Mit Case Study

BCG – Join BCG – Interview Prep – Practice Cases – Distribution†¦ http://www. bcg. com/join_bcg/interview_prep/practice_cases/dis†¦ The Boston Consulting Group Home > Join BCG > Interview Prep > Practice Cases > Distribution Strategy Distribution Strategy Crafting a Distribution Strategy for a Sugar Cereal Manufacturer Your client is the sugar cereal division of Foods Inc. , a U. S. -based distributor and manufacturer of packaged foods. According to the division president, Foods Inc. ‘s traditional strength has been with grocery stores, which still account for the majority of its $1. billion in sugar cereal sales. But Big M Mart, a discount chain, has been growing at a healthy rate of almost 15 percent per year and has now become Food Inc. ‘s largest customer. Your client is not sure how to react, and has asked BCG for assistance with its distribution strategy. Establish Understanding of the Case First, let me make sure I understand t he problem. Our client specializes in sugar cereals traditionally distributed through grocery stores. Sales to Big M Mart, a discount chain, have been growing at 15 percent per year, and the chain has recently become the largest distributor of the client's product nationwide.We are here to help evaluate the distribution strategy in light of Big M Mart's growth. That is correct. Could you explain to me how grocery stores differ from discount stores? Sure. Grocery stores generally specialize in food, as well as selling some household goods and over-the-counter pharmaceuticals. Discount stores, on the other hand, offer food alongside a wide variety of merchandise, including clothing, home electronics, and housewares. Does Big M Mart market its food products differently than do grocery stores? Discount stores advertise lower prices for a wide variety of foods, particularly staple, nonperishable foods.Could I take a moment to write a few notes to myself? Please feel free. Set Up the Fram ework Before making recommendations, I think we would need to evaluate whether sales growth at Big M Mart is good or bad for Foods, Inc. To do that, I would first look at how its sugar cereal performance at Big M Mart compares with that in other distribution channels. Second, I would look at its performance at Big M Mart in relation to competitors' performance. Next, I would determine what drives customer purchases. Finally, I would want to understand the supply chain.That certainly sounds like a reasonable approach. Let's proceed. Evaluate the Case Using the Framework 1 of 6 10/2/09 6:57 PM BCG – Join BCG – Interview Prep – Practice Cases – Distribution†¦ http://www. bcg. com/join_bcg/interview_prep/practice_cases/dis†¦ First, I would like to get a better sense of where Big M Mart stands in relation to our client's other distribution channels by examining the client's sales data and margins, by distributor. The marketing department does not hav e margins by channel, but tracks sales and volume for its top five distributors.What does this imply about Big M Mart as a distribution outlet? It looks as if the top distributors have been growing more important, but particularly Big M Mart, which is growing faster than all the others. This is particularly true when we look at volume, where Big M Mart's growth is much higher than that of the other four channels. And how could you interpret what these data says about margins? While the client's sales through other distribution channels are growing faster than volume, Big M Mart volume and sales growth are the same, so the average price paid by Big M Mart has remained constant.That implies that sales growth at Big M Mart could have negative implications for our client's margins. Next, I would like to look at how our client is doing in relation to the competition within Big M Mart. Have they been gaining or losing market share? How might you find that out? I would try to interview Big M Mart's purchasing personnel, since they would probably track those data for their own purposes. Why would they want to talk to you? How might you approach such an interview? I would approach the purchasing personnel and suggest that our client and Big M Mart work ogether to identify best practices to reduce costs and increase sales of sugar cereals at Big M Mart. Let's say in a perfect world you could get a breakdown of Big M Mart sales for the four largest competitors (see market shares below). 2 of 6 10/2/09 6:57 PM BCG – Join BCG – Interview Prep – Practice Cases – Distribution†¦ http://www. bcg. com/join_bcg/interview_prep/practice_cases/dis†¦ What can we infer about our client's competitors within this channel? Who should they be worried about? It looks like our client is losing market share, as is Tasty Breakfast, while Cereal Co. nd Private Label are gaining share. Private Label, however, looks to be growing from a very small base. I w ould like to explore why our client is losing market share to Cereal Co. at Big M Marts. Are their prices better than those of our client? After a period of price wars six to seven years ago that lowered industry margins, the cereal companies have refrained from price competition within the same channel. If prices are not driving the difference, I would look at other factors such as brand selection, percentage of shelf space, product placement, and in-store promotions.Visits to Big M Marts indicate that each name-brand company holds 30 percent of the shelf space, while private label has 10 percent. Cereal Co. brands, however, tend to be placed lower on the shelf than your client's products. Well, I suspect that children are a large target market for the sugar cereal manufacturers. The lower shelf placement could be especially important to children who are looking at the different types of cereals. Are there any other promotions? Some Cereal Co. brands have sales promotion tags, and the team notes that store flyers advertise specials on Cereal Co. rands for Big M Mart customer cardholders. So, even if all the companies are maintaining product prices, maybe Cereal Co. is strategically discounting prices to gain market share. It seems as if there is evidence of cooperation between Cereal Co. and Big M Mart. Do we know anything about their relationship? During earlier discussions with Big M Mart, you discovered that your client's competitors have 50 sales representatives dedicated to the Big M Mart account. Your client has seven. Cereal Co. appears to be dedicating more resources to its relationship with Big M Mart than our client is.This may explain its better product placement and promotion programs. 3 of 6 10/2/09 6:57 PM BCG – Join BCG – Interview Prep – Practice Cases – Distribution†¦ http://www. bcg. com/join_bcg/interview_prep/practice_cases/dis†¦ I think I have a good sense of distribution and competition. I would now like to look at the customers and understand why they select the products they do. One hypothesis I have is that shifting brand loyalties are hurting our client's market share at Big M Mart. That's interesting. What do you think might motivate purchases of sugar cereals?There are lots of factors, such as the games in the boxes, the price of the cereal itself, how it tastes. To better understand consumer behavior, we might conduct market research, possibly through focus groups, customer observation, and price sensitivity studies. BCG teams often do such research. Let's assume your team conducts some analysis. Your research concludes that most buyers tend to fall into two categories. Approximately 60 percent of buyers go straight to one cereal and grab it. We can call this group the â€Å"brand-loyal† shoppers.Another 40 percent of shoppers look at all the cereals and then select one that interests them. Let's call this group the â€Å"impulse† buyers. For the brand-loya l shopper, the priority would be product availability, while product placement would be important for consumers who like to shop around. Within these groups, are consumers price sensitive such that one brand can lure shoppers loyal to another brand? In general, your research indicates that consumers are not price sensitive and are extremely loyal to their preferred brand.But when the preferred cereal is unavailable, the brand-loyal customers will purchase discounted cereals approximately 35 percent of the time. Well, from that information, it appears that price is not a major driver of purchases unless the preferred cereal is out of stock. In these stock-out situations, you said, brand-loyal customers will purchase discounted cereals 35 percent of the time. What happens when the customer does not purchase a discounted cereal? In approximately 25 percent of cases, the customer walks away without purchasing any cereal at all.In the remaining 40 percent of cases, the brand-loyal custom er will act like an impulse shopper and select another brand. Interesting. It seems as if product availability could be a major driver of total cereal volume for Big M Mart. Of course, we would need to know how often stock-outs occur that cause consumers to walk away without purchasing cereal occur. Since I have a pretty good understanding of customer motivation, I'd now like to ask a few questions about the client's supply chain. I would want to talk to our client's distribution personnel to understand the distribution process and to determine how often stock-outs occur.Can you describe how our client's cereal is distributed at Big M Mart? Cereals are distributed from the factory to the distributor's warehouse twice monthly. The retailer then stocks the shelves itself. Do we have any knowledge about when the individual stores are out of stock? No, we do not, since our client only delivers to the warehouses and has no direct access to in-store inventory information. Since we identif ied product availability as a key success factor earlier on, I would want to make sure that the stores were stocking the product correctly.Let's say that in your earlier in-store investigations, you found out that Big M Mart stores averaged 15 percent of sugar cereal brands out-of-stock, across all brands. 4 of 6 10/2/09 6:57 PM BCG – Join BCG – Interview Prep – Practice Cases – Distribution†¦ http://www. bcg. com/join_bcg/interview_prep/practice_cases/dis†¦ Stock-outs would be a major problem for our client, since 60 percent of customers look for a specific brand of cereal and 35 percent of them would buy a discounted brand in a stock-out situation.Big M Mart would also have an incentive to reduce out-of-stock incidents, since 25 percent of the time, a brand-loyal customer will walk away without buying anything. Summarize and make recommendations Big M Mart is our client's leading customer, accounting for more than 20 percent of our client's su gar cereal revenue. Although sales to Big M Mart are increasing on an absolute basis, our client's margins there are lower than in its other channels and its competitive position is eroding in that channel. At Big M Mart, our client faces competition from both private label and Cereal Co. although the latter appears to be the greater threat. There appears to be a relationship between Big M Mart and Cereal Co. as evidenced by their joint promotions, the superior placement of the Cereal Co. product, and the substantial resources that Cereal Co. has dedicated to the Big M Mart account. We learned that 60 percent of customers are brand-loyal, implying product availability is most important. However, 40 percent like to try different kinds of cereal, indicating product placement is also important.Purchasers do not appear to be price conscious, unless the type of cereal they are looking for is out of stock, in which case there is a stronger tendency to base purchases on price promotions. I n terms of distribution, our client is making deliveries twice a month to Big M Mart's warehouses. Big M Mart, in turn, is responsible for stocking the shelves. We currently have no direct knowledge of when our client's items are out of stock at the individual stores, but there is evidence that stock-outs do occur with some frequency. Well, it sounds as if you understand the situation. What would you recommend the client do?The sales through Big M Mart appear to have a negative impact on the bottom line, as they have lower margins than sales through grocery stores. The client could work with grocery stores to ensure that they are able to compete effectively with Big M Mart in the sugar cereal market. This strategy could be risky, however, since Big M Mart is a large and important customer. Therefore, I would recommend that our client work more collaboratively with Big M Mart. To defend its current position at Big M Mart stores, the client should move toward a partnership with Big M Mart and dedicate more resources to the relationship.The customer and competitor data indicate that our client's first priority should be to improve distribution to ensure better product availability. In addition, it should push for product placement equal to, if not better than, that of its competitors. Why would Big M Mart be willing to enter into a partnership with Foods Inc? Foods Inc could offer to share its information about customer behavior to help increase revenues for both itself and Big M Mart. Stock-outs hurt Big M Mart in two ways. First, some brand-loyal customers simply walk away without purchasing cereal whenever their preferred brand is unavailable.Second, we know that other brand-loyal customers purchase lower-priced cereal whenever they encounter a stock-out of their preferred brand. Both of these instances lower Big M Mart's revenue. By eliminating stock-outs, Big M Mart could increase its sales by simply ensuring that customers don't walk away without making a p urchase. Converting these purchase occasions to sales would increase Big M Mart's sales of sugar cereals by more than 2 percent(1). Better availability also helps Big M Mart and our client increase their revenue by deterring the brand-loyal shoppers from trading down to lower-priced cereals.Recall that 35 percent of the brand-loyal shoppers purchase a discounted cereal if their preferred brand is not available. If improved distribution now makes the preferred brands more consistently available, the customers will pay a higher price for these products. Finally, we could use the information about consumer purchase behavior to help persuade Big M Mart to 5 of 6 10/2/09 6:57 PM BCG – Join BCG – Interview Prep – Practice Cases – Distribution†¦ http://www. bcg. com/join_bcg/interview_prep/practice_cases/dis†¦ share information about product availability in its individual stores.We could work with our client and Big M Mart to improve the current distri bution system to allow for more economical deliveries, while at the same time ensuring that our client's product is consistently available in the store. Thank you. Those sound like solid recommendations, but I would suggest that you fully understand the root cause of the stock-out situations and the cost to eliminate them before moving ahead. (1) 15 percent out of stock x 60 percent brand-loyal customers x 25 percent willing to forgo purchase = 2. 25 percent 6 of 6 10/2/09 6:57 PM

Kingdom Protista

Kingdom Protista: Characteristics Mostly unicellular, eukaryotic cells Reproduce asexually or sexually by conjugation Exhibit all three modes of nutrition Photosynthesis Ingestion Absorption Ultimately spawned all multicellular kingdoms Very diverse kingdom Difficult for taxonomists to agree on classification Diverse Modes of Nutrition Use diverse modes of nutrition Ingest food Absorb nutrients from surroundings Photosynthesis Protists that ingest food are typically predators Use extensions of cell membrane called psuedopods to surround and engulf prey item Diverse Modes of NutritionProtists that absorb nutrients directly from the surrounding environment can be Free-living types in the soil that decompose organic dead matter Parasites that live inside the bodies of other organisms, sometimes harming the host Diverse Modes of Nutrition Some protists have photosynthetic organelles called chloroplasts Photosynthetic protists are abundant in oceans, lakes, and ponds Free floating Mutuall y beneficial associations with other organisms: solar energy captured by the protist is used by host, which shelters and protects the protist Diverse Modes of NutritionPhotosynthetic protists are collectively known as algae Single-celled, non-photosynthetic protists are collectively known as protozoa Diverse Modes of Reproduction Most protists reproduce asexually by mitotic cell division Some also reproduce sexually Two individuals contribute genetic material to an offspring that is genetically different from either parent Occurs during certain time of year or circumstances (e. g. a crowded environment or a food shortage) Protist Reproduction Asexual Sexual (a) (b) Effects on HumansPositive impact – ecological role of photosynthetic marine protists (algae) capture solar energy and make it available to the other organisms in the ecosystem release oxygen gas Negative impact – many human and plant diseases are caused by parasitic protists Major Groups of Protists Protist classification is in transition Genetic comparison reveals evolutionary history of organisms Genetic, instead of physical features now separate protist species into different lineages Some physically dissimilar species are now placed in a common lineage The Excovates Lack mitochondriaTwo major groups Diplomonads: have two nuclei and move about by means of multiple flagella Parabasalids: live inside animals Parabasalids Mutually beneficial relationships with other species Parabasalid inhabits gut of termite Termite delivers food to parabasalid, which digests and releases nutrients to termite Parabasalids Harms host species Trichomonas vaginalis causes the sexually transmitted disease trichomoniasis Trichomonas inhabits urinary and reproductive tracts, using flagella to move through them Causes vaginal itching and discharge in females The EuglenozoansHave distinctive mitochondria Two major groups Euglenids Kinetoplastids Euglenids Single-celled, fresh-water protists Lack a rigid outer covering Best known example is Euglena Moves by whipping single flagellum Photosynthetic Some euglenids photosynthetic, others absorb/engulf food Euglenids Photoreceptor (eyespot) found in some euglenoids Provides for a way to sense location of light source Useful for photosynthetic euglenoids in maximizing photosynthesis Euglena : a Representative Euglenoid Flagellum Eye Spot Contractile Vacuole Stored Food Nucleus Nucleolus Chloroplasts KinetoplastidsAll species have one or more flagella Can be used for propulsion, sensing, or food gathering Many are free-living in soil and water Kinetoplastids Some species live in a symbiotic mutualistic association within another organism Some species digest cellulose in termite guts Trypanosomes live within tsetse flies and cause African sleeping sickness in fly-bitten mammals Trypanosomes infect the blood causing African sleeping sickness Trypanosomes in Blood The Stramenophiles Have fine, hair-like projections on flagella Mostly single-celle d but some multicellularSome are photosynthetic species Major stramenophile groups Water molds Diatoms Brown algae Water Molds Also known as oomycetes Long filaments aggregated into cottony tufts Many are soil and water-based decomposers Water Molds Profound economic impacts caused by water molds Late blight attacks potato plants (caused Irish potato famine in 1845) One species causes downy mildew (nearly destroyed French wine industry in 1870s) A Parasitic Water Mold Downy mildew on grapes Diatoms Found in both fresh and salt water Photosynthetic Produce shells of silica that fit togetherDiatomaceous earth is deposits of diatom shells (mined and used as an abrasive) Diatoms Part of floating phytoplankton community Important in absorbing CO 2 and producing O 2 Phytoplankton perform 70% of all photosynthesis Diatoms are important as food in marine food webs Herbivorous organisms â€Å"graze† on these â€Å"pastures of the sea† Brown Algae Form multicellular aggregates ( seaweeds) Superficially similar but not closely related to plants Contain brownish-yellow and green (chlorophyll) pigments producing brown/olive appearance Brown Algae Nearly all marineFound along rocky shores of temperature oceans Includes giant kelp Several species use gas-filled floats to support body Giant kelp forests provide food and shelter for sea animals Diverse Brown Algae Fucus sp. Giant Kelp The Alveolates Single-celled protists with small cavities beneath cell surface (alveoli) Comprise a distinct lineage Nutritional modes include photosynthetic, parasitic, and predatory The Alveolates Major alveolate groups Dinoflagellates Apicomplexans Ciliates Dinoflagellates Mostly photosynthetic Two whip-like flagellaMost species live in salt water Some species bioluminescent Certain specialized dinoflagellates live within coral, clam, and other protistan hosts Cell wall resembles armored plates Dinoflagellates & Red Tide Red Tide Dinoflagellates Nutrient-rich water causes populati on explosion called â€Å"red tides† Substantial fish kills result from oxygen depletion and clogged gills Oysters, mussels, and clams benefit from large food supply but may accumulate nerve poison Lethal paralytic shellfish poisoning in humans may result from eating these shellfishApicomplexans Also known as sporozoans All members are parasitic Form infectious spores Spores transmitted between hosts by food, water, or insect bites Apicomplexans Complex life cycle (e. g. Plasmodium- malarial parasite) Parasite passed to human by Anopheles mosquito Plasmodium develops in liver, makes spores in red blood cells (causing fever upon release) New mosquitoes acquire parasite while feeding on blood Plasmodium quickly evolves resistance to drugs Ciliates Inhabits both fresh and salt waterHighly complex unicellular organization Specialized organelles Cilia that propel cells through water at 1 mm/s Ciliates Examples of ciliate complexity Paramecium (contractile vacuoles, nervous system) Didinium (predator of other microbes) Paramecium has vacuoles and cilia The Complexity of Ciliates Macronucleus Micronucleus Food Vacuole Oral Groove Contractile Vacuole Cilia Food Vacuole forming The Cercozoans Cercozoans have thin, threadlike psuedopods, which extend through hard shells in some species Cercozoans includeForaminifera Radiolarians The Cercozoans Foraminiferans produce elaborate calcium carbonate shells with holes Deposits of fossilized foraminiferans form chalk Radiolarians have silica shells Heliozoans The Amoebozoans Amoebozoans move by extending finger-shaped pseudopods, also used for feeding Inhabit aquatic and terrestrial environments Generally do not have shells The major groups of amoebozoans are Amoebas Slime molds The Amoebozoans Amoebas Found in freshwater lakes and ponds Predators that stalk and engulf preyOne species causes amoebic dysentery The Amoebas The Slime Molds Distinctly unique lineage among protists Physical form blurs distinction between a co lony versus an individual The Slime Molds Two-phase life cycle Mobile feeding stage Stationary, reproductive stage forming a fruiting body Two main types Acellular Cellular Acellular Slime Molds Also known as plasmodial slime molds Composed of a thinly spread cytoplasm with multiple diploid nuclei Plasmodial mass feeds on bacteria and organic matter by engulfing them Acellular Slime MoldsCan form bright yellow or orange masses Dry conditions or starvation stimulate fruiting body formation Haploid spores produced Spores disperse and germinate into a new plasmodium The Acellular Slime Mold Physarum (a) (b) Cellular Slime Molds Live in soil as independent haploid cells Pseudopodia surround and engulf food (like bacteria) Cellular Slime Molds Food scarcity creates a pseudoplasmodium Individual cells release chemical signal if food is scarce Dense, slug-like aggregation of cells forms Slug† crawls towards light, forms a fruiting body Haploid spores produced are dispersed to form ne w single-celled individuals The Life Cycle of a Cellular Slime Mold Single, amoeba-like cells emerge from spores, crawl, and feed. When food is scarce, cells aggregate into slug-like mass called pseudoplasmodium. Pseudoplasmodium migrates toward light, forms fruiting bodies; produces spores. fruiting bodies spores nucleus The Red Algae Multicellular, photosynthetic seaweeds Pigments combined with chlorophyll produce bright red to black appearances Found exclusively in marine environmentsThe Red Algae Very common in deep, clear tropical waters Red pigments absorb deeply penetrating blue-green light Can therefore live deeper than other seaweeds The Red Algae Diversity of forms and uses Some species deposit calcium carbonate Some species harvested for food Energy captured by red algae important in food chains Products extracted from red algae include: Carrageenan (stabilizing agent) Agar (substrate for bacteria in petri dishes) The Red Algae Multicellular, photosynthetic seaweeds, rang ing in color from bright red to nearly black Live in clear tropical oceansSome species deposit calcium carbonate, which contributes to the formation of reefs Red Algae The Green Algae All species photosynthetic Both multicellular and unicellular species Found in both freshwater and marine environments Some form long filamentous chains of cells (e. g. Spirogyra ) Spirogyra: A Green Algae The Green Algae Some form colonies of clustered cells (e. g. Volvox ) Mostly microscopic forms but Ulva (sea lettuce) is a multicellular leaf-sized green algal seaweed The Green Algae Green algae are closely related to plantsThe earliest plants may have been similar to today’s multicellular green algae Protists and Life Marine phytoplankton: 70% of all photosynthesis Diatoms – abrasive products and oil reserves Sarcodines and limestone deposits Protists and disease Water molds – downy mildew, late blight of potato Dinoflagellates and â€Å"red tide,† shellfish poisoning Zo oflagellates – African sleeping sickness, Giardia Sarcodines – amoebic dysentery Sporozoans – Plasmodium and malaria Giardia: the Curse of Campers

International Law Dissertation Example | Topics and Well Written Essays - 750 words

International Law - Dissertation Example esolution is usually included in bilateral international treaties (BITs) which are intended to bind treaty states to commitments for protecting investors and their investments against hostile host state conduct in transactions under foreign direct investment (FDI).7 The proposed dissertation will analyse arbitral awards and their underlying decisions relative to investor/state arbitration with a view to identifying and analysing contributions to customary international law and the consequences for resolving investor/state disputes. In particular, the proposed dissertation evaluates the extent to which international commercial arbitration and more especially, ISA, has established or can satisfactorily establish the necessary legal protections for encouraging and facilitating FDIs. The proposed dissertation will argue that although there is no multilateral international legal instrument regulating FDIs and investor-state relations, international customary law and the principle of fair and equitable treatment included in BITs and emerging from investor/state arbitration has contributed to a sufficiently coherent body of law so that investors are accorded the protection necessary for investing abroad. It will be argued that although international arbitration in general including ISA is not regulated by a centralized forum by which binding precedents may be created, the cumulative effect of BITs and ISAs applying and interpreting BITs have resulted in a customs, norms and rules that have established a coherent body of law applicable to standards of treatment expected of host states.8 It will also be argued, however, that despite the emerging coherent body of law, there are challenges to overcome. For example, the different language used in BITs has resulted in arbitrators rending inconsistent or unclear decisions in their interpretation of protective clauses in BITs.9 Outline: In order to support the hypothesis that ISA or international commercial arbitration has co ntributed to a coherent body of law for promulgating FDIs, the proposed dissertation will be presented as follows: Part I: Introduction. The introduction establishes that over the last 20 years or so, there have been two interesting developments in international commercial transactions: an increase in BITs10 and a decrease in investor/state arbitration.11 The introduction then makes an undertaking that the dissertation will establish that the link between these developments is ISA in that BITs provide for arbitration of investor/state disputes and interpret and apply the level of protection provided for in BITs. Part II: This part of the dissertation

China Essay Example | Topics and Well Written Essays - 250 words

China - Essay Example Moreover, he also believed that everything in nature has two sides and that opposite sides complement each other i.e. the dialectical nature of things (Hansen, 2000). Therefore, Laozi emphasized on the fact that emptiness or nothingness is not emptiness or nothingness, but complements certain objects. Zhuangzi further emphasized on the issue of dialectic ism that was recognized by Laozi. He recognizes the nature as a movement, which has a different phenomenon in the world that is derived from and manifestations of nature (Hansen, 2000). In addition, Zhuangzi can be described as a renowned philosopher who praised human ambition, as well as imagination, which facilitated everyday thinking in order to understand how things interact in nature. Zhuangzi influenced the development of Daoism by emphasizing further on Laozi’s position on dialectic ism. By doing so, he introduced a new notion of self-transformation as the key precept in the Taoist process (Hansen, 2000). He also stated that it is essential to transcend all the dualities of existence. According to him, the way nature worked and reconciled the opposite sides showed how the Tao dualities were resolved in unity. On the other hand, Laozi influenced the development of Daoism by advocating for humility in leadership. He also promoted the development of anti-authoritarian movements that stressed on giving power to the weak (Hansen,

Classical Liberalism Essay Example | Topics and Well Written Essays - 750 words - 1

Classical Liberalism - Essay Example Locke attempted to protect some areas of personal life from governmental action. People should not be deprived of their property rights by the state. The acceptance of the government authority over people is to ensure that the latter protects their property and liberty. In ancient times, people enjoyed full-fledged freedom and liberty; and the state should endeavor to provide these rights (Stein 21). The Lockean perception states that the fundamental duty of the state is to protect private property. However, this theory has been discounted because the state has extended protection to only property that it creates and to the extent to which it deems to be sufficient. The state is the bestower as well as the depriver of property. Consequently, the restrictions imposed by the state on land use become an intrinsic part of the land (Epstein 129). A state that controls private property is akin to a dictatorship. Moreover, a state that strictly protects the right to private property, cannot address crises effectively. For instance, during times of war, natural disasters and economic depressions the state is empowered to control private property. However, classical liberalism requires the state to operate under certain limitations, while seizing private property. Therefore, a classical liberal society cannot survive in a real time environment and it cannot build gigantic projects like the Tennessee Valley Authority. Such classical liberal societies cannot deal with the Texas farmers in drought situations. There will be no technological advancement in a classical liberal society. It cannot launch expedition to outer space, and there would be no scientific experiments (Rockwell). The sole ruler of a society is its legislation, therefore, it is irrelevant as to who wins in the elections or who emerges as the president. Communities develop by themselves, and the future of the people is determined by their actions.

Masters Degree in Jazz Music Essay Example | Topics and Well Written Essays - 500 words

Masters Degree in Jazz Music - Essay Example What makes the situation even more distressing is the lack of recognition from the authorities. The government likes to promote Thailand as a land of traditional music and art. The traditional music is encouraged whereas the more contemporary forms are sidelined. As receiving recognition itself is very difficult in the country, the prospects of studying music are bleak. Further, the perception of music is distorted by the media. There is little or no understanding of the various genres of music even by some of the popular music companies in Thailand. Hence the people of my country do not place artists, especially musicians in high regard. My country, which is a developing one, considers engineering sciences, medical sciences, and other such fields to be more respectable than arts and music. The common misunderstanding of the people of Thailand is that there is no future in fields like music. The outlook is extremely narrow and their beliefs stem from their lack of knowledge of anything outside their immediate career interests. This disturbing misplaced sense of superiority among the supposedly more educated class of professionals in the various fields of sciences is more detrimental to the growth of the young musical talent in the country than anything else. These ideas tend to be passed down from the older generations to the younger ones thereby discouraging latter to seriously consider music as a full-time career. The youngsters shy away from music careers as they are afraid to be thought of lesser than their peers for their choice of career. In countries like the United States of America, children are given a free reign as far as their careers choices are concerned and person’s abilities and interested are given importance to. In Thailand, however, the people are driven by their traditionalist and conservative ideas.  Ã‚  

Communication skills Research Paper Example | Topics and Well Written Essays - 2500 words

Communication skills - Research Paper Example The media like television and newspaper has the ability to reach the public in a strong way and communication is the foundation to it. Good communicators are not born but they are made with the help of proper training and knowledge. It is not just easy to thrive in today’s a competitive and demanding world and communication is one factor which is under our control. If one can be best at its communication skill, then he can make things work at his will and favor. The importance of communication in this business world is so great that the success of a company and employee solely depend on it. To become a successful employee in any field, apt communication skill is of utmost importance. Good communication skill is immensely important to work jointly with employees of an organization. To be a good communicator one need to also be a good listener. In his books (Guffey,23)â€Å"Communication does not take place unless the sender encode meaningful message that can be decoded and und erstood by the receivers†. The Importance of Communication Skills The importance of communication skill is highest when it comes to any sphere of human life. When it comes to career, communication stands as a key factor in employee’s job placement, career advancement, performance and organizational success. In hiring a candidate, communication skill is considered as the most needed competency. ... It is very important for people to understand the skills and techniques required to communicate effectively to each other. According to (Wilhelm)â€Å"Communication has two parts. The first is the communicator and how effectively she can convey her message to the listener. The second is how well the listener of the communication receives the message. If there is misunderstanding, misinterpretation or confusion – then the communication is not successful†. The success can be achieved by the communicator only if he is well versed with communication skills and techniques. Communication skill is completely beneficial as it creates effective flow of thoughts and information from one party to another as well as minimizes the chances of confusion and misunderstanding. If a person lack in communication skill, then he cannot promote himself and also the organization he is working for. In communication there is exchange of information, thoughts and ideas with the help of dialogues and conversation. It is always a two way process which included gesticulation and vocalization. The importance and value of communication skill never can be disregarded. In all walks of life, good communication skill is extremely important. A poor communication skill can have a negative effect on both the professional and personal life of a person. A good communication skill is very essential in fields like medicine, teaching and media where in the main focus is on the understanding of patients, students and the mass population respectively. Even for a salesperson, communication is of key importance as his profession is entirely depended on his verbal skills. In the same way verbal communication is vital,

Managerial Economics and the Airline Industry Essay

Managerial Economics and the Airline Industry - Essay Example In line with the high unemployment rate in U.S., millions of the Americans could no longer afford to spend their savings on leisure purposes which will require them to spend a large portion of their savings on air fares. Based on the students knowledge on managerial economics, economic tools and concept will be used in weighing the pros and cons of recommended solution on how the U.S. airline industry could avoid filing bankruptcy. Considering the slow growth in the U.S. economic condition combined with the oil price hike in the global market, merger and acquisition should be approved by the U.S. Department of Justice in terms of minimizing and preventing the number of cases wherein these businesses would file bankruptcy. because of the continuously increasing oil prices in the world market. In line with this matter, the only way to make the airline operational cost to go down is by making its available human resources more efficient. The benefits of allowing business merger among the U.S. Airline Companies includes the fact that existing airline companies will be able to enjoy the benefits of economies of scale. In the process of merging two airline companies into one, the airline business will have more power in terms of maximizing the use of its human resources and assets aside from having a bigger market share in the industry. During the reorganizational process, airline managers will have the option to select and retain their best talents while laying off employees that do not have sufficient knowledge and skills in airline operations. By doing so, a newly merged airline company will be able to cut down its operational costs by making its human resources more efficient. Basically, cutting down the operational costs will enable the newly merged company to offer a more competitive air fare prices as compared to its competitors. For this reason, there is a

Sunnyvale Foods Marketing Analysis Essay Example for Free

Sunnyvale Foods Marketing Analysis Essay Sunnyvale Foods is a brand of canned and frozen fruits and vegetables as well as condiments. While the firm has been around for over a hundred years, their profits have started to decline. From the statement given by the former president of Sunnyvale Foods, it can be deduced that the firm has a production oriented marketing concept. Rather than producing to meet a need, the firm is producing in anticipation of a need. Sunnyvale Foods focuses on mass marketing, aiming at â€Å"everyone† rather than target marketing a specific group (i. e. -busy families). The marketing mix is comprised of the â€Å"four P’s†: product, place, promotion, and price. The product is a line of 65 food items. The place is limited to chain supermarket stores limited by their ability to carry the entire line of 65 foods that the firm produces. Promotion of the firm as described in the case was vague but did mention sales promotion such as manufacturer’s coupons. The price is in the range of competitors but in today’s rushed society, many food brands don’t have the advantage of customer loyalty which results in losing customers to either the store brand or another brand that is offered on sale. The main competitive advantage of Sunnyvale Foods is the history of the business. Based solely on their 127 year old business, their name has become a reputable one. Their primary disadvantage is their vast line of products. By producing 65 different products, they lose advantages found in economies of scale. Also, by having a strict policy requiring stores that carry their product to carry all 65 items, they are given a disadvantage by limiting their potential retailer pool and complicating the process of resupplying inventory. To be stuck in the middle mean to be between differentiations, focus strategy, and cost leadership. While Sunnyvale Foods is focusing on one specific niche of the market, their differentiation is limited to various types of the same product rather than new products. Also, as stated in the case â€Å"no company in the industry has much effect on the price at which its products are sold. † The primary issue with Sunnyvale foods is that the firm is marketing for a production in a marketing company era. While the firm was very successful in years past, the market has changed and that needs to be recognized in their marketing strategy beginning with becoming a marketing oriented firm and finding a target market.

Isolation of Recombinant Escherichia Essay Example for Free

Isolation of Recombinant Escherichia Essay One technique important in both genetics and biochemistry is the Polymerase Chain Reaction (PCR), first developed in the 1960s, and then automated in 1983. Current PCR technology was not developed until the discovery of thermostable polymerases, specifically Thermus aquaticus (Taq) polymerase (1). The protein Taq polymerase was first isolated from the extreme thermophile T. aqauticus, where extreme thermopiles are bacteria that live in temperatures at or above 45 °C. The Taq enzyme is a member of the DNA polymerase I family (2, 3). The interesting property of Taq polymerase is that it has a temperature optimum at 74-75 °C, allowing it the remain active in temperatures required for PCR double stranded DNA denaturation (3, 1) . The protein has an approximate molecular weight of 6263 kDa when isolated from T. aquaticus, and 94 kDa when isolated from recombinant Escherichia coli, and is still stable at temperatures of 93-95 °C, hence the thermostability of the enzyme ). Taq specifically lacks any proofreading activity in the 3’ to 5’ direction, and therefore has a relatively high error rate of single base mispairings of 1 error per Isolation of Recombinant Taq Polymerase for PCR 9000 nucleotides, as well as a frame shift error rate of 1 per 41,000 basepairs (5, 6). Taq polymerase has an activity that is highly dependent on the environment of which it is in as it is thermostable, and has differing activities at nearly all temperatures up to the point of denaturation. Taq specifically can add up to 1000 base pairs in length on a template in under one minute under typical PCR conditions. The enzyme has a specific activity of 200,000 units mg-1, and can add approximately form 60 nucleotides per second at 70 °C (7). The isolation of Taq is essential for the PCR reaction. The most important reason for Taq being used in PCR is the thermostability at high temperatures (95 °C). This allowed for the process of elongation, annealing, and denaturation to occur without the replacement of new enzyme, and thereby, was more efficient, faster, and cheaper because the reaction could be automated through the use of a machine known as a thermocycler which basically is just a machine able to change temperatures of an isolated environment rapidly (7). Prior to the discovery of Taq, PCR was done using Klenow fragments of E. coli DNA polymerase I at 37 °C. The lack of thermostability required replenishment of enzyme after each PCR cycle (8). One of the initial difficulties of Taq polymerase was the organism in which it was expressed in, T. aquaticus, as it was difficult to culture and produce large quantities of enzyme. E. coli bacteria were engineered to expressed the Taq polymerase gene to allow for retrieval of large quantities of enzyme ). The isolation of the Taq gene involved culturing T. aquaticus and then isolated the DNA of the cells through lysing, proteinase K addition, extracting of aqueous and phenolic phases, dialyzing of extractions, addition of SDS, and then centrifugation of solution to eventually retreieve the DNA of the organism as outlined in Lawyer et al., 1989. With the isolation of the 2401+ BP gene of Taq, the gene was incorporated into a 6.58 kbp plasmid (pLSG1). The gene was inserted 171 bp distal to the lacZÃŽ ± promoter/operator, and 109 bp distal to the BgII site, so the gene expression could be controlled through an inducible promoter. With the pLSG1 plasmid, the vector was introduced to E. coli bacteria to allow for plasmid uptake (4). Other experiments have been conducted towards the purification of Taq from recombinant E. coli. Specifically Engelke et al., 1990 developed a method for purfication of Taq. The E.coli strain 2 DH1 was used for the expression of the recombinant plasmid containing Taq polymerase. The bacteria were grown in 12 Litre batches of Luria Broth; using 1 mL of saturated DH1 culture and 80ÃŽ ¼g/mL of ampicillin. Isopropyl-1-thio-ÃŽ ²-Dgalactopyranoside (IPTG) was added to 0.5mM and the cultures were grown for 16-20 hours. The cells were harvested in 2.4 L of buffer A (50 mM TrisHCL, pH 7.9, 50 mM dextrose, 1mM EDTA) and collected via centrifugation, resuspended in Buffer A with 4mg/mL lysozyme and incubated at room temperature for 15 minutes. Buffer B (10 mM TrisHCl, pH 7.9, 50mM KCl, 1mM EDTA, 1mM phenylmethylsulfonyl fluoride (PMSF), 0.5% Tween 20, 0.5% NP-40) was added and incubated in 180 mL fractions, for 60 minutes at 75 °C in a water bath. The mixtures were centrifuged at 8000 rpm for 15 minutes at 4 °C. Taq then precipitated with polyethyleneimine (PEI) at room temperature, then isolated through centrifugation and suspended in buffer C (20mM HEPES, pH 7.9, 1 mM EDTA, 0.5mM PMSF, 0.5% Tween 20, 0.5% NP-40) containing 0.25 M KCL. PEI eluatents were diluted in 50mM KCL and buffer C and applied to a 150mL BioRex 70 ion exchanger column, and then eluated using 200mM KCL. The protein was dialyzed for 12 hours against two changes of 1 L storage buffer (20mM HEPES, pH 7.9, 100 mM KCL, 0.1 mM EDTA, 0.5 mM PMSF, 1mM dithiothreitol, 50% glycerol. The experiment resulted in 40-50 mg of protein per litre of cell culture (9). The methods used in this experiment differed in certain key aspects. First, Engelke’s experiment made use of a higher concentration of ampicillin. The IPTG was added to the same concentration, but was added after cell growth up to an optical density of 0.700. Instead of a water bath at 75 °C, this experiment made use of an air incubator for the temperature requirements. Engelke’s experiment made use of PEI to precipitate Taq, while this experiment made use of 30g of (NH4)2SO4 per 100mL of supernatant. Buffer C was not used throughout this experiment, and no ion exchange columns were used. The dialysis procedure was done for twice as long with twice as many changes of solution per 6 hours. The changes made from Engelke’s experiment offers a different method for protein precipitation. The method used by Engelke made use of PEI which is an affinity precipitation method versus a salt prec ipitation method. The PEI Isolation of Recombinant Taq Polymerase for PCR method has the major drawback through the lack of selectivity, and can often precipitate nucleic acids as well (10). This is why the BioRex column needed to be used. Ammonium sulfate has the advantage that the precipitation can be controlled based on ionic strength of species involved, as well as has no negative effects on the activity of the target enzyme. Salting out also has the advantage that only native state proteins are precipitated due to the hydrophobicity involved with native state proteins (10). Buffer C was not required for this experiment as no BioRex column was required. This experiment made use of various techniques and methods including: SDS-PAGE, differential centrifugation, Western Blotting, real time-PCR (rtPCR), PCR, agarose gel electrophoresis, and dialysis. Two important techniques were PCR and rt-PCR. PCR does not allow for the quantification of DNA amplicons as it is an end-point PCR, but it does allow for confi rmation of template duplication along with measurement of base pair length. Amplification of primer would confirm the presence of a thermostable DNA polymerase. The following agarose electrophoresis helps to find amplicon size which can tell us the activity of Taq, as well as the specificity, as one template should only return one band in PCR (7). rt-PCR allows for a quantitative assessment of PCR, and therefore the kinetics of the reaction, as it detects the amount of amplicons produced in the reaction. The point at which the standard curve reaches threshold in cycle number gives information on the activity of Taq, as a more active sample of Taq reaches threshold earlier. Melt curve analysis also provides information regards DNA amplicons in solution (11). The purpose of this experiment was the test the methods for the isolation of PCR grade Taq polymerase from recombinant E. coli using differential centrifugation, salting out, and heat denaturation following lysation of cells to potentially improve isolation of Taq from past methods. The presence of Taq will be confirmed through Western blotting, and rt-PCR and PCR reactions along with purity will be assessed through SDS-PAGE. The activity of Taq will be found through rt-PCR and PCR. Finding the most efficient method for the isolation of Taq offers a valuable reagent source for any PCR reactions required. The isolation technique would also be applicable to any thermostable proteins. 3 EXPERIMENTAL PROECDURES Isolation of Taq Polymerase Luria broth (500 mL + 100ÃŽ ¼g/mL ampicillin) was inoculated with 50 ÃŽ ¼L of frozen Taq polymerase expressing E. coli cell stock. Incubation was commenced for 12 hours at 37 °C until the Optical Density had reached 0.700. IPTG (0.5 mM or 0.112g/L culture) was added and the culture was incubated for 12 to 14 hours at 37 °C. The 50mL of cells were then centrifuged (4000 RPM x 15 minutes at room temperature) in an Eppendorf Centrifuge 5810, and 5 mL of buffer A (50 mM Tris-HCl, pH 7.9, 50 mM dextrose, 1mM EDTA) was used to suspend the separated pellet. The solution was then centrifuged again (4000 RPM x 15 minutes at room temperature) in an Eppendorf Centrifuge 5810 and the pellet was once again suspended in Buffer A, with an additional 20 mg of lysozyme added. The reaction was incubated for 15 minutes at room temperature. Following incubation, 5mL of buffer B (10 mM Tris HCl, pH 7.9, 50mM KCl, 0.5% Tween 20, 0.5% NP-40, 1mM PMSF, 1mM EDTA) was added and incubated at 75 °C for 1 hour in a New Brunswick Scientific-Innova 40 incubator shaker series, and shaken by hand approximately every 5 minutes. The solution was then centrifuged (15000 RPM x 10 minutes at 4 °C) in a Thermoscientific Sorvall RC 6+ centrifuge and using a 603s Delta Range 30g of (NH4)2SO4 per 100mL of supernatant (8 mL of supernatant equivalent to 2.4g (NH4)2SO4 ) was added and incubated for 10 minutes at room temperature and shaken on the Innova 40 incubator. The lysate was then centrifuged again (15000 RPM x 10 minutes at 4 °C) in Thermoscientific Sorvall RC 6+ centrifuge and the resultant pellet was suspended in 2mL of buffer A. The solution was then dialyzed in a Spectra/Por membrane tubing set at 6000-8000 Da molecular weight selection in 1 L of storage Buffer (50 mM Tris HCl, pH 7.9, 50mM KCl, 0.1mM EDTA, 1mM DTT, 0.5 mM PMSF, 50% glycerol) for 24 hours at 4 °C changing the buffer every three hours. The dialysis solution was then diluted in a 1:1 ratio of storage buffer and stored at -70 °C until needed. Protein Concentration Determination A Bovine Serum Albumin Bio-Rad assay standard curve was prepared (0 –0.3 mg/mL) using Isolation of Recombinant Taq Polymerase for PCR a 1mg/mL stock solution and an Asys Expert Plus spectrophotometer set at 620 nm. Bio-Rad assay was run in triplicate using 20ÃŽ ¼L of protein dilution and 150 ÃŽ ¼L of diluted Bio-Rad Dye Concentrate. 10x and 100x dilutions of the sample prepared previously were made and 20ÃŽ ¼L were used with 150ÃŽ ¼L of diluted Bio-Rad Dye concentrate. The solutions were incubated for 10 minutes and absorbances were tabulated. sandwich was then assembled with an additional ice block in the transfer apparatus. The apparatus was run at 180mA overnight in a refrigerator and the membrane was then stored in TBST buffer (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.1% Tween 20) and refrigerated. 24 hours prior to the primary antibody (Anti-Taq monoclonal Antibody (8C1)) addition, the membrane was blocked in 1 gram of Carnation nonfat dry milk (5% w/v) and 20 mL of TBST Buffer. The primary antibody in TBST with SDS-PAGE 5% w/v nonfat dry milk at a 1:800 dilution of A discontinuous polyacrylamide gel was antibody was added to the membrane and shaken prepared using a Mini-PROTEAN Tetra Cell for 1 hour at room temperature. The membrane was module. The casting stand was assembled for 1mm then washed three successive times for 15 minutes gel and filled with National Diagnostics 12% with TBST buffer at room temperature. The Resolving Gel (Protogel 2400 ÃŽ ¼L, Resolving Gel secondary antibody (Peroxidase-conjugated Buffer pH 8.8 1560 ÃŽ ¼L, dH2O 1974 ÃŽ ¼L, 30% w/v AffiniPure Goat Anti-Mouse IgG (H+L)) was then APS 21ÃŽ ¼L, TEMED 6ÃŽ ¼L), casted to 1 cm below top applied in TBST with 5% w/v nonfat dry milk at a of glass plate, and then 4% Stacking Gel (Protogel 1:2000 dilution of antibody and shaken for one hour 390 ÃŽ ¼L, Stacking Gel Buffer pH 6.8 720 ÃŽ ¼L, dH2O at room temperature. The membrane was then 1830 ÃŽ ¼L, 30% w/v APS 6ÃŽ ¼L, TEMED 3ÃŽ ¼L) was washed three successive times for 15 minutes with casted on top with a ten well gel comb. The TBST buffer at room temperature. The edges of the electrode set up was then submersed in 1 x Trismembrane were dried with a Kim and next 1mL of Glycine SDS Page Running Buffer. To each 20 ÃŽ ¼L Renaissance Western Blot Kit NEN Life Sciences of sample, 20 ÃŽ ¼L of Laemmli buffer (0.5 M TrisProducts, Cat. No. NEL 101 luminol reagent with HCl, pH 6.8, 4.4% w/v SDS, 20% v/v glycerol, 2% 1mL of oxidizing reagent were mixed together and v/v 2-mercaptoethanol, 10 mg/mL bromophneol then applied to the membrane. The membrane was blue) was added and boiled for 3 minutes and then then imaged with an exposure time of 40 seconds cooled for 5 minutes on ice. To one well 7ÃŽ ¼L of using AlphaEaseFC software. New England BioLabs Inc. Prestained Protein Marker (7-175 kDa) was added. To the following PCR/agarose gel electrophoresis wells 15 ÃŽ ¼L of standard Taq polymerase was added, A master mix for PCR was prepared (1x PCR along with 20ÃŽ ¼L of six different samples, with the buffer minus Mg, 0.2mM dNTP, 1.5 mM MgCl2, fifth being prepared the previous year with the same 0.5ÃŽ ¼M forward primer, 0.5ÃŽ ¼m Reverse Primer, 0.1 method of isolation as outlined previously. The gel ng Template DNA and Nuclease-free PCR water) was run at 200 Volts for 40 minutes, incubated in and 22.5 ÃŽ ¼L of master mix and 2.5ÃŽ ¼L of Taq fixing solution overnight and then stained with Bio- sample, the standard, or the Taq prepared a previous Safe Coomassie Blue for one hour at room year were added to PCR tubes and centrifuged temperature under agitation. The gel was then briefly on a Fisher Scientific Accuspin micro 17 analyzed used AlphaEaseFC software. just briefly using 1.5mL eppendorf tubes with no caps to contain the PCR tube. The PCR tube was Western Blotting then added to T3 Biometra Thermocycler and Using the method described above for SDSdenatured at 94 °C for 3 minutes and then 35 cycles PAGE, a SDS-PAGE gel was taken prior to fixing. of PCR with the denature 94 °C for 45 seconds, The gel was then transferred to transfer buffer anneal 55 °C for 30 seconds, and extension at 72 °C (20mM Tris-HCL, pH 8.0, 150 mM Glycine, 20% for 1.5 minutes. The sample was then incubated at Methanol). Immobilon-P transfer membrane with 72 °C for 10 minutes and then temperature was 0.45 ÃŽ ¼m pore size and Whatman paper were cut to maintained at 4 °C. The samples were then stored at the size of the gel. The membrane was wet with -20 °C until agarose gel preparation. A 1% agarose 100% methanol, then transferred to MilliQwater gel w as prepared through 1.5g of agarose (Sigma and soaked for several minutes. A standard blotting No. A-6877 Type II) to 150mL of Tris-Acetate4 Isolation of Recombinant Taq Polymerase for PCR EDTA (TAE) buffer. The solution was microwave for 1 minute and mixed until in solution. Once cooled to 60 °C, 7.5 ÃŽ ¼L of Biotium Gel Red Nucleic acid stain was added and mixed. The solution was then poured into the electrophoresis tray; a comb was installed, and set at room temperature. One Litre of 1x Tae buffer was prepared through dilution of 50x TAE buffer and then the solution was poured onto the electrophoresis tray to cover the gel in 1mm of buffer. 20 ÃŽ ¼L of PCR product prepared previously and 4ÃŽ ¼L of Gel Red dye were mixed and 20ÃŽ ¼L of each sample, the standard, and Taq prepared the previous year and Invitrogen life Technologies 1 Kb DNA ladder Cat. No. 15615016 was run at 150 Volts, 100 mA for one hour (or until dye reached the bottom of the gel). The bands were then visualized under 300 nm light and fluorescence was measured at 590 nm. The gel was analyzed using AlphaEaseFC software. concentration of the sample Taq was 1.88 + 0.11 mg/mL. The solution of proteins was not pure Taq as confirmed by the SDS-PAGE (Fig. 2) as various proteins created distinct bands (B to K excluding E). The standard Taq revealed only one band (A), indicating band E was most likely belong to Taq, as it was the darkest band in the gel. An analysis of the molecular weights of the bands through electrophoretic mobility (Tab. 3) showed the standard Taq having a molecular weight of 115.2 + 14.6 kDa, and the likely band (E) had a molecular weight of 113.4 + 14.3 kDa. There was a distinct distortion in the bands of the SDS page in all lanes with the exception of the standard Taq and the 2011 Taq (Fig. 3). The distortion is of a smile. The overall gel also has a large distortion, but of a frown. It would appear there was a similar protein to D E and F present in all samples, including the 2011 sample. The standard Taq did not contain the bands. Re al Time PCR The Western Blot (Fig. 4) revealed distinct A master mix for PCR was prepared (1x PCR bands; however, there were more than one band in buffer minus Mg, 0.2mM dNTP, 1.5 mM MgCl2, each lane with the exception of the standard Taq. 0.5ÃŽ ¼M forward primer, 0.5ÃŽ ¼m Reverse Primer, 0.1 Two distinct bands were present in 5, Taq, and 2 (b, ng Template DNA and Nuclease-free PCR water). c). The lanes of * and ? contained several bands To PCR tubes, 22.5 ÃŽ ¼L of Master Mix and 2.5 ÃŽ ¼L also. The overall gel also expressed a slight color of Taq sample or the standard Taq were combined, banding along the solvent front edge which is mixed through vortexing and then centrifuged with shown in both Fig. 3 and 4. The 2011 lane did not a Fisher Scientific Accuspin micro 17 just briefly appear to have any Taq present, as no band was using 1.5mL eppendorf tubes with no caps to distinguished. The entire ladder expressed some contain the PCR tube. The Taq samples were antibody activity. prepared in triplicates. 20ÃŽ ¼L of each sampled were The real time-PCR revealed a threshold reached then transferred to a 96-well PCR plate and then at 20 cycles, with the vast majority occurring at 24 sealed. The well was then placed in a BioRad CFX cycles. The melt curve showed an approximate connect Real Time System using the programing of melting temperature of 81 °C (Fig. 7). enzyme activat ion (95 °C, 30 seconds, 1 cycle), 40 The agarose gel electrophoresis revealed one cycles of Denaturation (95 °C, 1 second) and distinct band at approximately 5883.5 base pairs in annealing/extension (60 °C, 5 seconds), with a melt length. The brightest bands, and therefore the curve of (60-95 °C in 0.5 °C intervals, 3 seconds per highest quantities of Taq enzyme were found in the step, 1 cycle). The samples were then analyzed std., 2 and 4. When the base pairs specific activity using AlphaEase FC software. of the enzyme was calculated it was found to be 834.5 + 63.9 bp/min/ÃŽ ¼g of sample, or 3922.3 + 192.9 bp per minute. RESULTS The results of the Bio-Rad assay on the sample of Taq polymerases diluted to 10x and 100x revealed that the 10x dilution was far to concentrated and fell outside the linear curve of the Bio-Rad assay. The retrieval of protein from the Luria broth was found to be 300.8 + 17.7 mg protein per L of Luria broth. These results (Tab. 1) suggest the protein 5 DISCUSSION Through the analysis made through SDS-PAGE, the MW of the standard Taq was found to be 115.2 + 14.6 kDa and 113.4 + 14.3 kDa. This is different from the accepted literature value of 94 kDa (9). Even with error correction, the prot ein did not fall Isolation of Recombinant Taq Polymerase for PCR within the range of the accepted literature value. In total, the two proteins differ by 23% and 21% without error correction, or 21.2 kDa and 19.4 kDa respectively. In comparison to one another, the two bands have essentially the same molecular weight, indicating whatever error occured in the gel was equivalent on both the standard and the isolated Taq. One explanation for the difference in the molecular weights may be explained through the quantitiy of protein used. The darkest and thickest band ( E, fig. 4) likely belongs to the Taq protein. To get a more defined band, a dilution would be effective in making a higher resolution band (12). The amount of protein isolated per volume of Luria broth was determined to be 300.8 + 17.7 mg per L of Luria Broth. Quite obviosuly, there are issues both with the heating of the gel, and distortion of the bands into â€Å"smiles†. The distoration of the gel likely was caused by unequal heati ng of the gel causing the center of the gel to be hotter than the peripheries, as the walls of the apparatus act as heat sinks (13). The uneven heating can be removed by switching to a lower voltage for a longer period of time (12). The distortion of the protein bands within the individual lanes produced a smile structure. The distortion was likely caused by either an overloading of proteins, which can be solved by dilution of the protein sample, or was due to salt conditions of the loading sample. This step could be fixed through extra steps of dialysis to decrease salt content of the loading sample. (14). One final issue with the SDS-PAGE gel was the distance between bands. The target molecular weight was near 100 kDa, so the concentration of the gel could be decreased to allow for a higher resolution of the higher molecular weight proteins, or allowed to run for a longer period of time (14). A purity assessment of the isolated Taq enzyme can be made through the SDS-PAGE gel (fig. 2). Distinct banding occurs in ten different bands on the Taq lane, with 9 being distinct from Taq protein (E). This highlights that there were infact multiple proteins still present in the Taq solution. This would indicate that the heat shock portion of the methods was insufficient in denaturing all of the proteins in the E. coli, allowing for precipitation upon salting out. This is based on the extra protein banding only occuring for the Taq polymerases prepared for this experiment. A factor that could have also played a role was the incubation at 75 °C was continually 6 interrupted through the need to shake the reaction vessel thereby lowering the temperature of the solution. This was due to mechanical difficulties of the equipment. It would be best to find a working New Brunswick Scientific-Innova 40 incubator shaker series to improve the protein isolation. To decrease the protein impurities, an increased heat cycle could be implemented, as Taq is thermostable at 75 °C, and could sustain structure at that temperature for long durations (7). The ammonium sulfate salting out would be mor e efficent after an increased heat cycle as even fewer native state proteins would remain (10). Another method to decrease impurities would be to add a purification step using another specific property of Taq polymerase. This could be the isoelectric point. This could be done through ion exchange columnsor isoelectric focusing (12). The extra isolation step would significantly decrease the impurities, and increase the specific activity per mg of protein of sample.The impurities were likely a result of other proteins present in E. coli bacteria lysate that were relatively thermostable, as those proteins would be most probable (9). The isolation of Taq can be confirmed through the Western Blotting and PCR reactions (Fig. 4-7), as a distinct band in the Western Blot, and measureable amplicon replication in the PCR and rt-PCR. In the standard of Taq of the Western blot (Fig. 4) there is a distinct band. The same band in the channel containing the isolated Taq can be seen. The band occurs in the same relative vicinity as the Taq molecular weight band in the SDS-PAGE (Fig. 2) so would fit best fit the Taq enzyme. The banding of the blot shows a common band across all lanes that line up with the standard Taq, emphasizing the isolation of Taq. There is a hesitation in confirmation of Taq due to the extra protein banding in the prepared fractions, as these bands were not seen in the standard Taq. The banding would suggest proteins transferred from the gel to the membrane and was still able to bind to the primary antibody or secondary antibody. There are various possible explanations for this. First and foremost, the banding occurred in areas wherever protein was present (ladder and lanes). This would indicate lack of specificity in the primary antibody which is intended to only find full sequence Taq and bind to it (15, 16). Another problem may be due to lack of blocking solution binding to the membrane, or Isolation of Recombinant Taq Polymerase for PCR excessive washing removing blocking solution from the membrane. A final possible explanation may be binding of the secondary antibody to membr ane bound proteins with the exception of casein (the blocking protein used) (15, 17). Antibody specificity can be corrected by finding a new antibody, lack of blocking simply requires longer blocking periods or increased blocking solution concentration, and washing can be minimized to see resultant effect on the membrane. Each of the possible problems with the Western Blot would have to be tested by altering the procedure used above by one method (washing, antibody, blocking solution). The PCR results show template replication through thermocycling, which indicates the presence of thermostable DNA polymerases in the PCR tube. From this, it can be conferred that Taq polymerase was indeed isolated. Further confirmation could be made through further purification of Taq. This could be done through 2-D SDS-PAGE vs Isoelectric point electrophoresis using the isoelectric point of Taq and using the bands emphasized as Taq, and a lower concentration gel (12). Another method would be to analyze the gel bands through other methods such as mass spectropscopy or NMR (18). There wa s distinct differences between three sets of Taq polymerases: the standard, the sample prepared in the previous year, and the sample produced in this experiment. Most distinctly the proteins differ with respect to SDS-PAGE gels. Quite obviously, the purest of the enzymes was the standard Taq, followed by the 2011 sample, and the sample prepared in this experiment. The sample prepared through this experiment had a high amount of a salt concentration and resulted in distorted bands, along with numerous other proteins present in the sample. The enzymes also differed with respect to the Western Blot (Fig. 4). The 2011 sample failed to return 2 ° antibody response, indicating lack of Taq polymerase, or lack of primary antibody binding, while the standard and experimental sample both had representive banding. There may have been excessive blocking or drying of the lane containing the 2011 Taq, as the SDS-PAGE shows a representive band in the region of Taq, that is the darkest band in the lane (15). The protein concentrations as determined through the Bio-Rad assay (Tab. 1, Fig. 1) returned 7 drastically different results. The two protein concentrations differed by 2x concentration. The easiest explanation of thi s result is the 10x dilution was insufficient in reducing the absorbance to within the standard curve. Due to the absorbances being above the standard curve, the results are invalid, as the region in which the curve is linear is up to 0.5mg/mL (19). The 100 x dilution returned a result of 1.88 + 0.11 mg/mL. This coroborates the SDS-PAGE findings as the protein was not excessively overloading the lane. The SDS-PAGE could have been further diluted, but the concentration used was sufficient for the purposes of the experiment. In an analysis of the PCR results (fig. 7), the brightest fluorescence bands occurred in the std., 2 and (4/Taq) lanes. This would indicate the highest activities occuring in these lanes. When compared to the western blot, the darkest banding of regions of Taq (5,?,*) returned the bands with less fluorescence. This result shows that the amount of enzyme may inhibit the PCR reaction as the the bands with the highest recoveries returned the lowest fluorescence. With an assessment of the basepair length, reaction time, and amount of enzyme used, an approximately activi ty of 834.5 + 63.9 bp/min/ÃŽ ¼g of protein, or 3922.3 + 192.9 bp per minute. In comparison to the literature values of the protein, this is slightly above the 60 base pairs per second value, however, that was at 70  °C (7). The rt-PCR returned a consistent melting temperature of 81 °C (Fig. 6)for all amplicon samples indicating the lack of a primer-dimer formation. Threshold was initially reached at 20 cycles (Fig. 5), which an RFU value of approximately 9000. This indicated a high activity of the taq polymerase used, at least above 1.25 Units (20). Both PCR assays agree with one another. There was no primer dimer formation noted on the agarose gel, or the melt curve analysis. There was a high activity of the enzyme sample isolated as found through the bpmin-1 and cycle # of reaching threshold, however, between the two assays, the rt-PCR has the significant advantage of time, and no electrophoresis required. Currently, Taq is widely available and would likely be cheaper to simply purchase commercially. This experiment does however outline a method for thermostable protein isolation which could be used for the more recent and more valuable thermostable enzymes (Pyrococcus furiosus Polymerase) which Is olation of Recombinant Taq Polymerase for PCR are superior to Taq in both thermostability, and error rate due to proofreading ability (21). Overall, the purpose of the experiment was met. Taq was indeed isolated from a culture of recombinant E. coli. This was confirmed through the Western Blotting, and thermostable DNA activity in the PCR and rt-PCR. The purity was assessed and found to be below that of the methods used by Engelke et al., 1990. The purity could be increased through use of a cation exchange column (9). The length of heat denaturation and an automatic heat controlled shaker would help to remove excess proteins and improve purity. The length of dialysis time would need to be increased for less band distortion in SDS-PAGE, and either more selective primary antibody, increased blocking or decreased washing would be required for improved Western Blotting. For further experiments, it is suggested testing the new method modifications, and or implementing recombinant Pyrococcus furiosus Polymerase.

Automatic Number Pate Recognition System Information Technology Essay

Automatic Number Pate Recognition System Information Technology Essay Automatic number pate recognition system is a mass surveillance method that uses optical character recognition on images to read the license plates on vehicles. System might scan number plates at around one per second on cars traveling up to 100mph(160 km/h).they can use existing closed -circuit television or road-rule enforcement cameras, or ones specifically designed for the task. They are used by various police forces and as a method of electronic toll collection on pay-per-use roads and monitoring traffic activity, such as red light adherence in an intersection. ANPR can be used to store the images capture by the cameras as well as the text from the license plate, with some configurable to store a photograph of the driver. Systems commonly use infrared lighting to allow the camera to take the picture at any time of the day. A powerful flash is inclined in at least one version of the intersection-monitoring cameras, serving both to illuminate the picture and to make the offender aware of his or her mistake. ANPR technology tends to be region -specific, owing to pate variation from place to place. Some concerns about these systems have centered on privacy fears of government tracking citizens movements and media reports of misidentification and high error rates. However, as they have developed, the systems have become much more accurate and reliable. There is an increasing requirement to identify vehicles and track their location for a wide number of applications. These include: Congestion charging Several major cities around the world levy a charge a drive within them Car park management Using the number plate to identify the time of entry and departure of a Vehicle. Counter-terrorism Monitoring the arrival and departures of vehicles at major ports. Literature Review Our literature survey mainly focused on automatic number plate system research papers and its existing system along with its application, image processing technique and neural network recognition. These can be clearly illustrated as follows: Automatic number plate recognition system Javaanpr existing open source code in sourceforge.net Thesis describing research, image processing and neural networking technique along with its algorithm in pdf on javaanpr on sourceforge.net Image processing technique ImageJ -api based on java language for digital image processing Image editor -api based on java language made for image processing JAI api -java advance imaging for image processing from sunmicrosystem at java.sun.com. Opencv- Digital Image Processing (text book from library) Neural networking technique Introduction to java neural network second edition by jfheaton at heatonresearch.com Some ocr samples using neuralnetworking at sourcecode.com and its explanation Study on nepali ocr research conducted by madan puraskar guthi(yala Maya Kendra) Ocr sample developed by Google based for Linux available for windows on dot net (tesseract) Jooneengine-java api on neural network not so well developed and efficient at http://www.jooneworld.com Kohenen -java api on self organizing map applied to compress jpeg image. Somdemo-sample java program for illustration how self organizing map works. Program iterately train to converge with identical color from random samples Artificial neural network text book available at library (low price edition from pearson education. Neural networks systematic introduction by Raul Rojas(from lectures at free university at Berlin and later at the university of Halle) Automatic Number Pate Recognition system a)javaanpr Javaanpr open source available at sourceforge.net worked as prototype for building our Nepali automatic Nepali number plate recognition system. It also contain thesis in pdf format prescribing image processing technique and neural networking technique along with its algorithm. It works well recognizing foreign number plates contained as sample in the site. It was beautifully coded applying sophisticated and specialized algorithms for image processing and neural network technique. It also used xml files to save and retrieve neural network training data. Figure: sample javaanpr at sourceforge.net For more information-http://sourceforge.net Image processing Technique a)ImageJ 1.42 ImageJ was first developed on class files now available on GUI interface. User can just process image using various buttons and entries if prescription is required .programmers can develop own macros and plugins to achieve its intended function if required and compile there within and run the code. It is capable of processing both 2D and 3D interactive image processing. Figure. ImageJ graphical window interface For more information: http://rsb.info.nih.gov/ij/ b) Image editor Image editor was also found during search for image processing tool. It is also based on java language and available as java API, now class file are available with GUI interface easing its its manipulation. Image editor api seems inefficient and not so capable for our intended operation and not so much researched. C) JAI api the java advance imaging(JAI) API further extends the java platforms (including the java 2D API) by allowing sophisticated, high -performance image processing to be incorporated into java applets and applications.JAI is a set of classes providing imaging functionality beyond that of Java 2D and the Java Foundation classes, though it is compatible with those APIs. JAI implements a set of core image processing capabilities including image tiling, regions of interest, and deferred execution.JAI also offers a set of core image processing operators including many common point, area, and frequency-domain operators. JAI is intended to meet the needs of all imaging applications. The API is highly extensible, allowing new image processing operations to be added in such a way as to appear to be a native part of it. Thus, JAI benefits virtually all Java developers who want to incorporate imaging into their applets and applications. JAI features Cross-platform imaging Distributed Imaging Object-oriented API Flexible and Extensible Device Independent Powerful High Performance Interoperable Initially program coding was done in JAI Later it becomes little inefficient and we again go for another programming method. For further information-http://java.sun.com d) Digital Image processing (text book from library) e) Opencv The OpenCV implements a wide variety of tools for image interpretation. It is compatible with Intel ® Image Processing Library (IPL) that implements low-level operations on digital images. In spite of primitives such as binarization, filtering, image statistics, pyramids, OpenCV is mostly a high-level library implementing algorithms for calibration techniques (Camera Calibration), feature detection (Feature) and tracking (Optical Flow),shape analysis(Geometry, Contour Processing ),motion analysis (Motion Templates, Estimators ), 3D reconstruction (View Morphing),object segmentation and recognition (Histogram, Embedded Hidden Markov Models, Eigen Objects). The essential features of the library along with functionality and quality is performance. The algorithms are based on highly flexible data structures (Dynamic Data Structures) coupled with IPL data structures; more than a half of the functions have been assembler optimized taking advantage of Intel ® Architecture (Pentium ®MMXà ¢Ã¢â‚¬Å¾Ã‚ ¢,Pentium ® Pro, Pentium ®III, Pentium ®4). Why We Need OpenCV Library The OpenCV Library is a way of establishing an open source vision community that Will make better use of up-to-date opportunities to apply computer vision in the Growing PC environment. The software provides a set of image processing functions, As well as image and pattern analysis functions. The functions are optimized for Intel ® Architecture processors, and are particularly effective at taking advantage of MMX†ºÃ¢â‚¬ º Technology. The OpenCV Library has platform-independent interface and supplied with whole C Sources. OpenCV is open. Relation between Opens and Other Libraries OpenCV is designed to be used together with Intel ® Image Processing Library (IPL) And extends the latter functionality toward image and pattern analysis. Therefore, OpenCV shares the same image format (IplImage) with IPL. Also, OpenCV uses Intel ® Integrated Performance Primitives (IPP) on lower-level, if It can locate the IPP binaries on startup. IPP provides cross-platform interface to highly-optimized low-level functions that Perform domain-specific operations, particularly, image processing and computer Vision primitive operations. IPP exists on multiple platforms including IA32, IA64, And StrongARM. Source:-openCV reference manual.pdf Cmgui-wx-2(.net wrapper class) This openCV tool can be easily integrated with .net platform like c#, visual basic etc. Cmgui is an advanced 3D visualization software package with modeling capabilities.Cmgui is a part of CMISS, a mathematical modeling environment initially developed by the University of Auckland Bioengineering Institute.CMISS stands for Continuum Mechanics, Image analysis. Signal processing and System Identification. There are three major CMISS software packages. Broadly speaking the main areas each piece of software deals with are as follows: CM is used for computational modeling Unemap is used for signal acquisition and processing Cmgui is used for model visualization and manipulation For more information:-wiki/getting started with cmgui Neural Networking technique a) Introduction to java neural network by jeff heaton This book along with video lecture helped very much for us to understand neural networks and learn coding technique. It was published form Heaton research center and they have developed encog framework for neural network where programmer can build fast neural network prototype for fast testing and checking since easy and flexible. After parameters have been determined for best operation such as number of hidden layers and number of neurons in each layer coding can be done since it code will be inflexible for such modification. Book contained different chapters on various types of neural networks and also its application. Only first seven chapters are allowed to read online and rests are not. It provides all its source code on site which also helps in learning and testing. Same book is also available in c# language. For more information-http://heatonreasearch.com/ b) On the beginning of project research we also got OCR sample using neural network at sourcecode.com with explanation. It was written at c#, due to compiler problem I didnt stress here much. c) Nepali OCR For us it was good news and opportunity to study research on Nepali OCR conducted by madan puraskar guthi. Different research papers were available on the site along with image processing portion code used to fragment Nepali character Image written on java. It deals with problem issues and complexity faced on Nepali character like devnagari font. For more information -http:// d) OCR engine tessaract by Google This was used by Nepali OCR for its processing and it supports many languages like Hindi, Nepali, Urdu, arabi etc. we didnt research here much. Figure: segmented portion of Figure : Another segmented portion of For more information-make Google search for link d) joone engine joone engine as a api in hope for easy and efficient coding we consider but it seems unworthy for project work. For beginner liking to test some xor operations and similar may find at least satisfactory otherwise unworthy. For more information-http://www.jooneworld.com/docs/engine.html e)Kohenen This sample also seems beautiful in understanding self organizing map or kohenen network. Here it is used to compress jpeg image. It was programmed on seven packages. For more information-http: // f) som demo This sample tries to converge iteratively with similar colors from randomly scattered pixel colors based on Euclidean distance method. Figure: som before training Figure: som after training For more information-link available at reference http://www.ai-junkie.com/ann/som/ g) Artificial neural Network text book (library) h) Neural network systematic introduction (by Raul Rojas) This book is good for understanding neural network systematically and based on lectures at free university at Berlin and later at the University of Halle. For more introduction-reference at http://www.wikipedia.com/selforganisingmap Figure: sample kohenen neural network (3D kohenen feature map) Source: http://rfhs8012.fh-regensburg.de/~jfroehl/index.html Anpr system application around world Police enforcement Germany On 11 March 2008, the Federal Constitution Court of Germany ruled that the laws permitting the use of automated number plate recognition systems in Germany violated te right to privacy. Hungary Several Hungarian Auxiliary Police units use a system called Matrix Police in cooperation with the police. It consists of a portable computer equipped with a webcam that scans the stolen car database using automatic number plate recognition. The system is installed on the dashboard of selected patrol vehicles (PDA based handled versions exists as well) and is mainly used to control the license plate of parking cars, as the Auxiliary Police doesnt have the authority to order moving vehicles to stop. If a stolen is found, the formal police are informed. United Kingdom The UK has an extensive (ANPR) automatic number plate recognition CCTV network. Effectively, the police and security services track all car movements around the country and are able to track any car in close to real time. Vehicle movements are stored for 5 years in the National ANPR Data Centre to be analyzed for intelligence and to be used as evidence. USA In the USA, ANPR systems are more commonly referred to as LPR (License Plate Reader or License Plate Recognition) technology or ALPR (Automatic License Plate Reader/Recognition) technology. One of the biggest challenges with ALPR technology in the US is the accuracy of the Optical Character Recognition (OCR)-the actual identification of the characters on the license plate. From time to time, states will make significant changes in their license plate protocol that will affect OCR accuracy. They may add a character or add a new license plate design. ALPR systems must adapt to these changes quickly in order to be effective. In addition to the real-time processing of the license plate numbers, some ALPR systems in the US collect data at the time of each license plate capture .Data such as date and time stamps and GPS coordinates can be reviewed in relation to investigations and can help lead to critical breaks such as placing a suspect at a scene, witness identification, pattern recognition or the tracking of suspect individuals. Average Speed cameras Another use of ANPR in the UK, Italy and Dubai (UAE) is for speed cameras which work by tracking vehicles travel time between two fixed points ,and therefore calculate the average speed. These cameras are claimed to have an advantage over traditional speed cameras in maintaining steady legal speeds over extended distances, rather than encouraging heavy braking on approach to specific camera locations and subsequent acceleration back to illegal speeds. UK The longest stretch of average speed cameras in the UK is found on the A77 road in Scotland, with 30 miles (48 km) being monitored between Glasgow and Ayr. Italy In Italian highways has developed a monitoring system named Tutor covering more than 1244 km (2007). Further extensions will add 900 km before the end of 2008. The Tutor system is also able to intercept cars while changing lanes. Traffic control Many cities and district have developed traffic control systems to help the movement and flow of vehicles around the road network. This had topically involved looking at historical data, estimates, observations and statistics such as: Car park usage Pedestrian crossing usage Number of vehicles along a road Areas of low and high congestion Frequency, location and cause of road words The UK Company Traffic master has used ANPR since 1998 to estimate average traffic speeds on non-motorway roads without the results being skewed by local fluctuations caused by traffic lights and similar. The company now operates a network of over 4000 ANPR cameras ,but claims that only the four most central digits are identified , and no number plate data is retained. Electronic toll collection Ontarios 407 ETR highway uses a combination of ANPR and radio transponders to toll vehicles entering and exiting the road. Radio antennas are located at each junction and detect the transponders, logging the unique identify of each vehicle in much the same way as the ANPR system does. There are numerous other electronic toll collection networks which use combination of Radio frequency identification and ANPR. These include: Bridge pass for the Saint John Harbor Bridge in Saint John New Brunswick City link Eastlink in Melbourne, Australia Gateway Motorway and Logan Motorway, Brisbane , Australia Fast Trak in California ,United states Highway 6 in Israel Tunnels in Hong Kong etc Charge zones the London congestion charge The London congestion charge is an example of a system that charges motorists entering a payment area. Transport for London (TFL uses ANPR systems and charges motorists a daily fee of  £8 paid before 10pm if they enter, leave or move around within the congestion charge zone. Stockholm congestion tax In Stockholm, Sweden, ANPR is used for the congestion tax of cars driving into or out of the inner city must pay a charge, depending on the time of the day. Other uses ANPR systems may also be used for/by: Section control, to measure average vehicle speed over longer distances. Border crossings Fillings stations to log when a motorist drives away without paying for their fuel. A marketing tool to log patterns of use Traffic management systems, which determine traffic flow using the time it takes vehicles to pass two ANPR sites. Drive Through Customer Recognition, to automatically recognize customers based on their license plate and offer them their last selection, improving service to the customer To assist visitor management systems in recognizing guest vehicles. Circumvention Techniques (drawback) Vehicles owners have used a variety of techniques in an attempt to evade ANPR systems and road -rule enforcement cameras in general. These methods may be Increasing reflective properties of the lettering and so that system might no locate or produce high enough level of contrast to be able to read Use of plate cover or spray Use of dirt to smear their license plate or utilize covers to mask the plate ANPR imaging hardware The frontend of any Imaging hardware is image capturing device that is camera. Retroreflective camera returns the light back to the source and thus improves the contrast of the image. A camera that makes use of active infrared imaging (with a normal color filter over the lens and infrared illuminator next to it) benefits greatly from this as the infrared waves are reflected back from the plate. This is only possible on dedicated ANPR cameras, however, and so cameras used for other purposes must rely more heavily on the software capabilities. Figure: hardware components used in ANPR system Figure source-http://securityautomation.co.uk To avoid blurring it is ideal to have the shutter speed of a dedicated camera set to 1/1000th of a second. License plate capture cameras can now produce usable images from vehicles traveling at 120 mph (190 km/h).threshold angles of incidence between camera lens and license plate are also major consideration to avoid image distortion during installation. Manufacturers have developed tools to eliminate errors from the physical installation of license plate capture cameras. Research on down sampling character For neural network input character image is down sampled into matrix whose value is binary 1 or 0 according to Boolean property of character on matrix region. It showed that no of samples required is not fixed and it varies with thickness of font traced. Figure: down sampling image character o with 7*5 matrix Figure: downsampling same character image o (buffered) with 32 *35 matrix Research works on algorithms A new algorithm for character segmentation of license plate Character segmentation is an important step in License Plate Recognition (LPR) system. There are many difficulties in this step, such as the influence of image noise, plate frame, rivet, the space mark, and so on. This new algorithm presents character segmentation using Hough transformation and the prior knowledge in horizontal and vertical segmentation respectively. Furthermore, a new object enhancement technique is used for image preprocessing. The experimentation results show a good performance of this new segmentation algorithm. Algorithm (steps) Preprocessing Size normalization Determination of plate kind Object enhancement Horizontal segmentation using Hough transformation Vertical segmentation For more information:-a new algorithm for character segmentation of license plate.pdf an adaptive thresholding algorithm for the augmented reality toolkit It is well known that fixed global thresholds have adverse effects on the reliability of marker-based optical trackers under non-uniform lighting conditions. Mobile augmented reality applications, by their very nature, demand a certain level of robustness against varying external illumination from visual tracking algorithms currently AAR Toolkit depends on fixed-threshold image-binarization in order to detect candidate fiducials for further processing. In an effort to minimize tracking failure due to uniform shadows and reflections on a marker surface, a fast algorithm for selecting adaptive threshold values, based on the arithmetic mean of pixel intensities over a region-of- interest around candidate fiducials. Algorithm This works on a per-marker basis and evaluates the mean pixel luminance over a thresholding region-of -interest (ROI), which is defined as bounding rectangle around the markers axis -aligned corner vertices in screen space. If a marker has been detected in any given frame, its bounding rectangle will be used as thresholding -ROI prediction for successive frames. This method yields good thresholding level in practice, given sufficiently high video frame rates. Fig.1.reflection off a markers surface with adaptive thresholding (upper) and a global threshold (lower) For more information:-10.1.1.9.4636.pdf adaptive license plate image extraction This paper represents the automatic plate localization component of a car license plate recognition system. The approach concerns stages of preprocessing, edge detection, filtering, detection of the plates position, slope evaluation, and character segmentation and recognition. Single gray-level images are used as the only source of information. In the experiments Israeli and Bulgarian license plates were used, camera obtained at different daytime and whether conditions. Algorithm (step) preprocessing for plate candidate identification vertical edge detection rank filtering plate candidate segmentation vertical projection acquisition prime clipping of the plate plate skew evaluation horizontal segmentation plate candidate verification Cray-level distribution consistency considerations

Madness and Insanity in Shakespeares Hamlet - Insanity within Hamlet E

Insanity within Hamlet  Ã‚        Ã‚  Ã‚   Let us explore in this essay the real or feigned madness of the hero in William Shakespeare’s dramatic tragedy Hamlet.    Critical opinion is divided on this question. A.C. Bradley in Shakespearean Tragedy staunchly adheres to the belief that Hamlet would cease to be a tragic character if he were really mad at any time in the play (30). On the other hand, W. Thomas MacCary in Hamlet: A Guide to the Play maintains that the prince not only feigns insanity but also shows signs of true insanity:    Hamlet feigns madness but also shows signs of true madness) after his father’s death and his mother’s overhasty remarriage; Ophelia actually does go mad after her father’s death at the hands of Hamlet. For both, madness is a kind of freedom – a license to speak truth. Those who hear them listen carefully, expecting to find something of substance in their speech. Is it they, the audience, who make something out of nothing, or is it the mad who make something out of the nothing of ordinary experience? (90)    Hamlet’s conversation with Claudius is insane language to the latter. Lawrence Danson in â€Å"Tragic Alphabet† describes how Hamlet’s use of the syllogism is pure madness to the king:    From Claudius’s point of view, however, the syllogism is simply mad: its logic is part of Hamlet’s â€Å"antic disposition.† Sane men know, after all, that â€Å"man and wife is one flesh† only in a metaphoric or symbolic sense; they know that only a madman would look for literal truth in linguistic conventions. And Claudius is right that such â€Å"madness in great ones must not unwatched go† (III.i.end). (70)    Hamlet’s first words in the play say that Claudius is "A little more than kin and less t... ... Sons, 1899.    Felperin, Howard. â€Å"O’erdoing Termagant.† Modern Critical Interpretations: Hamlet. Ed. Harold Bloom. New York: Chelsea House, 1986. Rpt. of â€Å"O’erdoing Termagant: An Approach to Shakespearean Mimesis.† The Yale Review 63, no.3 (Spring 1974).    Foakes, R.A.. â€Å"The Play’s Courtly Setting.† Readings on Hamlet. Ed. Don Nardo. San Diego: Greenhaven Press, 1999. Rpt. of â€Å"Hamlet and the Court of Elsinore.† Shakespeare Survey: An Annual Survey of Shakespearean Study and Production. No. 9. Ed. Allardyce Nicoll. Cambridge, Eng.: Cambridge University Press, 1956.    MacCary, W. Thomas. Hamlet: A Guide to the Play. Westport, CN: Greenwood Press, 1998.    Shakespeare, William. The Tragedy of Hamlet, Prince of Denmark. Massachusetts Institute of Technology. 1995. http://www.chemicool.com/Shakespeare/hamlet/full.html No line nos.