Tone, Rhyme, and Irony in Thomas Hardy's the workbox Essay

Tone, Rhyme, and Irony in Thomas Hardy's the workbox - Essay Example The tone of the poem is set as aggressive and menacing in the first line of the poem with the forceful and demeaning words, â€Å"See, here’s the workbox, little wife,† (Hardy 374). The opening words of the poem, where the husband greets his wife, are seemingly innocent enough unless the reader close reads and notices an almost overpowering sense of control. The husband’s intentions were never to give a gift to his wife, but a reminder of an issue she has caused for him. The overall tone can be mistaken for a happy and light conversation between the man and his wife until further read into, when the reader can tell there is a hidden fact they both know of. The narrator announces in the aggressive tone that he has made something for his wife to take a look at. In the following stanza, it is revealed that the workbox the narrator constructed of polished oak is a present to his wife. Gifts signify gratitude and rewarding. However, the tone in which the husband opens to present his wife with the gift is aggressive and angry in its tone. The combined tone of anger and control shows that the giving of the gift is ironical because it is not a present the husband gives in joy and thankfulness to his wife. A sense of irony comes into play in the first stanza when the tone set by the first two lines is taken into consideration. The aggressive tone of the opening words along with the context that the rest of the stanza carries is enough to reveal a large amount of ironic quality.â€Å" ‘See, here’s the workbox, little wife, that I made of polished oak.’ He was joiner, of village life, She came from borough folk† Throughout the rest of the poem we learn details about the source of the materials and the wife’s past life which reveal the nature of the relationship through the real intentions of the husband and his wifes silence. Throughout the poem, irony intertwines in the theme to show that a gift from a loved one can be an act that is

Civil Rights during Truman, Eisenhower, Kennedy and Johnson Essay

Civil Rights during Truman, Eisenhower, Kennedy and Johnson - Essay Example Eisenhower was more â€Å"cautious† than Truman, who lost the re-election because of his strong advocacy for civil rights (Corse). Eisenhower did not think that legislation and court decisions could alter racial discrimination and prejudice, but he did order desegregation in public schools and the military (Corse). Southern politicians, nevertheless, doubted Eisenhower's cold feet on civil rights and took it as a stand for civil rights (Corse). In 1957, Governor Orville Faubus of Arkansas employed the National Guard to stop the desegregation of Little Rock Central High with the enrollment of nine Black students. Eisenhower placed 1000 paratroopers to protect the Little Rock Nine (Corse). Grassroots activism intensified during Eisenhower's administration, because of the slow improvement in civil rights (Corse). Some of the pivotal events are Rosa Parks' actions and her impact on the Montgomery Bus Boycott, the rising popularity of Martin Luther King, organization of the Souther n Christian Leadership Conference (SCLC) and the Student Nonviolent Coordinating Committee (SNCC), and the lunch counter sit-ins in numerous cities in the 1940s and 1950s (Corse). Kennedy tried to broaden the voting rights for Black people. Attorney General Robert Kennedy collaborated with SNCC and others to improve the registration of Black southern voters (Corse).Kennedy also selected numerous African Americans for Federal positions and made Thurgood Marshall a Federal judge. Activists felt that Kennedy lacked vigor and dedication to the civil rights movement, however, and the former continued direct actions, such as conducting freedom rides that stopped segregation in interstate commercial travel (Corse). on. After the bloody attacks of the Birmingham police against non-violent protesters in 1963, Kennedy was forced to call for the drafting of the civil rights legislation (Corse). He died shortly, nevertheless, which transferred his civil rights obligations to Johnson. Johnson was able to secure legislation through Congress that presidents before him were not able to do. He was able to help pass the Civil Rights Act of 1964, through votes from Northern Democrats and Republicans. This act made segregation in public places and transportation as illegal. It also made the Equal Employment Opportunity Commission that will fight and deal with job discrimination. It further protected the voting rights of minorities and banned gender discrimination. During this time, the Voting Rights Act of 1965 was also passed, which emphasized the right of minorities, especially Blacks to vote, and so this act also greatly increased the number of Black voters in the South. The Immigration Act of 1965 prevented discrimination in immigration, where preference for European immigrants was banned. Hence, civil rights movement was launched more actively during Truman's time and subsided somewhat during Eisenhower's and

Increase Crime Among Youth Essay Example for Free

Increase Crime Among Youth Essay 1. This is to confirm that we have allotted flat no. D4 / E 5 admeasuring 920 Sq. ft.(built up area) on the Survey No.49 A / 7 constructed by us to shri PARAG VIJAY MODI for a total consideration of Rs.12,00,000/-(Rupee Twelve Lac Only)Under an agreement for sale dated 20 /12/2012. 2. We confirm that we have obtained necessary permission / approvals sanction for construction of said building from all the concerned competent authorities and the same are in force. The construction of the building as well as of the flat is in accordance with the approved plans. We assure that the said flat as well as the said building and the legal appurtenances there to be not subject to any encumbrances, charge or liability or any kind whatsoever and that the entire property is free from encumbrances and marketable. We have a clear, legal and marketable title to the said property and part thereof. 3. Shri. PARAG VIJAY MODI has/ have paid the total cost of Rs. 5,90,000/-(Rupees Five Lac Ninety thousand Only). 4. Possession of the said Unit/Gala/Shop/ Premises / Flat will be given to Shri. PARAG VIJAY MODI . 5. We have hereby confirm that we have NO OBJECTION to your giving finance to Shri. PARAG VIJAY MODI his her mortgaging the said Flat to your Bank by way of security for repayment of such finance. Notwithstanding anything to the contrary contained in the said agreement. We hereby register the Banks charge in our books in respect of the said Flat. AND Shri PARAG VIJAY MODI will not be permitted to transfer, assign, sell off or in any other way/ manner deal with the said Flat prejudicial to the interest of the Bank, without the prior written consent of your bank. 6. We undertake to form a Co-operative Society of the Flat holders of the aforesaid building under the Maharashtra Co-operative Societies Act.1960 within two years from the due hereof. We also agree to inform and given proper notice to the Co. operative Society as and when formed, about the said flat being so mortgaged to your Bank. 7. We are aware that relying on what is stared herein above, you have agreed to give the finance to Shri. PARAG VIJAY MODI P.S. Your are requested to issue your pay order in favour of sarasvat Bank c/a No CAPUB /11 M/s Gagangiri Construction. Instruction: If No.5 is not applicable then cancel the same and renumber the remaining paragraph.

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.

Why Rome Fell (a Condensed Version) :: essays research papers fc

Why Rome Fell (a condensed version)   Ã‚  Ã‚  Ã‚  Ã‚  The sun had long ago set, the newborn moon peeked out from behind a scattering of thin, high clouds. From a vantage point atop one of seven hills I could see glimpses of how this great city must once have looked. The mammoth buildings seem to shed their long years and are once again as they were; huge, awe inspiring, it is as if a portal in time had opened and I am afforded a glimpse into what was Rome. What could have caused this once master of all cities to fall? This paper will attempt to describe some of the explanations generally accepted, or should I say argued, and possibly shed some light on what could have caused the fall of what was, unquestionably, the most powerful empire in history.   Ã‚  Ã‚  Ã‚  Ã‚  I feel that I must begin with the explanations given by Edward Gibbon. While few agree entirely with his logic, his Decline and Fall on the Roman Empire is certainly unavoidable in a paper such as this. His work could be best summed up by the word confusing. According to David Jordan, ‘the causes for Rome's fall march across the pages of the Decline and Fall, seemingly without pattern, and seemingly unrelated to each other. This quote taken from the seventh chapter of Jordan's Gibbon and his Roman Empire sum up my feelings concerning the work; however, I will attempt to show some of Gibbon's Causes for this decline. Two of Gibbon's causes are the political blunders of its emperors and their search for personal glory. These are especially obvious in his chapters on Constantine. In them Gibbon accuses the emperor of destroying Rome for his own personal glory. Another cause would have to be the anti-Roman nature of Christianity. Gibbons argues that the ‘insensible' penetration of Christianity was fatal to the empire by undermining the genius of a great people. On a pessimistic note, Gibbon also lists as a ‘causes' the inevitable collapse of all human institutions, some arguments on the corrupting nature of luxury, and some detailed reflections on the vanity of human wishes. While the arguments presented are lengthily backed, they seem to fail in explaining the true nature of the fall.   Ã‚  Ã‚  Ã‚  Ã‚  Others, many others disagree with Gibbon's explanations and proffer their own for approval. One such author is David Woomersley who in his work, The Transformation of the Decline and Fall of the Roman Empire , openly attacks Gibbon's work calling it ‘a blunt instrument with which to dissect these centuries.' That quote, taken from chapter sixteen, is one of many which show the violent disagreement of the two ideas.

The Hunchback of Notre-Dame Essay -- essays research papers

In this novel The Hunchback of Notre-Dame (1831), Victor Hugo talks about the life of his characters in the city of Paris. This story takes place in the late-fourteenth century. With inequality all around it was hard for a person to gain respect without good looks or social status. In this paper I will mainly discuss the story of Quasimodoe Esmeralda, and their struggle in this story Quasimodoe`s mother was a gypsy. She could not take care of him any longer so she left him in front of a church. Gypsies were not liked at this time, but the church was highly respected. Being it that Quasimodo`s mom had left him on front steps of the church the king could do almost nothing. Quasimodoe could not leave the church due to the fact that he was deformed and ugly. Quasimodo grew very lonely and bored up in that bell tower all by him self. He was looking down one day and set eyes upon a young woman named Esmerelda. On the day of fools Quasimodo saw this young lady and decided to go do! wn and take a closer look at this woman. It was a holiday, feast of fools, when he decided to go down. When the people saw him they were horrified. He got whipped and lectured by his master. Well, to make a long story short Esmerelda ends up dyeing, and Quaismodo runs away and is never seen or heard of again. They state that in the end they find two skeletons lying in each others arms. One the body of a lady (Esmerelda), and the other the body of a man who was deformed (Quaismodo). It also states that th...

Macbeth Relationship Analysis Essay

I found the relationship between Macbeth and Lady Macbeth very interesting in the play. In Act I they are completely devoted to each other. Love, respect and trust are the contents of their relationship. The trust in the relationship is revealed right at the beginning when Macbeth sends his wife a letter telling her about the witches and the predictions â€Å"This have I thought good to deliver to thee, my dearest partner of greatness, that thee mightst not lose the dues of rejoicing by being ignorant of what greatness is promised thee.† (I, v, 10-13). The affection between the two is clearly shown when Macbeth salutes his wife with â€Å"My dearest love† (I, v, 58) and also on the letter where he says â€Å"†¦my dearest partner of greatness†¦Ã¢â‚¬  (I, v, 11). The respect comes when Macbeth listens to his wife, which talks about the murder. He respects her opinion and gives her a polite answer â€Å"We will speak further.† (I, v, 71). In Act I, ii we see that King Duncan considers Macbeth a brave soldier and good man â€Å"O valiant cousin, worthy gentleman!† (King Duncan, I, ii, 24). This can be considered a weakness or, perhaps, strength in the relationship, it depends on the point of view. It is a weakness if we analyze Macbeth’s side. He is â€Å"†¦too full o’ the milk of human kindness†¦ that wouldst thou holily; wouldst not play false†¦Ã¢â‚¬  (I, v, 16 & 21). It is strength if we analyze Lady Macbeth’s side. She has a very strong nature and knows Macbeth’s weak points, therefore manipulating and controlling the relationship. She uses of a great argument in Act I, vii accusing Macbeth for not having the courage to do what he wants â€Å"Wouldst thou have that which steem’ st the ornament of life, and live a coward in thine own steem, letting â€Å"I dare not† wait upon