Mutant and Wild-type Yeast Strains via Mitochondria Proteins

Mutant and Wild-type Yeast Strains via Mitochondria Proteins Differentiating between mutant and wild-type yeast strains via mitochondria proteins    By: Jason Hoang Lab Partner: Daryan Chan Introduction Yeasts are important organisms due to their uses in everyday such as baking, making fermented foods and alcohol production (Steensels et al, 2014). Yeasts have been so widely studied that it was one of the first organisms to have its genome sequenced (Goffeau et al, 1996). Thus, Yeasts are more than capable of acting as a model organism for eukaryotes (Botstein et al, 2011). For this experiment we are working with Saccharomyces cerevisiae. The mitochondria is the powerhouse for cell, as it is the major production site of ATP for the cell. The inner mitochondrial space has an electrochemical gradient, from which ATP is generated by using 5 protein complexes create an electrochemical gradient to assist in ATP production (Alberts et al., 2015). The COX6 assembly accepts electrons from cytochrome c and uses oxygen as the terminal electron acceptor to make water (Alberts et al., 2015). ATP synthase then uses the resulting proton gradient made by those complexes to pump protons back into the mitochondria matrix and make ATP (Alberts et al., 2015). The objective of this experiment was to determine if a given yeast sample was a wild type or a mutant with no COX6 activity. One of the major techniques to be used in this lab is subcellular fractionation. This technique first lyses the cells and then uses centrifugal forces to separate particles by size (Alberts et al., 2015). The centrifugal forces results in the denser particles moving away from axis of rotation creating a pellet which contains the heavier particles and a supernatant which contains lighter particles (Alberts et al., 2015). Another major technique used was Gel electrophoresis. Gel electrophoresis is used so that a fraction with multiple proteins can be separated based on size and shape (Alberts et al., 2015). Protein fractions are loaded on to wells in the gel and an electrode is attached (Alberts et al., 2015). SDS page is popularly used because it can confer a negative charge and linearize proteins being run through the gel (Alberts et al., 2015). The proteins will run through the gel due to their negative charge (Alberts et al., 2015). A standard is used to provide a reference to determine the sizes of the sampl proteins (Alberts et al., 2015). One of the other major techniques used in this lab was western blotting. After proteins are run on electrophoresis a labelled antibody is exposed to the electrophoresed fractions in a process called immunoblotting in order to detect presence of a specific protein (Alberts et al., 2015). The gel is exposed to a membrane where a current is run to dive the proteins onto the membrane (Alberts et al., 2015). The membrane is then drenched in labelled antibodies to detect for a specific protein (Alberts et al., 2015). This process can detect very small amount of specific protein and is useful for detecting changes of concentration of a specific protein in a cell under various conditions (Alberts et al., 2015). To measure cytochrome oxidase activity in this lab, we looked towards Beers Law which states that the ability of a solution to absorb light at a single wavelength is proportional to the concentration of solute in solution (Lukofsky et al, 2009). This show that absorbance and concentration are linearly related. Therefore, this would allow us to determine the rate of cytochrome oxidase activity in a sample. Materials and Methods Experiment was performed according to protocols set by Department if Biology, Winter 2016, Biology 331 for Experiment 1: Subcellular fractionation of yeast cells, pg 2-8, Experiment 2: Yeast growth curve; light microscopy; protein determination, pg 1-4, Experiment 3: Polyacrylamide gel electrophoresis, pg 1-8, Experiment 4: Development of Western Blot; COX Activity Assay, pg 3-10, written by Dr. Dragana Miskovic where the experiment was performed with no deviations unless specifically noted (Miskovic, 2017) The only deviation occurred in experiment 2 where we had ran out of BSA STD and had to borrow from another group. The borrowed BSA STD was not tested to have exact concentration as specific in lab protocol and may have had different concentration. Results Experiment 1 Yeast Strain Sample: A2 Table 1. Masses recorded for Lysing Yeast Cells Section Items Mass (g) Mass of Centrifuge Bottle containing Yeast 159.2 Mass of Empty Centrifuge with Pellet 49.89 Mass of Pellet 3.23 Amount of STE solution needed to resuspend yeast pellet: 3.23g x 2= 6.46mL Table 2. Volumes recorded for the Aliquoting Yeast Subcellular Fractions Section Solutions Total Volume LPS 3mL + 3.7mL= 6.7mL HSS 3mL + 2.8mL= 5.8mL MITO 300ÃŽÂ ¼L + 300ÃŽÂ ¼L= 600ÃŽÂ ¼L It was also noted that after the MITO fraction was made the pellet was intact and not messy Figure 1: Drawings of 50/100/200 ÃŽÂ ¼L dye drops from pipetman and expected 1 mL dye drop Experiment 2 Part A Table 3. Concentration of Yeast Cell at Two Different Times Time OD600 Reading Concentration (cells/ mL) 2:47pm 0.021A 210,000 4:59pm 0.043 A 430,000 It has been determined that an OD600 value of 1.0 is thought to contain roughly 1 x 107 cells/ mL. An OD600 value of 0.021 A will contain a concentration of 210 000 cells/ mL. An OD600 value of 0.043 A will contain a concentration of 430 000 cells/ mL. Figure 2. This graph shows the change in absorbance of yeast culture at 0 and 120 minutes. Equation to represent growth is calculated and shown above. Calculating Doubling time Formula for growth of yeast is y=0.0002x + 0.021. given initial absorbance reading of 0.021, doubled concentration should give reading of 0.042. Therefore use y= 0.042, where x means time in minutes 0.042=0.0002x + 0.021 where x = 105. Therefore it was found that doubling time is 105 minutes. Part B Yeast cells dyed with methylene blue stain Figure 3. These are some of the cell types that were observed when the overnight culture was stained with methylene blue under 40x magnification. It was found that roughly a third had a stained positive for a nucleus. None of the cells appear to be multi-nucleate. No vacuoles were observe either Yeast cells dyed with neutral red Figure 4. These are some of the cell types that were observed when the overnight culture was stained with neutral red under 40x magnification It was found that over 90% of the cells stained positive for a nucleus. Many of the cells appeared to be multi-nucleate and budding as well. It appeared that 1 or 2 vacuoles appeared to be detected per cell. Part C Table 3. Absorbance Results for the BioRad Protein Determination Assay 1 2 3 4 5 6 7 8 9 10 A 0.27 0.191 0.21 0.201 0.196 0.37 0.397 0.404 0.369 0.036 B 0.246 0.24 0.303 0.192 0.226 0.245 0.272 0.372 0.252 0.035 C 0.23 0.263 0.248 0.294 0.037 0.036 0.036 0.036 0.036 0.036 D 0.256 0.227 0.25 0.277 0.035 0.035 0.035 0.035 0.035 0.034 E 0.246 0.182 0.242 0.215 0.474 0.362 0.306 0.389 0.482 0.035 F 0.289 0.349 0.285 0.246 0.299 0.264 0.347 0.738 0.203 0.036 G 0.203 0.254 0.321 0.249 0.035 0.035 0.035 0.037 0.035 0.036 H 0.2 0.261 0.263 0.274 0.034 0.034 0.035 0.035 0.035 0.034 11 12 A 0.036 0.037 B 0.035 0.035 C 0.037 0.038 D 0.035 0.044 E 0.036 0.035 F 0.035 0.035 G 0.035 0.035 H 0.035 0.034 Figure 4. This graph shows absorbance readings of standard solution using BSA at different concentrations. Calculating concentrations of LSP, HSS, and Mito. Equation for concentration of solution based on absorbance reading was determined based on above graph. Equation yielded was y=0.1264x + 0.2159 Average absorbance readings: LSP was 0.245, HSS was 0.249, and MITO 0.289. Using found readings as y for above equation we calculated concentration of proteins in each sample. LSP: 0.245 = 0.1264x + 0.2159Therefore x = 0.230 mg/mL HSS: 0.249 = 0.1264x + 0.2159Therefore x = 0.262 mg/mL MITO: 0.289 = 0.1264x + 0.2159Therefore x = 0.551 mg/mL Dilution factor needed to get fraction to 2ÃŽÂ ¼g/mL LSP: (0.230 mg/mL)(0.1mL)=> (0.0230 mL)(1/x mL)(10 dilution factor) = 2mg/mLTherefore x= 0.115 mL HSS: (0.262 mg/mL)(0.1mL)=> (0.0262ÃŽÂ ¼g)(1/x mL) (10 dilution factor) = 2mg/ mL Therefore x= 0.131 mL MITO: (0.551 mg/mL)(0.1mL)=> (0.0551ÃŽÂ ¼g)(1/x mL) (10 dilution factor) = 2mg/ mLTherefore x = 0.2755 mL Experiment 3 Figure 5. PVDF Membrane after proteins are transferred over from gel after electrophoresis. Our group is left side (D.C, J.H) Experiment 4 Figure 6. Membrane after detecting solution had been added over 10 minutes ago. Bands on right hand side are the standard Figure 7. This graph shows the distance travelled by each protein in the standard mix against the Log(Mw) on semi log paper Table 4. Cytochrome c Oxidase (COX) Activity Assay Sample Absorbance At 0 sec (OD) Absorbance after 20 sec (OD) Change in absorbance Change in Concentration (ÃŽÂ ¼mol/mL) COX activity (ÃŽÂ ¼mol/ L/min) Blank 0.525 0.525 0 0 0 LSP (1) 1.259 1.253 0.006 0.2143 0.6429 LSP (2) 1.272 1.264 0.008 0.2857 0.8571 HSS (1) 0.493 0.491 0.002 0.0714 0.2143 HSS (2) 0.496 0.491 0.005 0.1786 0.5257 MITO (1) 0.553 0.557 -0.004 -0.1429 -0.4286 MITO (2) 0.537 0.535 0.002 0.0714 0.2143 Table 4. This shows the Sample calculation for COX activity Change in absorbance = Absorbance at 0 sec Absorbance after 20 sec 1.259-1.253 = 0.006 Change in concentration ΆA = ÃŽÂ µ x b x Άc 0.006 = 28mM-1-cm-1 x 1 cm x ΆcTherefore Άc = 0.0002143 mM => 0.2143 ÃŽÂ ¼molAssuming volume of assay is 1.0 mL, change in concentration is 0.2143 ÃŽÂ ¼mol/mL COX activity COX activity = change in concentration / time 0.2143 ÃŽÂ ¼mol/mL / (1/3 min) = 0.6429 ÃŽÂ ¼mol/mL/min Figure 8. Graphical representation of COX activity in LSP fractions Figure 9. Graphical representation of COX activity in HSS fractions Figure 10. Graphical representation of COX activity in MITO fractions Discussion Galactose was used over glucose as a carbon source for our yeast cells. This is because we wanted to determine if the mitochondria was functional in our yeast cells. Different yeast strains will use different metabolic pathways in presences of each. When glucose is used as a carbon source the yeast cells will generate ATP via fermentation, whereas when Galactose is used the cell will perform oxidation. This is important to observe as different yeast strains will have varying levels of cytochrome c usage based on that. To visually determine if cytochrome c will be utilized by the cell we can look at the fractionation experiment earlier. When separating for the MITO fraction if one had found a messy pellet it would have indicated that the mitochondria was not intact while solid pellets would indicate the mitochondria was intact. If one did find a messy pellet it could have been the result of differences in fractionation techniques, cells being lysed prior, or something had disturbed the cell in transport. For our experiment we had found the mitochondria to be intact, which is a strong indicator that the mitochondria for our sample was present. To reach cytochrome c oxidase (COX) we used diferential centrifugation which seperates objects based on size and density, where larger molecules such as the intact cells will settle at the bottom of a tube while mitochondria which is smaller would remain in supernatant. This is also why we had separate centrifugations, to get samples with intact cells and samples with intact mitochondria. Density gradient centrifugation is also a widley used technique that seperates based on density. In that case we would see multiple bands form in tubes with densest molecules gathering at the bottom and bands above it with less dense molecules. Experimentally we found yeast doubling time to be 105 minutes (1.75 hours) when inoculated in YPD (1% yeast extract, 1% peptone, 2% glucose). It has been determined in many other experiments that Saccharomyces cerivisiae has a doubling time of 1.69 hours (Deak, 2008). The difference could be attributed to many factors such as environment (amount of light, heat, and etc) and growth substrates used. But the difference is not very large and would still be considered to conform to literature results. During the methylene blue staining of yeast cells it was noted that roughly a third of yeast cells contained a nucleus but it did not seem to be multi nucleate. While the neutral red stains showed that many cells appeared to be budding with one or 2 vacuoles present per yeast cell. These findings fall in line with what is normally expected from yeast cells as they do have vacuoles in their cells (Armstrong, 2010). Furthermore results also fall in line with yeasts having nucleuses but not being multi nucleated (Roberts and Ganesan 1959) One thing that may have affected a major portion of the experiment was determining the concentration of each respective LSP, HSS, and MITO fraction and diluting it to 2mg/mL. it is important to note that during pipetting steps to get each sample that the suspensions be homogenous beforehand otherwise you may be taking up different components of the fraction and missing others depending on how deep the pipet was inserted. During the remainder of the experiment it was found that after gel transfer to PVDF membrane and during western blotting that very few to no proteins were showing up. If low concentration of protein was a factor then it would most likely be traced back to this step. Many reasons can be attributed to this for instance, poor pipetting technique, the fractions were not homogenized properly before pipetting or even the dilution factor could have been incorrect. As noted during the material and methods during the preparation of experimental samples which would to create o ur protein concentration standard curve we had run out of BSA STD and required taking some from another group. When we created our protein concentration standard curve it came out completely odd, having unexpected drops in absorbance readings. The expected result was a linear curve where a higher BSA STD concentration would have led to a higher absorbance readings. Due to the change in BSA STD this may have had a different concentration due to being taken from a different location in its container it could have had a different concentration. Thus causing inconsistencies for our standard curve. As the standard curve was deemed incorrect afterwards any protein concentration calculations based on it would have been flawed, leading to incorrect dilutions. If the dilutions been calculated incorrectly, as they most likely were, there is the chance that the protein fractions would have been over diluted leading to not enough protein to be present for visible bands for the gel electrophores is and western blotting. For the gel electrophoresis SDS was included in solubilisation buffer to give proteins inserted into the wells a negative charge so that when a charge was applied they would run to the other end of the gel and to help unfold the protein so that it would be able to go through the gel. For this experiment a 12% gel was used in the interest of saving time because a 15% gel would have caused the proteins to go through it slower leading to a lower resolution of identifiable protein bands. The purpose of transferring proteins from gel to PVDF membrane was to be able to visualize the movement of proteins on the gel after electrophoresis. To accomplish this we applied Ponceau stain to the membrane to increase the resolution of the bands and to ensure equal amounts of proteins are loaded onto the gel (Al-Amoudi et al., 2013). It was found that after proteins had been transferred to PVDF membrane that we had very few bands show up for the solubilizing buffer lane, both LSP sample lanes, and both HSS sample lanes. Bands did appear for both MITO samples, however, it appears that got smeared across the gel, bleeding over to other wells. This could have been the result of diluting samples in the wells for reasons noted above, the SDS gel would have been poorly constructed and contributed to the smearing, and poor electrode contact on the gel might have blocked the gel from having proper electrical charge. Issues could have also arisen during transfer of proteins from gel to me mbrane. Air bubbles could have been present during transfer which would have prevented any protein from being transferred as proteins cannot move through air. Additionally, poor folding between membrane and the gel could have attributed to smearing of MITO samples. The purpose of the western blotting was to be able specifically detect for the presence of biotinylated COX proteins. In order for a cell to express a biotinylated protein it needs to be able to take up foreign DNA, be able to properly fold COX-biotin fusion protein, the cell needs to be able to recognize the BSS signal fused to C terminus, and be able to translate COX and biotin together (moving stop codon so that it doesnt not stop halfway across the other.) It was found that after western blotting our membrane with protein fractions that no bands had appeared even after 10 minutes of membrane being in contact with detecting solution. This led to Figure 7. The chart showcasing the relative distances that proteins have travelled is blank as a result. This would imply that when the blocking solution was added that it managed to block the entire membrane (and any present proteins included) from interacting with the probe. However Tween-20 was used to wash excess reagent. So the milk most likely would not have been able to bind to any protein after introduction of Tween-20. Therefore the lack of data could be attributed to low concentrations of protein on membrane for reasons as noted above. Referring to Figure 5 the only proteins that were found on the membrane after were MITO which shows that there would have been no LSP or HSS for probe to bind to, whereas for present MITO sample the concentration may not have been high enough and as a re sult some of it could have been washed out by the methanol step causing concentration of MITO to be so low that it could have been blocked by the blocking solution. There is also a possibility that our yeast samples were not able to biotinylate the COX protein at all which could explain why there were no bands occurring Looking at COX activity graphs for LSP, HSS, and MITO they seem to follow what is expected except for HSS. COX was used as an identifying marker for identifying subcellular fractions containing COX because it is an integral membrane protein for the inner membrane space. If COX activity is present then that would indicate that the mitochondria is intact and functioning. These samples should have seen increased COX activity as cytochrome c was introduced into the fractions which provide electrons to the COX protein allowing it to pump proteins and reduce oxygen to water. Both MITO and LSP experienced increased COX activity as shown by figure 8 and 10 respectively. This falls in line with what was expected with the MITO fractions experiencing higher levels of COX activity then the rest as the cytochrome c had less of a distance to travel to reach inner mitochondrial membrane space than LSP. LSP should have a signal because it would contain intact yeast cells which have mitochondria Albe rts et al., 2015). Therefore LSPs rate of COX activity should be lower because the cytochrome c would have harder time reaching mitochondria. This is shown by Figure 10 having steeper reaction times than Figure 8 and 9. This reaction utilized Deoxycholate (DOC) to speed up the reaction which is why it was only done in 20 second intervals as DOC solubilizes with cytochrome c so that it can enter the mitochondria to interact with COX. If reactions were tested too long after DOC was added then the reaction would have finished before being able to measure absorbance. The one that did stand out was the HSS fraction which appeared to experience negative COX activity or none at all. This was expected as it should have all the remaining parts of the cell that werent the mitochondria, lysosomes and peroxisomes Alberts et al., 2015). This would indicate that these samples did not have an intact mitochondria with a COX protein to interact with cytochrome c. this could be explained by the In conclusion it was found that our yeast strain A2 is the wild type strain. This is because during initial centrifugation the resulting pellet was solid indicating intact mitochondria. Furthermore during COX assay the MITO strain indicated an active COX as shown by its increase in activity, proving that A2 was in fact a wild type strain with functioning mitochondria. References Al-Amoudi, M.S., Salman, M., Al-Majthoub, M.M., Adam,Abdel Majid A., Alshanbari, Naif A., Refat, Moamen S., (2013) Res Chem Intermed 41: 3089. doi:10.1007/s11164-013-1417-4 Alberts, B. et al. (2015). Molecular Biology of the Cell Sixth Edition. New York, NY: Garland  Science, Taylor Francis Group. Armstrong, John. Yeast vacuoles: more than a model lysosome. Trends in Cell Biology 20.10 (2010): 580-85. Web. 13 Mar. 2017. Botstein, D., Chervitz, S. A., Cherry, J. M. (1997, August 29). Yeast as a Model Organism. Retrieved March 12, 2017, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3039837/ Deak, Tibor. Handbook of Food Spoilage Yeasts, Second Edition. Contemporary Food Science (2007): 50-51. Web. 13 Mar. 2017. Glerum, Moira Miskovic, Dragana (2017). Biology 331 Advanced Cell Biology Laboratory Manual Winter 2016. Biology Department, University of Waterloo, Waterloo. Goffeau A, Barrell BG, Bussey H, Davis RW, Dujon B, Feldmann H, Galibert F, Hoheisel JD,  Jacq C, Johnston M, Louis EJ, Mewes HW, Murakami Y, Philippsen P, Tettelin H, Oliver SG  (1996) Life with 6000 genes. Science 274, 546 Lukofsky, David, Jonathan Bessette, Heejeong Jeong, Elsa Garmire, and Ulf ÃÆ'-sterberg. Can precursors improve the transmission of energy at optical frequencies? Journal of Modern Optics 56.9 (2009): 1083-090. Web. 13 Mar. 2017. Roberts, C., and A. T. Ganesan. The occurrence of multinucleate giant cells in yeasts. Antonie van Leeuwenhoek 25.1 (1959): 97-107. Web. 13 Mar. 2017. Steensels, Jan, Tim Snoek, Esther Meersman, Martina Picca Nicolino, Karin Voordeckers, and Kevin J. Verstrepen. Improving industrial yeast strains: exploiting natural and artificial diversity. FEMS Microbiology Reviews 38.5 (2014): 947-95. Web. 13 Mar. 2017.

Report on Adidas PEST and SWOT Essay

In this report I have tried to sketch a complete organizational picture of the adidas company. This include of current position of the company and its market share though out the world. To explain it the company’s SOWT and PEST analysis is briefly elaborated. A contrast of the Adidas with its competitors is added and the internal structure of the company is tried to shown in graphical snaps. The future strategies of the company are also listed that what kind of strategies formulated for adidas by their top managers. 2. Addias: Adidas is a sportswear manufacturing company that was started by Adolf Dassler. Adidas group has several brands including Adidas, Reebok, Taylor Made-Adidas and the Rockport. The company has spread its wing to incorporate other productions including handbags, shirts, spectacles, watches, balls, and sportswear. It is the largest company that sells footwear in the European market and has gained a significant market share at the global platform. Adidas has had a remarkable sale and has risen to the competition at the global scale with other international footwear companies. McDonald & Milne, (1999) state it in their journal. 2.1 Mission and vision of adidas: As the company is leading the global business of manufacturing and distribution of sports wear and accessories primarily to improve the sportsmen lifestyle. Adidas Group focuses on the objectives and puts total commitment towards strengthening the brand names. That’s why the mission and vision statement of the company is defined according to global context. Lussier & Kimball (2009) have both shown it in their work. 2.1.1 The Mission statement: â€Å"Our mission is to become the best sports brand in the world. To that end, we will never equate quantity with quality. Our founder Adi Dassler was passionate about sports. For Adidas, the athlete came first. He gave those on the field, the court and the track the unexpected and the little differences that made them more comfortable and improved performance. This is our legacy. This is what the brand stands for. This will never change.† 2.1.2 The Vision Statement: â€Å"To make adidas, products as high as sky and as wise as an owl† 2.2 The Functional over view of the company: The company operates in different segments under the executive brand name across the globe. These segments include retail and wholesale. The wholesale segment plays the leading role in the distribution of products from both Adidas and Reebok centers to all retail stores across the globe. On the other hand, the retailers are solely responsible of meeting with the demand of customers at the retail shops across the global market. Plunkett, ( 2008) explain it in his research. The Current Brand Division of Adidas Group: 2.3 The Strategy of Adidias Group: The adidas Group strives to be the global leader in the sportswear industry with brands built upon a passion for sports and a sporting lifestyle. We know that a profound understanding of the consumer and customer is essential to achieving this goal. To anticipate and respond to their needs, they continuously strive to create a culture of innovation, challenging there selves to break with convention and embrace change. By harnessing this culture, they push the boundaries of products, services and processes to strengthen their competitiveness and maximize the Group’s operational and financial performance. This, in turn, will drive long-term value creation for their shareholders. 3. The SWOT Analysis: Following is the brief SWOT analysis of the company in global context. 3.1 Strengths: Adidas is the world’s second largest maker of athletic footwear, apparel and equipment by Sales with revenues of $15,333.30 million Adidas group’s leading market position is built on its portfolio of strong brands, which enhances adidas market position and boosts its top line. Adidas’ portion of own retail has grown substantially giving adidas more brand control. Adidas operates over 2,200 stores for the adidas and Reebok brands worldwide. Adidas own†retail business includes: – e†commerce, – Mono branded stores run by retail partners, – Shop in shops established with key accounts, – Joint ventures with retail partners, – Co branded stores with sports organizations or other brands. 3.2 Weaknesses: China negatively impacted by clearance of high excess inventories following the Beijing 2008 Olympic Games which subdued consumer demand in 2009. Adidas outsources 95% of production to independent third†party suppliers in Asia, including 32% from China. Merchandise procured from foreign manufacturers is a weakness because it gives adidas less control over the product quality. †¢ Concerns over unsafe Chinese consumer products †¢ The Consumer Product Safety Commission (CPSC) has issued alerts and announced voluntary recalls by US companies on numerous products made in China. Vendor and manufacturer failures to achieve and maintain high manufacturing standards could result in manufacturing errors resulting in: †¢ Product recalls or withdrawals, †¢ Delays or interruptions of production, †¢ Cost overruns 3.3 Opportunities: Adidas has sponsorship agreements for major sports events across the globe †¢ Japan Football Association until March 2015 †¢ Australian Olympic Committee until 2016 †¢ Secured sponsorship rights to the 2014 FIFA World Cup Extended its partnership with: †¢ UEFA for the UEFA EURO 2012 †¢ UEFA EURO 2016 football championships †¢ UEFA Champions League Signed an 11†year global merchandising partnership agreement with the NBA †¢ Makes adidas the official uniform and apparel provider for the NBA, WNBA and the NBA Development League Official Sportswear Partner to 2012 Olympics in London. Sponsorship of major sports events helps the company strengthen its profitability and enhance its brand recall among consumers. 3.4 Threats: Widespread counterfeits deprive revenues for the company and can dilute the adidas brand image. The market for sports apparel and footwear has declined in 2009. †¢ Athletic footwear declined 3.2% in 2008 & 1.4% in 2009 †¢ Decline due to 3% decline in men’s footwear segment †¢ Active apparel declined by 4% †¢ Sports use apparel decreased by 5.5% in 2009. Lussier, R. N , & Kimball, D. C. (2009) explain it in their analysis report . 4. The PEST analysis: The external impact of adidas to worldwide is explained here. 4.1 Political: Adidas generates policies and monitor hazardous substance to protect human health and environment and also it protects the rights of its employees by following labor laws on country specific way. Adidas a multinational company need to consider the global political state as well as domestic political issues. It is important to monitor the government’s laws which affect the business of adidas. Since the political arena has a huge influence upon the regulation of public and private sector businesses, and the spending power of consumers and other businesses it has come necessary for adidas react dynamically and efficiency for these issues. The government enters in to any trade agreements adidas needs to keep it on track since it may affect the business. Government taxation policy plays a major role in company’s profit that’s why the efficient management of the adidas is always emphasizing on it. 4.2 Economical: Adidas as a multinational company maintain its strong economical growth year by year. In 2008, the adidas Group again delivered a strong financial performance. The product sales and the profitability gowned up in line with management’s initial expectations. Currency-neutral sales increased by 9%. And the double digits sales increase in adidas segment had the biggest impact on this development. Adidas managed to reduce its production cost with improving the product quality by locating the factory plants in Asia. 4.3 Social: There are no barriers for Adidas products like raise, age, religion, and lifestyle, it is always in fashion with special design in any product. Adidas focus in people who like sports and athletes, almost everybody beyond the boundaries. 4.4 Technological: Adidas introduces modern approaches to doing new and old things, Adidas join into technology by make up the world’s first â€Å"smart shoe†, adding a microchip inside the shoe and wireless mp3 player. Adidas uses hot melt system of production which is environment friendly. 5. Conclusion: The sportswear industry has developed into being more than just selling sportswear- and equipment. Due to heavy competition, organisations need to differentiate themselves and focus on both product attributes and brand values when creating brand strategies. Therefore, we can finally conclude that adidas brand themselves through personality traits and value propositions. However, the self-expressive, emotional, and functional benefits of the brands are somewhat diverse as they brand themselves through different personalities and therefore have different brand strategies. 6. References: McDonald, M. A., & Milne, G. R. (1999). Cases in sport marketing. London: Jones & Bartlett Learning. Lussier, R. N., & Kimball, D. C. (2009). Applied sport management skills. London: Human Kinetics. (Plunkett Research Ltd & Plunkett,2008). group.com/en/pressroom/assets/Resource_Center/adidas_Group/pdfs/Factsheet_Herzogenaurach_en.pdf Lussier, R. N., & Kimball, D. C. (2009). Applied sport management skills. London: Human Kinetics.

Comic Art The Seduction of the Innocent Essay - 3314 Words

Comic Art: The Seduction of the Innocent In 1991, at the 13th Annual World Fantasy Convention, an issue of the comic book series The Sandman was selected by a panel of experts in the field as the Years Best Short Story. This was not the first time that a comic book has been nominated for a prestigious literary prize (the first and only previous one being Art Spiegelmans retelling of the Holocaust in animal fable form Maus for the National Book Critics Circle Award in 1987), but it was the first to have won. The ensuing uproar at the awards ceremony and the umbrage that many took at a mere comic book winning instead of a standard-print story resulted in the rules of the awards being changed. Henceforth, no comic book could be†¦show more content†¦But as Neil Gaiman, writer of The Sandman once remarked at being told that he did not write comic books, he wrote graphic novels, that it was like someone being informed that she wasnt actually a hooker; that in fact she was a lady of the evening (Bender, 4). This person, t he editor of the literary review pages of a major newspaper, had obviously heard positive things about Sandman; but he was so stuck on the idea that comics are juvenile he couldnt deal with something good being done as a comic book. He needed to put Sandman in a box to make it respectable (Bender, 4). What makes comics different from the respectable fields of literature or fine art? Comics rely on a combination of words and pictures to bring across a message, to tell a story. To even use the medium is an exercise in creativity itself. The possible combinations of text and pictures are endless, and to create a coherent meaning and consistent tone of narrative from the manipulation of text boxes, word and thought balloons, panels and artwork is no easy task and require a strong mastery of the medium. Neil Gaiman is one such master. His series The Sandman has won numerous accolades (besides the World Fantasy Award earlier mentioned) since Sandman #1 was first published in 1989. It has been recognised as a groundbreaking title in comics. The story of how a god-like being, the personification of dreams andShow MoreRelatedThe And The Dark Knight And Shows Like The Walking Dead Comic Book Based Properties1688 Words   |  7 PagesComic books, in todays rise of all that is geek it is har d to avoid hearing about the next comic that is being adapted into a movie or TV show. With films like The Avengers and The Dark Knight and shows like The Walking Dead comic book based properties are taking over most of pop culture today. Yet, despite all the attention on these properties there is still an overwhelmingly popular misconception that comics are â€Å"kids stuff.† Yet, unbeknownst to the overall public in western society comics areRead MoreArgumentative Essay On Comic Books1642 Words   |  7 PagesEver thought how dumb comic books or graphic novels were growing up? Why such avid readers were called (myself included) nerds? Yet were smarter than you? Think back to how geeky their way of speaking was and they touched on such complexities even for a book! Shortly, reasoning will tell you why comic books are the best. Over the years, from the 20th to 21st century, research and documentation has been stacking up to prove that comic books make their readers smarter. Comics make you want to readRead MoreThe Discussion of Homosexuality in Comic Books1980 Words   |  8 PagesThe discussion of homosexuality in comic books has a long history, reaching back to 1954, when German-American psychiatrist Frederick Wertham published Seduction of the Innocent, a book that warned about the negative effect of popular literature. The book was a minor bestseller and was taken seriously at the time, creating alarm in parents with the claim that reading the crime-, superhero- and horror comics that contain descriptions of violence, sex, drug use and other adult themes, children willRead More Tennysons Merlin and Vivien Essay1646 Words   |  7 Pageswhich attacked the corruption of Camelot (Ricks 92). One of the most intriguing chapter of the Idylls is Merlin and Vivien, a narrative poem with both allegorical and moral points (Reed 48). Merlin and Vivien was written to demonstrate how comic expectations are foiled by ironic actuality (Kincaid 177). Merlin and Vivien is said by many to be one of the most ill-tasted chapters of The Idylls. Jerome Buckley comments that Merlins yielding to the seductive wiles of Vivien is... the Read MoreA Semi-Brief History of the Visual Narrative2279 Words   |  10 Pages American culture simply would not have been the same without it. The visual narrative, under hundreds of misnomers, combines the power of written word with the added impact of image to create an entirely new aesthetic experience--that of literary art. Since the beginning of human memory, there has been a tendency to communicate with images as often as, if not more than, words; lucky for us, Images are an international language, according to Marjane Satrapi (Foroohar 58). The earliest evidenceRead MoreSociety s Influence On American Culture1865 Words   |  8 PagesMaddie Fuller HNRS A192 Research Analysis Dr. Stone Since their conception, comic books have exhibited significant influence on American culture, especially through characters and storylines developed during times of war. Although the ideologies have changed and shifted over time, it is no secret that the writers have a message they are attempting to convey, especially through mainstream superheroes. Andrew Bolton’s definition of a superhero is, â€Å"American utopianism as expressed in the DeclarationRead MoreEffects of Reading Comic Books5082 Words   |  21 PagesEffects of Reading Comic Books on the Young Reader OUTLINE TITLE: EFFECTS OF READING COMIC BOOKS ON YOUNG READERS INTRODUCTION I. HISTORICAL OVERVIEW A. Rise of the Comic Strip B. The Comic Book Superheroes C. The Philippine Comic Book Industry II. COMIC BOOK CHARACTERISTICS A. Unique Characteristics B. Parts of the Comic Book C. Comic Book Genres III. EFFECTS OF COMIC BOOKS A. Negative Effects of Reading Comic Books B. Positive Effects of Reading Comic Books CONCLUSIONRead MoreJoker and Batman Essay9227 Words   |  37 PagesBatman (originally referred to as the Bat-Man and still sometimes as the Batman) is a DC Comics fictional superhero who first appeared in Detective Comics #27 in May 1939. He has since become, along with Superman and Spider-Man, one of the worlds most recognized superheroes.[1] Batman was co-created by artist Bob Kane and writer Bill Finger, although only Kane receives official credit for the character. Batmans secret identity is Bruce Wayne, billionaire industrialist, playboy, and philanthropistRead MoreEssay on Defining Lolita: the Novel and the Name2268 Words   |  10 PagesFor me a work of fiction exists only insofar as it affords me what I shall bluntly call aesthetic bliss, that is a sense of being somehow, somewhere, connected with other states of being where art (curiosity, tenderness, kindness, ecstasy) is the norm (314 - 315). He sees his novel in simple terms: art. Whether it be the novel Lolita or the name Lolita, a sexual meaning has been given to the word Lolita, and this is largely due to the strong sexual overtones used by the novels main character HumbertRead MoreAmerican Civil Rights Movement Essay15820 Words   |  64 Pages One of the most popular producers of silent cinema was David York Griffith. shot 61 short films. most promising american producers. =Shakespeare of Screen. a genius producer, a founder of new cinema language. innovator in the sphere of actor art. G. specialized in melodramas, he shot comedies, historical films, thrillers, westerns, filmings of Bible and different works of literature. Before 1913 there existed an opinion, that the spectator can not watch the film longer than 15 minutes. He shot

What Does a Strong College Applicant Look Like

Many of the most selective colleges in the United States reject far more students than they accept, so its only natural to ask what qualities and credentials the admissions folks will be looking for. What makes one applicant stand out while another gets passed over? This series—What Does a Strong College Applicant Look Like?—addresses this question.   There is no short answer. A strong college applicant can be outgoing or reserved. Some successful applicants lead from the front, some from behind. Some show remarkable academic skills, while others have impressive talents outside of the classroom. A college might be impressed with one applicants theatrical accomplishments, while another might have been too busy with a job to get involved in after-school extracurricular activities. This is as it should be. Nearly all colleges believe that the best learning environment is one in which students have diverse talents and backgrounds. The admissions folks are not looking for a specific type of student, but a wide range of students who will contribute to the campus community in meaningful and different ways. When applying to college, you need to tell your story, not try to conform to some type of mold you think the college prefers. That said, strong college applicants do need to prove that they are well-prepared for college and will enrich life on campus. The categories explored here will help you think about the defining features of a successful college applicant. The Defining Features of a Strong Applicant At 99% of colleges, your schoolwork trumps every other piece of your college application. The  first section, A Solid Academic Record,  looks at the elements that make up  a good academic record. If youve taken AP and Honors courses that have weighted grades, its important to recognize that many colleges will recalculate those grades to create consistency across the applicant pool. Whether a college is highly selective or not, the admissions folks are going to want to see that youve completed a sufficient college preparatory core curriculum. The  second section on Required Courses  looks at the types of math, science, and foreign language classes colleges like to see in an applicants high school transcript.   The best academic records reveal that applicants have taken the most challenging courses available at their schools. If you have a choice between an elective course and an Advanced Placement course, youd be wise to take the AP course if you are applying to selective colleges. The admissions folks will also be impressed if youve completed an  International Baccalaureate (IB) curriculum. As youll learn in the third section, successful completion of AP or IB courses is one of the best indicators of college preparedness. Your high school curriculum and grades arent the only academic measures used by colleges. The  fourth section covers the role of  Test Scores  in the admissions process. A good SAT score or good ACT score can strengthen an application significantly. That said, there are plenty of ways to compensate for low SAT scores, so less-than-ideal scores dont need to sabotage your college ambitions. Academic preparation, of course, is not the only defining feature of a strong college applicant. Colleges want to admit students who lead rich lives outside of the classroom and who bring their interests, talents, and experiences to the campus community. In the  fifth  section, Extracurricular Activities,  youll learn that  the best extracurricular activities are those that reveal your depth of interest and leadership skills. Colleges recognize, however, that extensive extracurricular involvement isnt an option for all applicants, and that work experience can be equally valuable. The best college applicants continue growing and learning in the summer, and the  final section,  Summer Plans,  looks at some of the best summer plans for high school students. The most important strategy here is to do  something. Whether that be travel, a job, or a creative writing camp, youll want to show the admissions folks that you use  your summers productively. A Final Word on Strong College Applicants In an ideal world, an applicant shines in all areas: she earns a straight A average in an IB curriculum, gets near perfect ACT scores, plays lead trumpet in the All-State Band, and receives All-American recognition as a star soccer player. However, the great majority of applicants, even those applying to top schools, are mere mortals. As you work to make yourself the strongest applicant possible, keep your priorities in order. Good grades in challenging courses come first. A weak academic record will almost certainly land your application in the rejection pile at highly selective colleges and universities. SAT and ACT scores matter at most colleges, so its worth putting in some effort with a review book to prepare for exams. On the extracurricular front, what you do doesnt matter nearly as much as how you do it. Whether it be a job, club,  or activity, put in your best effort and stick with it. Most importantly, realize that there are many kinds of strong applicants. Try to resist comparing yourself to your classmates, and avoid the trap of trying to second guess what you think a college is looking for. Put your heart and effort into being your best self, and youll be positioning yourself well for the college admissions process.