightenment’ era in the United States. Marketers are beginning to focus on an emerging market known as the Hispanic/Latino community. The sheer strength of the Hispanic market can no longer be avoided as marketers are realizing that traditional methods of reaching a generalized market segment do not apply to the complex Hispanic culture. In the last half of the 20th century, the size of the Hispanic market in America grew exponentially. Traditional attempts to capitalize on the Hispanic market failed in large part to stereotypes and cultural myths. A new focus was necessary to attract, reach and retain the market. In order tackle this potentially lucrative market, marketers need to understand the cultural attributes that define the Hispanic market. Their primary focus is to understand the statistical values that characterize the group. Census figures over the last thirty years clearly illustrate a pattern of growth, not only in population, but in wealth and education as well. Clearly, this is becoming a stronger, savvier and better-educated market. As marketers become better acquainted with the Hispanic market, they have found several attributes that are typical of the Hispanic culture and influence in America. They first need to understand that the term â€Å"Hispanic† is a broad generalization of several cultures and races, each with distinct characteristics and values. Once an understanding that many subcultures encompass the Hispanic community, marketers can disseminate the target market and address those characteristics shared amongst the Hispanic community. Level of acculturation, brand loyalty, language, religion and a strong sense of family are those shared traits that need further study in order to properly understand what Hispanics believe, care for and personify. Once a deeper comprehension of the Hispa... ...d Asians. New York, NY: American Marketing Association. 1987. Guernica, Antonio. Reaching the Hispanic Market Effectively; The Media, the Market, the Methods. New York, NY: McGraw-Hill Book Co. 1982. Larmer, Brook. Latino America. Newsweek, July 12, pg. 50-58. 1999. Noriega, Chon and Ana M. Lopez, Eds. The Ethnic Eye: Latino Media Arts. Minneapolis, MN: University of Minnesota Press. 1996. â€Å"Riverside† Webster’s Tenth New Collegiate Dictionary. 1998 Rodriguez, America. Making Latino News; Race, Language, Class. Thousand Oaks, CA: Sage Publications. 1999 Roslow, Peter, and Janel Therrien Decker. A Guide to Building Market Dominance: Case Histories in Hispanic Marketing. Roslow Research Group Inc., 1998. United States Census Bureau. â€Å"Census 2000†. Washington: Census.gov. 2002. http://www.census.gov. (30 Sep. 2002) United States Census Bureau. â€Å"Historical Income Tables-Households†. Census.gov. 2002. http://www.census.gov/hhes/income/histinc/h05.html. (30 Sep. 2002) Valdes, M. Isabel. â€Å"Marketing to American Latinos; A Guide to the In-Culture Approach†. Ithaca, NY: Paramount Market Publishing, Inc. 2000. Whitefield, Mimi. â€Å"Mining the Market† The Miami Herald 17 Oct. 2001, C1+

Genetic Enhancement of a Child’s Memory: A Search for a Private and Pub

Genetic Enhancement of a Child’s Memory: A Search for a Private and Public Morality ABSTRACT: Prospects of human genetic modification raise the question of genetic enhancement of memory. A moral framework that takes into account the tension between the roles of parent and citizen on the question of genetically enhancing a child’s memory is presented. Weaknesses of both moral liberalism and moral communitarianism are addressed: a tyranny of a powerful minority of liberalism, while a tyranny of orthodoxy and a tyranny of perfectionism plague different forms of communitarianism. A position is advanced that draws on the strengths of both a Rawlsian form of contractarianism and a moderate version of communitarianism. I argue that genetic enhancements of memory in children pose such serious wrongs and threats to general well-being that the practice should be decided from behind a Rawlsian veil of ignorance. With the cards down, as Ronald Green describes the veil of ignorance, a basic right to nondiscrimination on the basis of genotype would be negotiated. With this right in place, conflicts between the parental role and the role of citizen would be managed by the negotiated prohibition of parental decisions genetically to enhance the memory of children. Let me imagine myself some years from now as a citizen and a parent — who also happens to be a philosophy teacher — facing the question of whether I should choose various enhancements for my young child. Orthodontics, music lessons, soccer leagues, and genetic enhancement of an average memory are among the practices I am considering. I soon discover an internal tension. Ronald Green, in an article called "The Rawls Game," (Teaching Philosophy, 1986, 9:1, 51-60) provides an el... ...eligious intolerance, new definitions of what it means to be a human person have been created. In a hypothetical, unanimous agreement to prohibit genetic enhancement of a child’s memory I would join a citizenry that exercises parental autonomy and — in the face of a volatile new technology — defines a new way of understanding what it means to be a human parent. My choice as a parent to serve as a link between past and future human generations prompts me to pursue a perspective of fairness in the application of this new technology — a technology that incorporates self-interest and benevolence but makes neither self-interest nor benevolence my primary motivation as a parent. I could tell a coherent story to my child if I were able to relate to that, with the cards down, people unanimously placed genetic enhancement of a child’s average memory off the political agenda.

Manage Own Performance in Business Environment Essay

1.1Outline ‘guidelines, procedures and codes of practice relevant to personal work. There are procedures that need to be followed relating to various aspects of the job including correct procedures to greet visitors, answer the telephone, dealing with incoming and outgoing mail, taking minutes for team meetings as well as other procedures. 1.2Explain the purpose of planning work and being accountable to others for own work The purpose of planning before attempting any work is to create a realistic time frame in which you wish to complete the work to a good/high standard and if you’re an employee, the employer has a right to check if work is up to standard 1.3Explain the purpose and benefits of agreeing realistic targets for work The purpose for agreeing realistic targets for work is to keep everyone on task and focused on accomplishing a target that is obtainable, not out of reach. This way, everyone can work towards effectively reaching targets efficiently instead of struggling to achieve the impossible. The benefits include fast and reliable compliance and completion of tasks at hand 1.4Explain how to agree realistic targets When speaking to a senior employee you will gain respect by being straight about how much you can do. If you set too easy a target for yourself you won’t be pushed to improve yourself and it will be clear to your senior employee or your employer. If you make it impossible to reach they will be unimpressed by your inability to work out how long you need and missing the deadline 1.5Describe ways of planning work to meet agreed deadlines First you need to prioritize your work. That is, place the most important job at the top; the least, at the bottom. When judging priorities, you need to do several things: firstly you need to determine what is required in the given task. This is the number of jobs that need to be done. Second you need to figure out what is required. If you’re doing something that’s not necessary, eliminate it. If you’re doing something that’s necessary but is not required of you personally, you need to delegate it. And lastly if someone can do a certain job better than anyone else, delegate the job to that person . 1.6Explain the purpose of keeping other people informed about progress The reason why you keep colleagues up to date with progression is so that they know what targets to achieve and whether or not they can meet the deadline on time also it helps to outline and set objectives. If you’re behind on a piece of work it is also helpful for colleagues to know progression so they could pick up the slack 1.7Explain the purpose and benefits of letting other people know when work plans need to be changed It respects people’s time and allows people to be prepared for work, mentally and physically. It is respectful of the other person’s time to give them a good idea of what and how you want them to do the task and when they need to start. If these plans change, respect demands that you inform others involved of the changes to these plans 1.8Describe types of problems that may occur during work There are many different types and severities of problems that you come across in your working life for example bullying, disgruntled workers and harassment are some of the major examples. But problems such as the printer running out of toner, work not saving properly and power cuts are less severe but can also cause workplace stress. 1.9Describe ways of seeking assistance with getting help to resolve problems Any technical fault that involves your computer you should report this to your computer technician if your company has one. Government and large companies mainly have a team of technicians to help different problems out. However if the problem is more severe i.e. harassment, then you should report to your superiors and tackle the problem professionally. 1.10Explain the purpose and benefits of recognising and learning from mistakes The main purpose of recognising mistakes is to learn from them and to try and prevent them from happening again therefore the next time a situation arises you will have the knowledge of the previous times to prevent you making the incorrect decision and therefore being successful. The benefits of learning from mistakes are obviously you don’t make the same mistake time and time again and as a result of this you will be more successful at what you do. 2.1 Explain the purpose and benefits of agreeing and setting high standards for own work The purpose of agreeing and setting high standards for work assures that each person tries their hardest and always reaches for new heights. By setting a high bar there is no room for excuses and the atmosphere in the workplace becomes excellent behavior and work gets completed with ease. Everyone benefits from setting high expectations for themselves. Everything can be done better, faster, and more efficiently. 2.2 Describe ways of setting high standards for work You can set yourself high standards of work by putting 100% effort into every task you take on. By putting through high standards of work every time, you and the others around you will continuously demand and expect high standards from you all the time. Also if you always try and find ways of how you can improve your work then you automatically raise the bar for yourself. 2.3 Explain the purpose and benefits of taking of taking on new challenges if they arise. The purpose of taking on new challenges w they arise is vital to success. By not challenging yourself, the same pattern of mediocrity or  self defeating attitude persists. You cannot grow without moving onto bigger, better, and more challenging tasks. The benefits therefore are then self growth opportunities and a gaining of new skills and confidence. Also in the workplace it is especially important to take on new challenges as it shows to your employer that you are capable and willing to new things. 2.4 Explain the purpose and benefits of adapting to change The purpose of adapting to change technically is so you don’t get left behind and this is same in business, if you fail to adapt to any sort of change then you won’t be successful completing the task when the time comes. The main benefit of adapting to change

Chocolate and Like Water for Chocolate Essay

In Chocolat, I learned that food has magical power that engages and connects people and brings them into good relations. Vianne and her daughter were not welcomed in a conservative and religious town at beginning, however her chocolate had magical power to melt those peoples’ cold attitude and they became drawn into her chocolate, even that stubborn pastor Reynaud who had strong hostile feeling against Vianne did so at the end. I liked the part that Reynaud couldn’t resist to break in her store and try all the chocolates before Easter Sunday. It is like one of my dreams. I roll in chocolates. I imagine myself in a field of chocolates, on a beach of chocolates, basking-rooting-gorging. I have no time to read the labels; I cram chocolates into my mouth at random† (Harris 312). I believe his attitude also influenced and changed town people since he is the symbol that his faithful people ought to believe in and follow in their town. I also enjoyed seeing the relationship between Vianne and Roux. With similar characteristics they both have, such as free minded, not religious like town people, move from a place to another and somewhat isolated from society, it is natural that they feel close each other. Especially, the night they spent together is one of my favorite parts, because it was described beautifully and romantically. â€Å"The garden was still warm in the glow of the braziers. The mock oranges and lilacs of Narcisse’s trellis blanketed us beneath their scent. We lay on the grass like children. We made no promises, spoke no words of love, though he was gentle, almost passionless, moving instead with a slow sweetness along my body, lapping my skin with fluttering of the tongue. [†¦] For the moment, simple wonder; at myself lying naked in the grass, at the silent man beside me, at the immensity above and the immensity within. We lay for a long time, Roux and I, until our sweat cooled and little insects ran across our bodies, and we smelled lavender and thyme from the flower bed at our feet as, holding hands, we watched the unbearable slow wheeling of the sky† (Harris 289-290). In Like Water for Chocolate, I learned the method of Magic Realism and enjoyed reading several themes which were described with Magic Realism. Magic Realism is an aesthetic style or genre of fiction in which magical elements blend with the real world. The story explains these magical elements as real occurrences, presented in a straightforward manner that places the â€Å"real† and the â€Å"fantastic† in the same stream of thought. I enjoyed reading this novel from very beginning with Tita’s dramatic birth in kitchen. Her tide of tears on her birth becomes lots of salt to be used for cooking later on. â€Å"Tita was literally washed into this world on a great tide of tears that spilled over the edge of the table and flooded across the kitchen floor† (Esquivel 6). â€Å"That afternoon, when the uproar had subsided and the water had been dried up by the sun, Nacha swept up the residue the tears had left on the red stone floor. There was enough salt to fill a ten-pound sack-it was used for cooking and lasted a long time† (Esquirel 6). I like this part because Tita not only has a big passion over cooking, but also she could produce an ingredient –salt by her own, which has an important role later on. I enjoyed reading the part that the wedding cake Tita made for her sister makes every single guest feels longing, intoxicated and frustrated at the wedding. Tita’s love over Pedro was so strong and her poison: tears in the cake made everyone become sick. â€Å"The moment they took their first bite of the cake, everyone was flooded with a great wave of longing. †¦] But the weeping was just the first symptom of a strange intoxication- an acute attack of pain and frustration- that seized the guests and scattered them across the patio and the grounds and in the bathrooms, all of them wailing over lost love† (Esquirel 39). Watching both films also helped me understanding and picturing each scene clearly. Now I am enjoying the third novel, The Edible Woman, because this novel is written in modern plot and describes women’s conflicted feeling in modern society through food and cooking.