Comparing Machiavelli And Machiavelli s Views On Glory,...

Hobbes and Machiavelli were both advocates of self-preservation in their philosophies, although Hobbes placed higher priority on the idea of self-preservation more so than Machiavelli. Their views on glory, courage, and honor differ greatly from each other and as a result affect the level of emphasis they place on the value of self-preservation. Hobbes believed that self-preservation was the reason for entering a social contract and that fear of a violent death was the driving force behind leaving the state of nature. Machiavelli argues that the point of Government is for self-preservation. Machiavelli and Hobbes shared the same views on that the Sovereign or Prince should implement rules that benefit them first while having the best interest of the people in mind. The difference being that the sovereign for Hobbes should exude the collective will of all the people while Machiavelli would argue for a Prince who would make more difficult decisions that might not be in line with the wants of the people in order to achieve certain ends. If fear is what keeps the people alive for Hobbes; than Machiavelli on the use of fear through punishment on behalf of the prince is in line with his philosophy... â€Å"The bond of love is one which men, wretched creatures they are, break when it is to their advantage to do so; but fear is strengthened by a dread of punishment which is always effective† The use of fear by the Sovereign runs very powerfully with Hobbes so that the people willShow MoreRelatedStephen P. Robbins Timothy A. Judge (2011) Organizational Behaviour 15th Edi tion New Jersey: Prentice Hall393164 Words   |  1573 Pagesand permission should be obtained from the publisher prior to any prohibited reproduction, storage in a retrieval system, or transmission in any form or by any means, electronic, mechanical, photocopying, recording, or likewise. To obtain permission(s) to use material from this work, please submit a written request to Pearson Education, Inc., Permissions Department, One Lake Street, Upper Saddle River, New Jersey 07458, or you may fax your request to 201-236-3290. Many of the designations by manufacturersRead MoreDeveloping Management Skills404131 Words   |  1617 Pagesbuilt-in pretests and posttests, focus on what you need to learn and to review in order to succeed. Visit www.mymanagementlab.com to learn more. DEVELOPING MANAGEMENT SKILLS EIGHTH EDITION David A. Whetten BRIGHAM YOUNG UNIVERSITY Kim S. Cameron UNIVERSITY OF MICHIGAN Prentice Hall Boston Columbus Indianapolis New York San Francisco Upper Saddle River Amsterdam Cape Town Dubai London Madrid Milan Munich Paris Montreal Toronto Delhi Mexico City Sao Paulo Sydney Hong Kong Seoul Singapore

The Ultimate Basic Essay Outline Trick

The Ultimate Basic Essay Outline Trick The Upside to Basic Essay Outline Outlines can also function as a brainstorming tool that can provide you a notion of what things to write about. They can be useful for any paper to help you see. What the In-Crowd Won't Tell You About Basic Essay Outline Introduction essay paragraphs are central in any bit of writing for the reason they give readers advance info about the content and the intention behind the material. The conclusion provides the last say of the paper. Besides helping you organize your thoughts, an essay outline also enables you to plan the distance of your paper. The aim of your essay is defined by the sort of paper you're writing. The Fight Against Basic Essay Outline Employing an essay outline can help you organize your thoughts from starting to end and makes certain you don't leave any essential parts from the middle! Again, the more work that you do in the outline, the less work you must do when you're writing u p the very first draft. Thus what you present and how you do it is dependent upon the outline you have structured at the very start. An outline can help you organize your principal suggestions and determine the order in which you're likely to write about them. What You Should Do to Find Out About Basic Essay Outline Before You're Left Behind Mom Blog If you're a mom who enjoys writing, then a Mom Blog might be the perfect part-time business enterprise. The conclusion Generally speaking, different families elect for the divorce option for unique explanations. Essay outline for college plays an important function. The title is the aim of the research and the outline should guide the student to attain the objective. Getting the Best Basic Essay Outline Main body Arguments ought to be expressed in detail in the primary body. Classroom is going to be kept clean during and following class. Students lead busy lives and frequently forget about an approaching deadline. You may al so Speech Outline Template. The Honest to Goodness Truth on Basic Essay Outline Then, when the simple framework is down, you might find it a lot simpler to flesh things out. The draft goes together with the essay structure so the content can flow in a systematic method. There's a range of theories and approaches that have been propounded to date, to understand and locate an excellent idea about behaviour of various organizations. If you're writing a background synthesis, in some instances it could be appropriate that you offer you an interpretation of the material or have a position (thesis). Basic Essay Outline at a Glance Outlining makes it simple for the writer to create the points flow in the post. With essay outline, essays will stop writer to get off topic or jumping from 1 argument to a different argument that doesn't relate with what you're discussing. An outline for an informative essay ought to be developed while the writer is conducting his research since it will enable them to remember the most crucial points. Writing a great essay demands certain skills that is able to make your thoughts read and voice heard in an ideal way. The Nuiances of Basic Essay Outline Writing an essay outline can be as simple as you would like to make it. Writing an essay in mla format is comparable to writing any other kind of essay. Writing an outline is a rather effective method to think through how you'll organize and present the info in your essay. Before you start writing an outline for the essay, ensure you comprehend the assignment. The last paragraph of the essay stipulates the conclusion. An essay outline is a fundamental element for each and every essay. This essay outline can help you write more effectively. The 5 paragraph outline works with almost all kinds of essay writings. As you progress through school, you're going to be asked to compose essays. Find strategies to compose a simple essay and learn what kinds of essays are. Most essays, but do follow the exact same very basic. When you first begin writing essays in school, it is not uncommon to have a whole topic assigned to you. For instance, lets assume the goal of your essay is to compose an overview. Basically, the pieces of an essay proved just 3 divisions. It's crucial that you learn early on how best to compose effective essays that communicate clearly and accomplish certain objectives. After the term analytical essay is mentioned it could sound like an intricate kind of essay.

Dickens inform Essay Example For Students

Dickens inform Essay Throughout the opening chapters of Hard Times Dickens clearly expresses his concerns for the education system at the time. Set in the 1850s the middle of the Industrial Revolution Hard Times describes the school as a rationalist place were the schoolroom is a monotonous vault were children are taught only facts The way that Dickens describes the education clearly shows he is against it and that a romanticist education is better. The reader is forced to agree with him. The first paragraph of Hard Times uses the repetition of the Facts. Dickens dislike for the rationalist education is shown in the first sentence, where he uses a large capital letter in the phrase Now what I want is facts. The word now is an imperative, which shows that Gradgrind is very controlling. By using the phrase the reader is almost immediately aware of Gradgrinds fascination with Facts and that he has no reasoning and us always right, even when he is wrong. Dickens continues to show his anxiety for the education system by using the phrase plant nothing else and root of everything else. This informs the reader that Gradgrind sees the children as flowerbeds full of weeds and ones that need Facts and a rationalist education to become something in life. He also believes that without Facts the children will just remain young and hopeless for the rest of their lives. At the start of the second paragraph Dickens turns his description to the actual room in which the children are taught. He describes it with a metaphor, calling it a plain, bare, monotonous vault. This gives the image of a large room in which the children are locked up. It is described as a vault because Gradgrind considers the children valuable to the future of the country and he doesnt want them to get any romanticist ideas from the outside world. The words plain, bare and monotonous make the school seem very boring and emphasises at how factual Gradgrind and the education is. The schoolroom described in the novel contrasts with classrooms nowadays. In the book is a dull place now classrooms are bright and full of colour. Dickens then turns his description to Gradgrind, although he doesnt mention Gradgrinds name in chapter 1 as soon as you see the name in chapter 2 you can tell instantaneously tell that the speaker is Gradgrind. By repeating the word square in his description of Gradgrind, Dickens gives us the impression that Gradgrind is very factual as squares are considered quite factual shapes. When Dickens uses the metaphor two darks caves to describe Gradgrinds eye the reader gets the idea that Gradgrind is exceptionally sinister and that his rationalist view on life has caused him to lose all emotion and life. Dickens re-enforces everything he has said about the school and Gradgrind in chapter one by describing Gradgrinds clothes. Square coat, square legs, square shoulders nay his very neckcloth. Dickens is very clever in this phrase as he uses Satire to make us laugh at Gradgrind. By describing him as square shows that Gradgrind is very factual and re-enforces that he has a rationalist view on education. Dickens finishes chapter one by describing the children. He describes them as little vessels that are ready to have imperial gallons of full of facts poured in them. By using the metaphor little vessels Dickens give the impression that there are a lot of children in a small space sitting in silence. By exercising the word imperial before gallons helps to give of the impression that the school us factual. It shows Gradgrinds obsession with facts, he cannot just have gallons, as that is not factual enough. They have to be metric or imperial. .uf6315f560a5a011a46440c667dd76ce9 , .uf6315f560a5a011a46440c667dd76ce9 .postImageUrl , .uf6315f560a5a011a46440c667dd76ce9 .centered-text-area { min-height: 80px; position: relative; } .uf6315f560a5a011a46440c667dd76ce9 , .uf6315f560a5a011a46440c667dd76ce9:hover , .uf6315f560a5a011a46440c667dd76ce9:visited , .uf6315f560a5a011a46440c667dd76ce9:active { border:0!important; } .uf6315f560a5a011a46440c667dd76ce9 .clearfix:after { content: ""; display: table; clear: both; } .uf6315f560a5a011a46440c667dd76ce9 { display: block; transition: background-color 250ms; webkit-transition: background-color 250ms; width: 100%; opacity: 1; transition: opacity 250ms; webkit-transition: opacity 250ms; background-color: #95A5A6; } .uf6315f560a5a011a46440c667dd76ce9:active , .uf6315f560a5a011a46440c667dd76ce9:hover { opacity: 1; transition: opacity 250ms; webkit-transition: opacity 250ms; background-color: #2C3E50; } .uf6315f560a5a011a46440c667dd76ce9 .centered-text-area { width: 100%; position: relative ; } .uf6315f560a5a011a46440c667dd76ce9 .ctaText { border-bottom: 0 solid #fff; color: #2980B9; font-size: 16px; font-weight: bold; margin: 0; padding: 0; text-decoration: underline; } .uf6315f560a5a011a46440c667dd76ce9 .postTitle { color: #FFFFFF; font-size: 16px; font-weight: 600; margin: 0; padding: 0; width: 100%; } .uf6315f560a5a011a46440c667dd76ce9 .ctaButton { background-color: #7F8C8D!important; color: #2980B9; border: none; border-radius: 3px; box-shadow: none; font-size: 14px; font-weight: bold; line-height: 26px; moz-border-radius: 3px; text-align: center; text-decoration: none; text-shadow: none; width: 80px; min-height: 80px; background: url(https://artscolumbia.org/wp-content/plugins/intelly-related-posts/assets/images/simple-arrow.png)no-repeat; position: absolute; right: 0; top: 0; } .uf6315f560a5a011a46440c667dd76ce9:hover .ctaButton { background-color: #34495E!important; } .uf6315f560a5a011a46440c667dd76ce9 .centered-text { display: table; height: 80px; padding-left : 18px; top: 0; } .uf6315f560a5a011a46440c667dd76ce9 .uf6315f560a5a011a46440c667dd76ce9-content { display: table-cell; margin: 0; padding: 0; padding-right: 108px; position: relative; vertical-align: middle; width: 100%; } .uf6315f560a5a011a46440c667dd76ce9:after { content: ""; display: block; clear: both; } READ: An Inspector Calls decrease the dramatic tension EssayThe name of chapter two is very important as by calling it murdering the innocents Dickens is referring to the biblical story where King Herod ordered all baby boys to be killed for his own gain. This relates to the metaphorical killing of the students in the school.  At the start of chapter two Gradgrind is described as a man of facts and calculations, this re-enforces the idea that he is very factual. Gradgrinds name also shows this because by using the word grind, Dickens gives the reader the image of someone grey, dull and very grinding every time they read it.

The Marketing Plan of Honda free essay sample

The mobile phone and phone device market in Vietnam is developing and with the high growth rate of phone users, Vietnam becomes a potential market of phone company. First introduced in 1996, until now Nokia is one of the popular brands in Vietnam. Understanding customer needs and want is a key for success of Nokia. Nokia has many line products that can be listed out as E series, N series, 1200, Lumia†¦ Nowadays, digital technology and smart phone seem to be more popular. Nokia Vietnam has a wide range of products and it is necessary to identify which areas that could be improved. Thus, Nokia’s trend is smart phone which be integrated many applications, running on Windows operating system for phone. With the same macro-environment, there are many competitors such as Apple with Iphone, Samsung with Galaxy, Sony Ericsson with Xperia. There are also external and internal factors that affect the company and create Strength, Opportunities, Weakness and Threats and by analyzing these factors Nokia can figure out marketing strategy and with the development in Vietnam, Nokia is reaching their specific strategy that called â€Å"global supply chain strategy†. We will write a custom essay sample on The Marketing Plan of Honda or any similar topic specifically for you Do Not WasteYour Time HIRE WRITER Only 13.90 / page Table of contents Company profile Nokia, a Finnish company was established in 1865 and over the past 150 years, Nokia has evolved from a riverside paper mill in south-western Finland to a global telecommunications leader connecting over 1. 3 billion people. In 1998, Nokia was selected the world’s biggest mobile phone manufacturer (Nokia’s official website, 2010). In 2010, Nokia becomes a leading company in digital technology industry, including mobile phones, telecommunications networks, wireless data solutions and multimedia terminals with about 27% market share of World mobile industry. Nokia has three main product categories: mobile phones, telecommunications networks and software’s solutions. Recently, this group has met many challenges and income loss. According to the technology page on British newspaper – The Guardian, Nokia has lost the â€Å"King† position in mobile phones area. The revenue in the first quarter of 2012 of Nokia has dropped 29% over the same period last year, down to 9. 7 billion USD. In Vietnam, Nokia was first introduced in 1996 and until now it’s still the most favourite brand by occuping almost market share. In 2011,  Nokia  also  announced it will  build a factory  mobile phones  in Vietnam. The company said  the new plant  would be worth  200 million  Euros  and  will be  located near Hanoi. The  new production  will  comeintooperation  from  2012. The  Nokia  has factories   Brazil,  Mexico,  Finland,  Hungary, Romania, Britain, China, India  and Korea. Thus,  a factory in  Vietnam is  the 10th  country  of  the Finnish company. Vice President, Nokia said  the factory  in our country  will  play a major role  in connecting  billions of people on  the  internet. Company analysis Mission statement Nokia’s mission is simple: Connecting People. The goal is to build great mobile products that enable billions of people worldwide to enjoy more of what life has to offer. The challenge is to achieve this in an increasingly dynamic and competitive environment. Everyone has a need to communicate and share. Nokia helps people to fulfill this need and we help people feel close to what matters to them and focuses on providing consumers with very human technology NOKIA Nokia exists to connect people with innovative, quality, up to date products that are easy to use. Nokia is a world leader in mobile communications, driving the growth and sustainability of the broader mobility industry. Nokia connects people to each other and the information that matters to them with easy-to-use and innovative products like mobile phones, devices and solutions for imaging, games, media and businesses. Nokia provides equipment, solutions and services for network operators and corporations. † ( N okia. com) Company objective The opening ceremony took place on Apr 23rd in Bac Ninh and according to our source the Nokia’s new factory will produce cheap mobile devices to serve the growing markets with localized functions. Vietnam factory will be the third large on the the world, after other factories in India and China which focus on smart phone production. From Nov 2011, this group founded Nokia (Vietnam) LLC towards the objectives to open production units in this country. Currently, many divisions are in the process to recruit young local talents to ramp up the pre-production process. Environment Analysis Macro-environment are the major external and uncontrollable factors that influence a company’s decision making and affect its strategies and performance. Analysis factors helps Nokia determine their target and make suitable strategies. The tool used to scan the business environment is through PEST analysis. Demographic factors Vietnam is the country with high population and almost are the young. This is plays an important role in determining major customers and deciding orientation. Mobile phone usage in Vietnam is big and getting bigger. Nielsen’s 2008 Vietnam Mobile Insights Report shows mobile use is spread across all age with a slight skew to the 25-34 years old, followed by the 15-24 years old and 35-44 years old. From teenage to over 60s, it seems everyone has his own mobile phone. In fact, 74% of people in Ho Chi Minh City and Hanoi City; 58% of people in urban and 37% of people in rural areas own cell phone. With 54% of the population under 25 years of age, it’s no surprise that the young in Vietnam are hungry for new technology and keeping up with the latest product. Moreover, almost people are in working age so their need to communicate is high. Not only call and message, they but also check mail, update information wherever and whenever. Hence, the smart phone has a chance to development. Economic factors The global crisis also influenced in cell phone industry. It makes this market production down and the revenue decrease. But Vietnam economy grows faster and faster, the need of smart phone is not climb down. The growth rate of this kind of phone in 2011 is 44% and is predicted to become 51% in 2012. Personal and household incomes have increased significantly in recent year, leading to much higher consumption of consumer products and services, including mobile phone. Most people use their phone for business and spend less than 200,000VND per month using prepaid card. Social Factors Vietnamese consumers are resilient and despite the global economic crisis, they remain relatively positive. Vietnam consumers are ranked the 9th most confident in the world according to Nielsen’s Global Consumers Confidence study released in November 2008 Technological factors Research and development plays a key role in success of any company. The more technological advance the firm is, the better they have advantage to capture the market. As Nokia is well-known for its wireless technology and is among the leader. They are using their advance technology in the service provider market and provide strong network range to bring Vietnamese consumers high speed Wi-Fi production for working purpose. Competitor Analysis Competitor analysis  in  marketing is an assessment of the strengths and weaknesses of current and potential competitors. This analysis provides both an offensive and defensive strategic context to identify opportunities and threats. In the market, there are many companies those have the same product or same target with Nokia, both in mobile field and in software supply field. Thus, an analysising well competitor plays a vital role to Nokia survival and development. Indirect competitor In the phone field, Nokia has to compete with home phone company, internet phone company such as Ifone, Sky phone or home phone product from Siemens, LG†¦ In the software supply, App Store is a big competitor for Ovi Store. In photo sharing, Ovi Share is competiting with Dropbox and is said to be stopped in the near future. Direct competitor Vietnam is considered be one of the potential markets in the world so many companies and groups invested there. In Vietnam, there are more than 20 brands of mobile phone and these figures is increasing. Nokia has 3 major types of mobile phone product: classical series (1200, 110i, 1208†¦), original series (5300, 2700, E series†¦), smart phone series (N series, Lumia†¦). Each type has specific competitors. In smart phone sector, Samsung and Apple are 2 major competitive. Nokia, Samsung and Apple are the top 3 handset makers in the world Apple Apple certainly is the phone manufacture that receives the largest amount of buzz, but it is still a newcomer in the cell phone market. Companies like Nokia and Samsung have been creating handsets for much longer and they have built a reputation on people mind. The fact is Nokia and Samsung are the top two cell phone producers in Vietnam and Apple is maybe at the third place. Obviously, Apple produce Iphone, but both Samsung and Nokia create multiple types of phone. For example, Samsung produced Samsung Galaxy Note, Samsun Galaxy Nexus while Nokia has Lumia 800, Lumia 900, N9†¦ In the beginning of 2012, Apple started combining with 3 largest telephone network groups in Vietnam: Mobiphone, Vinaphone and Viettel in distributing Iphone in Vietnam. Thus, Vietnam people can buy Iphone with the best price and have no worry about fake product and also get many services from distributer. In spite of high price, iphone has other strong point such as IOS and Mac OS which is simply, smooth, good battery energy and powerful processing; good and luxurious design, huge apps store. Hence this smart phone becomes popular in a developing country like Vietnam. Samsung Speedy is a key to success of Samsung Electronics Company, which is known as its theory â€Å"Sashimi†. That is to apply the principle of continuous improvement technology and quickly launch new products to win customers’ attention. Galaxy is the smart phone line product of Samsung running on Android, which includes more than 20 products with many kinds of price for different customers. Having the same design with Iphone, but with the cheaper price and also give favor to customer such as free application on Android market, Samsung is going to be the first choice for Vietnamese people. Although the price is cheap and the design is good, Samsung products are reflected with many mistakes in operating system, camera†¦ Product is not long-lasting, is the weakness of Samsung. Positioning map Iphone4 High price Galaxy Note Galaxy Nexus Lumia 800 N8 High quality

Reflective Essay

Reflective Essay Reflective Essay Reflective Essay Amanda Williams HCS/455 September 29, 2014 Nicolette Hamilton Reflective Essay A health care policy is crucial for the industry. This will allow the industry to make changes that may be necessary for the benefit of the consumer. The first step in the policy making process is to figure out what the policy is going to be and what it is going to cover. As an example, a health care policy would be the creation of more employment opportunities for health care professionals. This would involve creating more facilities in rural areas. After the policy has been formulated it is time to start the implementation process. At this time the policy is sent to the appropriate division within the Health and Human Services. â€Å"The US government’s principal agency for implementing many of the health laws is the US Department of Health and Human Services (HHS)† (Shi, 2014 pg. 39). In this division the rules and regulations are made on how the policy will be enforced. For example, if each state is required to report the number of facilities, the requirement will be made to do so. The implementation stage of the process is putting the policy into action. During this stage stakeholders have responsibilities as well. Stakeholders would consist of the government that provides the funding for the policy, the individual states will ensure the policy is enforced, and facilities or organizations using the funds to expand. The failure or success of a policy is

When to Abbreviate, Etc.

When to Abbreviate, Etc. When to Abbreviate, Etc. When to Abbreviate, Etc. By Mark Nichol When is it appropriate to abbreviate words? The answer to this question, as with many matters in writing, is not a simple one: It depends on type of content and the degree of the content’s formality. In technical publications and scholarly journals, abbreviation of statistical information or references to dimensions and durations abounds. Furthermore, the American Medical Association’s manual of style dictates that periods be omitted in many abbreviations. However, in general content intended for professional publication, consider whether to abbreviate, especially in contexts in which multiple various abbreviations might be distracting. Social titles such as Mr., Mrs., and Ms., are usually superfluous altogether but are abbreviated when they appear, except in generic usage such as â€Å"Hey, mister!† Doctor is abbreviated before a name but otherwise spelled out, as is saint. (Note, however, that cities and other geographic designations differ in using Saint or St.; consult a resource to verify the correct style for a particular location.) Military and quasi-military ranks are spelled out or abbreviated depending on context, but as with other titles, they should be spelled out in isolation (for example, â€Å"The captain returned the salute†). Regarding i.e., that abbreviation and its close cousins e.g. and etc. are convenient, but they are no improvement on the English equivalents (â€Å"that is,† â€Å"for example,† and â€Å"and so on,† respectively). Style for scholarly journals is to use the abbreviations in parentheses and spell out the English phrases outside parentheses, but this distinction is not recommended for general-interest publications; avoid them altogether. Names of countries and other geopolitical entities are usually spelled out as nouns but abbreviated as adjectives (â€Å"the United States,† but â€Å"the US economy†); note in the previous example that periods in such designations, as in most other capitalized abbreviations, are unnecessary. Designations of thoroughfares, like many other words, can be abbreviated in lists or in graphics where space is at a premium, but generally spell out such terms as avenue, road, and street even when they are part of an address. Titles of senior corporate executives- CEO, COO, and CIO, for instance- are almost always abbreviated in all references, but VP (â€Å"vice president†), SVP (â€Å"senior vice president†), and the like are generally spelled out in all instances. Corporate terms such as PR (â€Å"public relations†) and HR (â€Å"human resources†) can be spelled out or abbreviated depending on context; they’re likely to be spelled out in a formal report and probably will be abbreviated in a casual reference in a mass-market book. Academic degrees are often abbreviated after a person’s name, but it’s better to refer to someone receiving a bachelor’s or master’s degree or a doctorate rather than a BA, an MA, or a PhD. Likewise, â€Å"curriculum vitae,† or the plural form â€Å"curricula vitae,† is preferable to CV. References to media such as CDs and DVDs are ubiquitous (or at least were before they began to give way to online access to music and films), and there’s no need to spell those initialisms out. (You’d likely have to look up that DVD originally stood for â€Å"digital video disc,† though the second word has since been supplanted by versatile. And who knows, or cares, that URL stands for â€Å"uniform resource locator†? The initialism will do in all cases.) However, words for parts of a book or other printed publication should be spelled out (for example, â€Å"In my copy, chapter 6 starts on page 47†), and MS or ms, for manuscript, should be used only in informal contexts. No. is sometimes used as an abbreviation for number in phrases such as â€Å"No. 1†; it’s a compromise between spelling the word out and using the number symbol (#). The abbreviation for versus, vs., is acceptable in informal content, as is OK. (Okay is a common variant, but the initials are more accurate; the most likely derivation of OK is the jocular misspelling â€Å"oll korrect,† the only survivor of a short-lived flurry of such locutions coined during the nineteenth century.) Terms of distance and duration, such as foot and hour, are generally spelled out when accompanying numerals except in technical writing, as are designations such as Celsius and Fahrenheit; the same is true of phrases such as â€Å"miles per hour† and â€Å"pounds per square inch.† However, the abbreviations am and pm, often capitalized and/or with periods, are always acceptable, though a phrase such as â€Å"one o’clock in the morning† is appropriate for a casual reference in fiction or nonfiction. Abbreviations are much more likely to be employed in ephemeral publications such as newspapers than in more durable materials such as books, though formality varies widely in the latter format; online usage differs as well. Ultimately, the careful writer will consult a style guide appropriate to the type of print or online publication for guidance or at least will consciously consider the visual and cognitive impact of abbreviation. (See this post for more guidance on abbreviations, and search for â€Å"abbreviations† on DailyWritingTips.com for additional assistance.) Want to improve your English in five minutes a day? Get a subscription and start receiving our writing tips and exercises daily! Keep learning! Browse the Style category, check our popular posts, or choose a related post below:30 Synonyms for â€Å"Meeting†50 Synonyms for "Assistant"Personification vs. Anthropomorphism

The Past and Future of Energy Usage Essay Example | Topics and Well Written Essays - 1500 words

The Past and Future of Energy Usage - Essay Example Two works that have recently been published that attempt to replace this rhetorical sideshow with legitimate information is Vaclav Smil's Energy at the Crossroads and Alfred Crosby's Children of the Sun. Crosby's work offers a natural history of energy consumption from the development of fire and cooking all the way to third millennial technological possibilities such as the holy grail of nuclear fusion. Smil's presents a more detailed analysis of the economic and energy situation of the past century focusing specifically on the statistical utilization of hydrocarbon technologies in various countries. Their respective accounts and arguments give a good overall picture of the natural evolutionary, economic and environmental impact of the past, present and long-term future of energy. The primary thrust of Crosby's argument is that the story of technology in tandem with humanity has always been about finding novel ways of exploiting the same source of energy: the Sun. As humanity has expanded and become more socially and culturally sophisticated technology has responded by finding consistently more efficient ways to harness the sun's powers, indirectly through wood and biomass and in more recent times, directly through wind and solar power utilization. However, unlike other analyses Crosby places humanity within nature versus against nature, and thus his work is a "natural history" of energy consumption rather than a litany of environmental offenses we have committed against nature. Of course, Crosby is sympathetic to the Malthusian end game at stake. He reveals through historical examples that every crossroads or crisis point, humanity has found a way to transform technologically the way in which we utilize energy to avoid the collapse of civilization. One speci fic example of this was the dependence on whale oil in the 18th century. "Humanity's yearnings for the convenience of light at night, though implemented through a primitive technology, were endangering a whole species of Cetacea."1 The two options to resolving this yearning included conservation, unthinkable by all except for the most progressive of individuals, or finding a substitute, which turned out to be kerosene and "the history of human exploitation of sun energy took another violent turn."2 The situation is much the same, today's whale oil is petroleum, with two "minor" complications here in the 21st century 1) the global political and economic dynamic have interposed agendas on technological transformation, and 2) hesitancy in adopting conservationist practices has placed a significant stress on the planets resources locally and globally. The question of Oil is more than just a question of natural history; it has become the bellwether issue of our time. Many would assert that the current situation in the Middle East is directly related to this question, and the political catchphrase of the decade has been "reducing our dependence on foreign oil." Smil's statistical analysis of oil usage is thorough and he means to tackle the popular debate regarding the "Peak Oil" theory. Peak Oil offers that at some point in the near past or near future3 maximum oil production will be reached followed by terminal decline. The consequences of failing to find a viable alternative energy form or

Revelation and the Apocalyptic Tradition, Jesus' Letters to the seven Essay

Revelation and the Apocalyptic Tradition, Jesus' Letters to the seven Churches, visions and signs from heaven - Essay Example Bible says the ‘Jesus sent seven letters from heaven’ through John to seven churches on earth. John wrote the letters as said by Jesus to us (Letters to the Seven Churches of Asia, Revelation 2 & 3). All seven letters follow the same pattern and refer to actual historical circumstances in those churches in the first century that first praises the church, then criticizes for their work, giving them warning and finally encouraging to continue their good work (Harris, Ch.20).The Revelation was a special vision given by Jesus to John which interprets the history which had happen and reveals the prophecy that will happen (Letters to the Seven Churches of Asia, Revelation 2 & 3). Jesus, while instructing John to write the letters, appeared to John showing signs like double edged sword, seven stars, keys, seven lamps. Harris (20) interprets the symbols as referring to the events in first century; future predictions which will take place or occur at a particular place and time. The seven lamps symbolize seven churches, where the letters were to be sent. The seven churches were Ephesus, Smyrna, Pergamum, Thyatira, Sardis, Philadelphia and Laodicea. The seven letters explain the significance to doing good and benefits of overcoming the bad. Referring to the first letter written to Ephesus, Jesus writes to the angel ‘announcer’ of the church ‘people’ of Ephesus expressing his happiness over their hard work and patience for not giving up their faith but criticizes the same church for not showing eager love as in the past. But the same does not prevail now and threatens to steal the lamp from the church if eager love does not exist and promises fruit from tree of life for overcoming the obstacle (The Letter to the Church at Ephesus). Second letter written to Smyrna reveals true love of God to the church. Jesus writes that ‘He is the first and He is the last’ and praises the church of Smyrna for their faith in Christ despit e horrible torture carried out by the Roman and Israelis for not following their God. Jesus criticizes those persecuting the people of church and assures that the people who will face the atrocities and win, will be crown of life. This refers to a place in heaven with real riches. The letter also refers to the second death which will not die which means ‘the lake of fire’ i.e. our soul which will never die (Revelation 2: Jesus’ Letters to Seven Churches). The letter to Pergamum explains the true faith of Christianity. As usual letter starts praising the people for their faith in God but criticizes them for following two religions, eat food sacrificed to idols, comprise with religious duties. Jesus warns them to accurate the doctrine of Christianity and threatens to come to church with double edged swords which are His ‘words’. While encouraging, Jesus ensures to give them MANNA, special bread from heaven and a name of white stone (Letters to the Seve n Churches of Asia, Revelation 2 & 3). In its letter to Thyatira, Jesus appears in eyes with fire and shining bronze feet praising the church for its increasing greatness of deeds, faith, service to people, love while warning them not to follow Jezebel, the lady who is spreading sex sins and destroying churches. The letter reveals that Jesus has patience and power look through the hearts of people to inspect their soul and encourages the church to follow true religion and the one who overcome will receive God (Letters to the S

Management and Chemical Engineering Essay Example for Free

Management and Chemical Engineering Essay I have completed my Bachelors of Technology in Chemical Engineering at Indian Institute of Technology, Guwahati, India (IITG). I am seeking a position in your organization in order to enhance my skills and practical experience, while being resourceful and innovative. I have worked hard in my undergraduate studies, striking a balance between compulsory courses like Fluid Mechanics, Process Equipment Design, Process Control and Instrumentation, Chemical Engineering Thermodynamics, related wet lab courses and supervised research under the guidance of Dr Chandan Das (Associate Professor, Department of Chemical Engineering, IITG) Dr AK Golder (Associate Professor, Department of Chemical Engineering, IITG). My Bachelor Thesis Project was on Biosorption of heavy metals to purify industrial effluents under the guidance of Dr. Chandan Das (Associate Professor, Dept. of Chemical Eng., IITG) and Dr. AK Golder (Associate Professor, Dept. of Chemical Engineering, IITG). When I look back now, I realize the role of an inspiring mentor in a student’s life. Dr. Das and Dr. Gloder gave me a whole new direction to explore my research skills and the interest in reading research articles while doing my undergraduate courses in mainstream Chemical Engineering. After graduation I joined Jindal Steel and Power Ltd as a Project Engineer where I practiced what I learnt during my educational life. Post completion of one year, I joined SPI Pvt. Ltd. As an Assistant Manager. I choose this as apart from technical skills, I also wanted to gain a good understanding about the business processes and its various elements. While working there my interest became more focused towards managerial skills. It was there where I decided to go for my higher education in Masters in Management as managerial knowledge via a reputed and esteemed university will help me to learn a wider scope of business management skills which was missing in my technical career. In my education thus far, I have been actively involved in sports and co-curricular activities including making manual robot, organized Rock-o-Phonix for three consecutive years (2008, 2009 2010) and represented IIT-Guwahati in Inter IIT sports meet in Football which has helped me to develop upon simplicity in approach, patience, team work, confidence, loyalty, ability to manage loads of stress and adaptability. To work using the in-hand knowledge and indigenous creativity simultaneously is my biggest asset. Education I believe is not just excelling in courses that we take and cracking their exams but a continuous process of development and self-realization. Not just asking â€Å"what to do† or â€Å"how to do† but â€Å"who am i† and â€Å"what is my purpose† too. My long term career goals are to become socially responsible individual and to get the dream job of working in the field of interest. If given the opportunity of pursuing the graduate study in field of management I would be getting involved in a project that will help me grow intellectually and professionally and result in useful outcomes.

The Impact of Television on American Society Essay -- essays research

The Impact of Television on American Society   Ã‚  Ã‚  Ã‚  Ã‚  What role does television play in society? For decades we have seen many parts of our world rapidly going through changes in technology. Today’s society has been transformed by means of communication and the available information through mass media. Most Americans rely on television for news, sports, and entertainment. Television is just one of the many examples of how technology has changed our lives. Since the invention of the television in the early 1900’s, it has played a very important role in our lives. Having a television set in the home has become very essential in today’s society. We depend on it to entertain us with its sitcoms and to inform us about current world issues. The problem is that sometimes what we hear or see on television is not always accurate or correct. Sometimes news stories give us the wrong information and we believe that it is true because it is being said on television. They may give you a story, but they may lea ve out some details to prove their point or to make the story more interesting. Americans are among the most ill-informed people in the world. Television, in other words, is not the great information machine. It is the great disinformation machine. TV tends to mislead us with its stories and in many ways we allow ourselves to be fall behind in the world as we tend to want to watch the entertainment shows and not the news broadcasts. Thus, we are not informed about what is going on in o...

Dow Pbb

Dow Chemical By Kai Draaisma Laurens de Blij Diedrik Oost Eva Sloff Lesley Flohil 13-11-2012 Duisenberg School of Finance Executive summary 1. Summary of Facts. With an annual revenue of $20. 2 billion Dow held the leading market position worldwide in ethylene and polyethylene (exhibit 1b). To consolidate all of Bahia Blanca’s polyethylene activity under Dow’s control a three-stage plan was developed. The first stage involved taking control of PBB, the second stage involved acquiring Polisur’s two polyethylene plants, and the third stage involved building a new ethylene cracker.To determine a proper risk premium Dow had to consider possible country risks. Currency and government policy risk may have had an impact because of possible future crises and changing government policies relevant to foreign investments. Furthermore, because of the ending of the convertibility law Dow also had to consider a possible exchange-rate risk. Dow created a holding company, Dow In vestment Argentina, to invest in the project, then they had to decide whether they will use general corporate funds or raise debt for the PBB bid. 2. Statement of problem . 1General analysis Rational for Acquisition of PBB Dow Chemical’s corporate strategy is to look for horizontal- and vertical integration, achieve technological leadership and gain international presence. Dow will benefit from a more integrated production process by lowering their cost of production. PBB will also create stable supplies of ethane for Dow allowing them to achieve operating rates near 100%. Cracking ethane in the region will result in lower operating risk for Dow meaning the supply/input risk for the production of Polyethylene.An increase in the production output of Dow will allow them to benefit from economies of scale. Overall Dow will gain technology leadership and operational excellence from adding PBB to their company and enhance them with a costleader position in the market required to c ompete with the government-subsidized chemical complexes. Risk measurement In this section we provide a brief risk analysis. Table 1 is designed to give a compact overview of the different risks, possible mitigating actions and whether the risks are integrated in the cash flow or discount rate.Not every risk was applicable in the acquisition of PBB. The risk that influenced our cash flows the most is the currency risk caused by a possible liquidity crisis in Argentina. Argentina has experienced eight major currency crises (tijd/reference)and there is a risk of a new currency crisis in the future. When this happens, Dow will not be able to sell its output to the home country and will have to increase its export. Revenue de We have also taken into account that the convertibility law that now provides stability could eventually stop. This will also have a significant impact on inflation.Operating risk is the second risk with a large impact on projected cash flows. This is mostly the ri sk of not meeting the full operating capacity. For the first 3 years we keep the operating rate of the plant at 65% and from 1995 we increase the operating rate by 5% each year. Other risks had less of an impact on our cash flows. Information risk was less of an issue because Dow was already present in Argentina and PBB was important to the government of Argentina. Corporate Governance risk in Argentina is not expected to have a significant additional impact on cash flows.This risk is already included in the discount rate for similar projects in the US. Table 1: Risk overview| Risk| Mitigating actions| Cash Flow (CF) or Discount rate (DR)| Corruption risk| 1. Have a large bank loan from a national bank. 2. Government stake of 49% here is a mitigating factor | DR| Expropriation risk| Same actions as for corruption risk| CF (tax wil increase 5%)| Operating risk (only post/ completion risks )| n/a| CF| Sovereign risk| Through the convertibility law, the peso has been anchored to the US D to prevent hyperinflation. | DR| Currency/ Foreign exchange risk| 1.Dow is a well diversified investor, therefore this should be mitigated. 2. Buy put options on the Arg peso. | CF| Corporate Governance risk| Give shares to management to align incentives. | DR| Repatriation risk| Current political climate is stable, not likely that Arg will impose limits on fund outflows. | DR| Information Risk| Dow Chemical already has presence in Argentina, therefore are more likely to receive high quality information. | n/a| Adjusted discount rate All risks that are not used for adjusting cash flows are integrated in the adjusted discount rate.These risks include but are not limited to corruption risk, information risk, hyperinflation, sovereign risk, repatriation risk and other residual risks. These risks are partly mitigated, however all the different risks combined constitute a premium which we will add on to the the given discount rate for similar projects in the US of 8%-10%. A reasonable assumption for the combined premium will be [ ]. The auumed premium results in a discount rate of [ ] which we will use in our further valuation. 4. Recommendation Bidding decisionCompeting bidders need to comply with certain covenants (exhibit 6). Competitors Perez Companc S. A. and Copesul do not comply with the requirements of having a statement of net worth of at least 5 billion. Even if the two companies would merge they do not meet the the requirements. This makes Dow the sole bidder for PBB. This has a profound impact on the internal valuation. Appendix 1 Risk Information risk. When you are doing a cross-border valuation you have to take into account that there may be information risk.This includes the risk of outside investors getting the wrong information because of poor disclosure in the financial statements. However since Dow entered Argentina in 1957 we assume they are familiar with the market and accounting rules, therefore information risk for this project is low as Do w chemical has already collected country experience in Argentina via Dow Quimica. Because this is difficult to quantify we have taken this into account in our discount rate. Corruptionrisk. The risk of corruption includes the illegal payments and favors outside the rule of law.Corruption exists to some extent in all countries, but there are large differences across countries. According to the Global Corruption Index by Transparancy international, the level of corruption for Argentina in 1995 was given a 5. 24 out of a possible score of 10. This gave them a ranking of 24 out of 42 countries surveyed. All of which were assumed to be developed countries. However, Dow somehow secured itself thanks to PBB’s importance and the Argentine government’s intention to keeps 49% which is an advantage and not a disadvantage.The Argentine government will also appreciate the jobs being created in the later project stages, if successful, and a good relationship with the government will likely also be beneficial when discussing with YPF about long-term ethane supply. Mitigation: Part of this risk can be mitigated by taking a loan in Argentina to finance the project as the government will not want Dow to default on their debt resulting in an overall bad outcome for Argentina’s economic situation. Furthermore part of this risk is mitigated as the government has a stake in the company so it is also in their interest to keep the company strong.Currency risk: As Argentina has experienced 8 major currency crises in the past there is the risk of a new currency crisis occuring in the future. Even though the convertibility law took away some of the risk, Dow will still have to bear in mind that when a crises happen, they will not be able to sell their output to the home country and will have to increase its export. This risk is taken into account in the cashflows. Expropriation risk. Political instability or expropriation is small as they company has had a stable dem ocratic government past 12 years.However, changes in the policy of the government will cause a risk for Dow and their foreign investments. At that moment there were no restrictions on borrowing abroad and there were no limits imposed on payments of foreign loan principal and interest. If something changes in this regulations it may have an effect on the acquisition of Dow. For this reason we will also include this risk in our discount rate. Furthermore the government has no limits on cash flows going out of their country but can eventually decide to do so.We consider the risk of the government taking over the company to be low as the production of polyethylene and deriving ethylene from hydrocarbon crackers is complex. This is a process for skilled people and not just anyone can therefore run the company properly. The risk of expropriation is therefore low. As the production of ethylene and derivatives such as polythylene is a highly complicated process, requiring large captial inve stment and a high operating leverage, not many companies will be able and willing to take over the business.Furthermore as Polythylene is a commodity product that trades globally with a narrow price range. Mitigation:Dow can decrease the risk exposure by taking on a loan in Argentina to finance their project. This will decrease the risk exposure as Dow can only default on their debt when the government decides to raise taxes, which will not lead to a company default. Foreign Exchange Rate Risk. Foreign Exhange rate risk will be low because of Dow Chemical’s international presence and the assumption that shareholders are sufficiently diversified. The convertibility law which ensured a fixed Argentine peso and U.S. dollar at exactly one already decrease the foreign exchange rate risk. However we need to take into account the risk that the convertibility law might come to an end. Mitigation: The risk can be decreased by setting up binding financial contracts with the governmetn to make sure the deal will happen. Furthermore Dow could decide to place a put option on the peso to protect themselves when the peso collapses. Operating risk Pre-completion: as the project is already completed there is no risk concerning the technology risks or other completion risk.Post-completion: we have included the post-completion risk in our cashflows as we run the risk of not meeting the full operating capacity. For the first 3 years we keep the operating rate of the plant at 65% and from 1995 we increase the operating rate by 5% each year. As the company provides their own input this risk does not need to be taken into account. Operating risk would likely matter but we handled that risk by adjsuting down our casfhlows in stage 1 so we do not take it into account for adjusting our cost of capitalSovereign risk Politcal events suchs as wars, labor strikes, terrorism and changes in laws can happen in any country you work with and therefore needs to be taken into account. Sinc e it is hard to quantify this risk we have handled it through the discount rate. Appendix 2 | | Source: Erb, Harvey Viskanta, ‘Poltical risk,Economic risk and Financial risk’, Fuqua School of Business Working Paper No. 9606, 1996. | ——————————————– [ 1 ]. For broader explanation of the different risks see Appendix 1 [ 2 ].According to the Global Corruption Index by Transparancy international the level of corruption for Argentina in 1995 was given a 5. 24 out of a possible score of 10. This gave them a ranking of 24 out of 42 countries surveyed. All of which were developed countries more or less. Source: http://archive. transparency. org/policy_research/surveys_indices/cpi/previous_cpi [ 3 ]. See Appendix 1 for an explanation of the mitigating factors of the before mentioned risks [ 4 ]. This conclusion was reached byTaking the ‘total assets’ as net worth on the financial statements of Perez on Copesul (exhibit 8a &8b).

WeChat: Connect with Friends

Some of the website use their own language, and some use English. The most activity page Is Weight Indonesia; It has more than 800,000 Likes and 63 thousand talking about this. The second one is Weight India. It has more than 747 thousand likes and 7 thousand talking about this. Other pages have about 20 thousand likes. Some activity pages, like India, Malaysia, have different cover pages; but some, like Hong Kong, Australia, have the same cover page and most of the content are the same. If there is a need to separate Faceable by countries, It should every country character.If It makes every hint the same, it Just need one Page. Because It has 1 1 pages, I want to choose two typical ones of them. The Weight India page Is the most activity one. And it almost update twice a day. At first glance, we can see almost every post has picture with Weight icon. It is a good way to give new users the impression of Weight. The photos can help you know what they post. You even have no need to see the worlds up the photos. Weight has a function that people can build fan page in Weight about some topic, which Is Interesting or useful. Then people can get newest Information from that page or we can call It group.On their Faceable, they always post some Weight group, and promote you to follow the Weight ID, Like PER Cinema, or Colors TV. I think the reason why there are not so much replies is also because when people find a group on Weight, they would like to have a talk on Weight. It is a good way to appeal people to use it, but it also needs to create conversation on its Fan page. So that others who do not use Weight can know the opinion from those who use It. Those opinions will be the first suggestion for them to use It or not. To promote Itself, Weight posts their function and explain how to have fun with them.The conversation under this kind of poster has not so much replies, but if some one says he has problem with Weight, Weight will reply him with the solution. I think that is not enough for Weight to have interaction with people. They can lead some interesting topic on Faceable, not only introduce the function or follow some groups. Another one I want to choose Is Weight Taiwan. I choose this one because the posters they have on their Fan page. It not only talk about Weight Itself, It also post the conversation. However, Weight Taiwan does not have so many fans. It is not so activity like Weight India.It may Just post one for one or two days. That will not help it show up in public. Maybe because it is an App, so when people download and use it, they may not pay so much attention on its Faceable. No matter which Fan page, it always do not have so many replies. It is an important point they need to develop. I think when people have more replies and more share on Faceable, other who never heard about it can see. Now Weight is expanding its market, so it needs more activity. Another point I want to mention is that it has 11 Faceable Fan pages, but I think it need a main page for all over the world.Then on the main page, Weight can put the links of different countries. This can help people who do not in these countries can like Weight page and pay attention to what happen. Twitter: The same as Faceable, it also have individual page for different countries. Here is the problem, it do not have a list on its website of all Twitter it has. It has about 146 thousand followers, and it updates frequently. It rewets others' Twitter. Most of them are those people's opinion about Weight, like they can have group talk with friends or he can have free video chat with friend in Beijing.I think it is a very good way to promote their product. In this way, when people want to see the react of Weight and they find its Twitter and see these Tweets, they will think it is a useful and convince App. Here I also want to mention the Weight India. It has fewer followers than its main page, but it has about 10 times tweets. Weight is a Chinese app, wh en it wants to open it foreign market, India is their first choice. Now, India is the second biggest user of Weight, and because China use it own social media, like Webb, Renee. Then India is very activity on its promotion.On Weight India, it wets average 3 times a day. It will post some important event on both Faceable and Twitter with photos. Weight India is the only one has a Weight background picture. From this detail, we know Weight India is doing diligently and attentively. It has hash tag, and people always talk about how they use Weight; or when someone builds a group, he or she may washrag Weight. I think Weight can rewet this kind of tweets. It can help people expand their group, make those who have same interesting come together, also it can tell them use Weight. It can kill three birds with one stone.Not only its own hash tag, it also uses other hash tag to connect with other activities. Blobbing: When I searching its blob, I find it do not have a link to its blob. Final ly, I find All About Weight blob. It does not seem like the official blob post because it has no background, no other information. It looks like very simple and I do not think they pay attention to this platform. Under the title, it has one sentence: now we are here with a link. There Just one more blob, and others are exactly the same. If two places have the same articles, why it need to separate readers to different platform.Some posts re even in Spanish. Spanish is the second language in US, but it can't Just post in Spanish. They can have two posts with same content in different language. The I do not think it can typically representative the main idea. It talks about new emotions for Weight, why it needs to have tagged all, about. The emotions are different, but the tags are the same. At the top, it has a sentence saying: All you need to know about Weight: Readers from 154 countries as of May 2013. Where are the readers? There is no comment, no evidence to provide the dates. An other reason why there is no comment maybe the article is too short.The article is like posts on Faceable, not many words, Just a picture and several words to explain. When people first see on Faceable, they may want to comment, but if they see it again on blob and without any new, they do not want to comment. The article is not â€Å"news† for them. As I know from class, Weight India hires writers to writer about Weight every month, maybe the blob is on their country website. Actually I also see articles and news about Weight, but it does not have a specialized platform to collect this article to let people know comprehensive Weight. I find their official blob website, but it is aChinese website. I want to write about it because it has some good points. The articles are formal and have many replies. The Weight team posts these articles. All the topics are people concerned and they like to give opinion about these topic. It does not have tags, but it has classification. Then people can know the article is about function, tips or update. I hope they can work on this platform because from the replies, we can know there are many people following Weight and they must want to now information from official website. It they use it, they also need to change or update some things.It needs IRS or other ways to share the article, and tags for easy to search. Linked: In my point, Linked is a social media for career or company. Weight do not use Linked so well. I find a Weight Marketing Linked page. It has a big problem that it does not use it in English. I think if it wants to open its market, the first thing it needs to change is the language. I also suggest they can have a list of employees, especial like the CEO. Then people can see the size of the company or if they want to find a job in Weight, they can know who need to connect with.Weight almost posts rarely, ND I find some article even with strange pictures. The pictures have noting to do with the topic. In my opinion, the picture is also a media tool, and it can tell people directly. Maybe because the fan page name is Weight Marketing, it Just posts articles about marketing. It should be emphasis marketing, but not only marketing. It also can post the App itself or some interesting things happen with Weight. Under their products and services, there are Just two articles. From my experience of using this product, I think it can have more pages talk about this product specifically.In its Faceable, when it updates its App, it will have a post on Faceable to help people get new function. Linked can help people know what you have, so I think it is important to have a complete introduction of the function. It has Linked group with 161 members, but Just few of them are working at Weight. It is really not an activity group, and the nearest post is amounts ago. Weight need to pay attention to Linked because it is a good social media to promote itself. In its group, it needs to add more employe es and post more about Weight. Otherwise, the members talk try to make friends there.I know most of employees are Chinese, and they do not use Linked. However, if Weight want to have more users, it needs to take full advantage of social media. They can't be stuck by Chinese social media and culture. Youth It has Weight India channel on Youth. It has four parts, Weight TV Ad, Stars on Weight, Weight Global, and Weight Feature Tutorials. In TV Ads part, the most popular video has more than 3 million views, and the average view is 100 thousand. The ads are interesting though it speak Hindi and people also reply they think it's funny and amazing, and they want to use this app.It means the ads do a good promotion. Though I do not understand what they are talking, but I can know the story from their action. Weight use small interesting stories to tell people Weight is a very convince way to communication and many India stars are using it. Each part has about 50 videos. I can't tell whethe r it is lot or litter for it because I think as ad it needs to update and it will have new ads coming. When a new advertising showing up, if it can inform people to use it, then it is successful. Form the reply, we know the video catch audiences attention and they will try this app.I also think it can have a broad let audience put their own video. What does user do with Weight? It also can be interesting. Conclusion I think there has big space to improve its social media. First thing is its website. When people want to know a product. He must go to its website first. In Yacht's website, it does not have all social media it has. It also does not have a list of which countries can use its product. As I mentioned before, it need to have a main page for Faceable, Twitter. On the main page, it should have links to separate pages. The language is also a big problem.Weight tries to be an international app, so it needs to unify its language in English. To translate the media, it Just needs to have language selection at the top. Among all the platforms, I think they use Faceable and Twitter best. They update everyday and have reaction with followers. If I grade this company, I will it a B- or B. As a company, its social media cannot Just about one or two countries. India does very well, the Faceable, Twitter, Youth, and blob. It is activity on these platforms and does an effective promotion. However, in other countries Faceable, they seems like not pay attention to social media.Again the Linked is about the company; maybe costumers will not check Linked. They still need to fill out it. When people access to some technical, they may want to find who produces it, and who are in the team or what do the team create before. It should have a Weight Linked page, not Just marketing page. Its main market is in China, and Chinese has Webb. At first I do think it need to promote it in China because almost every phone has Weight. But now I think whether it is the first communicati on tool in China, it will need promotion and social media. It needs to have something new to keep people sing it.

Free Essays on Black English Or Standard English (June Jordan)

Black/Standard English Black English or Standard English? That was the question June Jordan ¡Ã‚ ¦s students were faced with. They wanted to write a letter to the police about the brother of a fellow student, Willie Jordan that had been killed by cops. Should the students use Standard English and write a professional letter which contains no feeling and might not get the point across? Or should the letter be written in Black English, where there is the possibility that it will not be understood or disregarded. And while discussing the topic of Black English, ask yourself, while these students are fighting racism, are they also supporting it? At the State University of New York at Stony Book, Professor June Jordan taught a course called  ¡Ã‚ §The Art of Black English ¡Ã‚ ¨. This class was focused on teaching students the language of African Americans who first came to this country. This language was titled  ¡Ã‚ §Black English. ¡Ã‚ ¨ A group of rules were established, titled  ¡Ã‚ §Guidelines for Black English ¡Ã‚ ¨, one of which states:  ¡Ã‚ §Clarity: If the sentence is not clear then it is not Black English. It is a language that is kept simple and strays from many of the rules of Standard English. Instead of using different verb tenses, both past and present tense are written the same. For example,  ¡Ã‚ §He goes to the store „ ³ He go to the store and He went to the store „ ³ He go to the store ¡Ã‚ ¨ Due to the topic of this letter, the students feel that it needs to be written in Black English. They want this letter to have feeling and contain all emotion that the family of Willie Jordan experienced. An excerpt from the letter sates  ¡Ã‚ §Reggie, like many brother and sister, he a victim of brutal racist police ¡Ã‚ ¨. This statement, if you do not know any Black English, can still be understood. It makes a statement about police brutality in the black community and ties it back to the brutality of blacks in the history of the country. If the st... Free Essays on Black English Or Standard English (June Jordan) Free Essays on Black English Or Standard English (June Jordan) Black/Standard English Black English or Standard English? That was the question June Jordan ¡Ã‚ ¦s students were faced with. They wanted to write a letter to the police about the brother of a fellow student, Willie Jordan that had been killed by cops. Should the students use Standard English and write a professional letter which contains no feeling and might not get the point across? Or should the letter be written in Black English, where there is the possibility that it will not be understood or disregarded. And while discussing the topic of Black English, ask yourself, while these students are fighting racism, are they also supporting it? At the State University of New York at Stony Book, Professor June Jordan taught a course called  ¡Ã‚ §The Art of Black English ¡Ã‚ ¨. This class was focused on teaching students the language of African Americans who first came to this country. This language was titled  ¡Ã‚ §Black English. ¡Ã‚ ¨ A group of rules were established, titled  ¡Ã‚ §Guidelines for Black English ¡Ã‚ ¨, one of which states:  ¡Ã‚ §Clarity: If the sentence is not clear then it is not Black English. It is a language that is kept simple and strays from many of the rules of Standard English. Instead of using different verb tenses, both past and present tense are written the same. For example,  ¡Ã‚ §He goes to the store „ ³ He go to the store and He went to the store „ ³ He go to the store ¡Ã‚ ¨ Due to the topic of this letter, the students feel that it needs to be written in Black English. They want this letter to have feeling and contain all emotion that the family of Willie Jordan experienced. An excerpt from the letter sates  ¡Ã‚ §Reggie, like many brother and sister, he a victim of brutal racist police ¡Ã‚ ¨. This statement, if you do not know any Black English, can still be understood. It makes a statement about police brutality in the black community and ties it back to the brutality of blacks in the history of the country. If the st...

Revamp Your Resume Administrative Assistant

Revamp Your Resume Administrative Assistant Resume formats are as varied and individual as the people behind them. Your top priority  is to always make sure that your resume shows the best of you: your experience, your goals, your skills, your education, and anything else exceptional or essential you bring to the table.Check out this template from the folks at Best-job-interview.com and see how you can add your individual stamp to your CV for administrative assistant positions.YOUR FULL NAME  Your mailing addressYour phone numbersYour email addressObjective(Optional; may not be necessary if youre responding to a job posting.)Example 1: Seeking a position as an administrative assistant in a busy environment.Example 2: Administrative assistant position in a professional company.Example 3: To secure the position of administrative assistant in an established company.Profile Statement(This is particularly useful if they havent requested a cover letter or theres no place to upload one.)Example 1: A number of years experience as an administrative assistant in a fast paced, professional environment.  Proven computer skills with good working knowledge of different computer applications.  An independent worker who is able to multi-task and meet deadlines efficiently and accurately.  Strong problem-solving skills and initiative evident in the implementation of efficient data management systems.Example 2: A proven record of efficiency in establishing, organizing, and managing office procedures. Proficient in a wide range of computer applications. Solid bookkeeping skills, excellent scheduling skills, and a strong background in customer relations. Recognized for the ability to manage multiple tasks and projects and successfully meet deadlines. A self-directed worker who enjoys a fast-paced work environment.Example 3: Outstanding office skills and knowledge of office management procedures. Provided administrative and secretarial support to a large department, managed a number of simultaneous projects, and me t deadlines consistently and accurately. An independent worker recognized for a proactive approach to problem solving. Proven experience in a demanding work environment dealing successfully with competing needs and different challenges.Work ExperienceAdministrative Assistant,  Premier Pharmaceuticals, Houston, TX;  January 2012 presentProvide full secretarial and administrative support to the 10-person sales departmentManage customer requests for information prepare departmental correspondence, documents, reports, presentationsSchedule and coordinate meetings and appointmentsDraw up and distribute minutes of meetingsPlan and arrange travel itineraries organize functions and eventsTrack expense claims and prepare expense reportsSet up and maintain customer data management systemsAdministrative Co-ordinator,  Buckleys Property Management Company, Houston, TX;  August 2008 November 2012Provided full range of secretarial and administrative support to the Managing Director plus 5 employeesScheduled meetings, appointments and property viewingsCoordinated company events and functionsHandled incoming calls and correspondencePrepared correspondence, documents, newslettersLiaised directly with maintenance contractors, clients, tenantsProcessed invoices and prepared paymentsEducationDallas Baptist University, Dallas, TX,  Bachelor of Arts 2008Technical SkillsMS Word, Excel, Outlook, Powerpoint, AccessTyping skills (include WPM rate if known)Excellent spelling and grammar skillsCore CompetenciesOrganizational and planning skillsCommunication skillsInformation gathering and management decision-makingProblem-solvingFlexibilityReliabilityTeamworkNotice this template is heavy on bulleted lists. For visual variety, one option Id recommend is choosing a paragraph format for either the position descriptions or the core competencies section.

Understanding and Preventing School Violence Annotated Bibliography

Understanding and Preventing School Violence - Annotated Bibliography Example Consideration given to the previous cases of school violence and how they have created a sense of fear.   â€Å"According to 2001 polls, more than 50 percent of parents with children in grades K-121 and 75 percent of secondary school students2 now think that a school shooting could occur in their community,† (Juvonen, 1).   The efforts taken by schools to combat the issues surrounding the problem.   These methods would, â€Å"..include the use of metal detectors, the presence of security guards on campus, rules and regulations regarding student conduct and dress, profiling of potentially violent students, anti-bullying instructional programs, and counseling and mediation,† (1).   Providing of facts surrounding the study of school violence.   Emotions felt on the part of the stakeholders most concerned with school violence.   More work is needed to be done, in order to better understand school violence.   The need to make the best possible choices.   With the high statistical data available, there would be plenty of information to show the need to do something.3.  Katers, Nicholas. â€Å"School Violence: The Culture of Violence in America.†Suite101.com 6 October 2006: 1-2Closer examination given to an issue that would have been widely discussed, yet still needing to achieve a solution.   â€Å"The recent spate of school shootings across America has spawned a revisited argument about how to solve the problems of school violence once and for all,† (Katers, 1).   An issue that has remained present within the school system at a continued rate.

Negotiation in China Research Paper Example | Topics and Well Written Essays - 1250 words

Negotiation in China - Research Paper Example   Many entrepreneurs have strived to make their ways in China with different investment intentions that include creating a partnership with Chinese firms. Negotiations are never easy since there are special insights that are required for proper negotiations processes. There exist a number of complications that hinder negotiations inChinaa which are largely based on the complicated Chinese culture. Better negotiation styles and skills are therefore very necessary in bringing competitive advantages and benefits. Chinese culture has very great influence on her business negotiations. China’s culture and behavior should be viewed differently by investors since the economics within Greater China become more internally linked together. To create competitive advantage among foreign investors going to china, there is a dire need to train them in international manners and customs so that they can learn a better negotiation style that is appropriate for China. The collectivism, hierarc hical, relationship and the haggling culture of Chinese people should be well understood particularly their long courting, formal and the fact that they prefer drawing on intermediaries when it comes to negotiation process. Dr. Robert Grosse authorized this research to further investigate the business managers’ perceptions regarding specific characteristics needed for successful negotiation in China. Dr. Robert B. Rogow, Dean of the College of Business and Technology at Eastern Kentucky University, will receive the finalized report on March 6, 2013.

Ibn Ishaqs Life of Muhammad Essay Example | Topics and Well Written Essays - 500 words

Ibn Ishaqs Life of Muhammad - Essay Example One of the rulers continued in power â€Å"†¦until God sent Muhammad the prophet. I was told on the authority of al-Zuhri that he said Chosroes wrote to Badhan the following letter: ‘I have been told that a man of the Quraysh has come forth in Mecca asserting that he is a prophet.’†1 Muhammad grew up in the â€Å"brackish settlement† of Mecca, as Muhammad ibn Abd Allah.2 His father, allegedly, died before he was born. Then, to make matters worse, when Muhammad was only six years old, his mother died, leaving him an orphan. He became a shepherd and worked for his uncle. This left Muhammad in a difficult place until he became a camel driver for caravans up until about age 40. He also struggled with poverty for much of this time. Muhammad’s life was to end in triumph, however—even though it had begun with unusual sorrow. Muhammad received a vision from an angel giving him the dictum of Islam, and Ibn Ishaq recounts Muhammad’s feelings as he prepared to leave for what is now called Medina from his home city of Mecca (this journey would be called hijira). It was â€Å"†¦unusual for Ibn Ishaq to give such an intimate account of Muhammad’s state of mind [where he was praying].

Toxicity and Autoactivation of Baits Experiment

Toxicity and Autoactivation of Baits Experiment Abstract Alternate splicing in exon 47 of the Purkinje cell calcium channel generates a splice variant with a five base pair insert (ggcag) before the stop codon in rat. This five base pair change the open reading frame of the exon 47 for resulting in an extended C-Terminal. Novel protein interaction at this region was hypothesised. Yeast Two Hybrid System was employed to screen against cDNA library to check for any protein interaction with 5 base pair insert region of exon 47. This project aimed to test the toxicity/ autoactivation of the baits in the yeast and to find the minimum concentration of 3-AT (3-amino-s-triole) at which it inhibits the HIS3 gene. The experimental result shows that there was no leaky expression of the HIS3 gene. The autoactivation/toxicity test results showed that the baits are less toxic than the control bait. The growth of non-interacting colonies in the Triple Drop Out media revealed that a more defined media should be used, demanding the repetition of experiment to obtain more convincing results. 1. Introduction 1.1. Nervous System The human nervous system consists of the Peripheral Nervous System (PNS) and the Central Nervous System (CNS). The PNS is formed of the cranial nerves and the spinal nerves. The central nervous system consists of the brain and the spinal cord. The brain can be divided into three major parts cerebrum, cerebellum and the brain stem. The cerebrum is divided into frontal lobe, parietal lobe, occipital lobe and the temporal lobe. The main function of cerebrum includes controlling of sensory organ, motor function, consciousness and imagining. The cerebellum is a uniform structure and its function is essential in movement and co- ordination of organs. The brain stem is made up of the mid brain, the pons and the medulla. The main functions of brain stem are transmission of information to and from the brain (Bear et al, 2001; Purves et al, 2004 and Thompson,1993). 1.2. Cells of CNS The brain consist mainly two types of cells nerve cells or neuron cells and the glial cells. The neuron are involved in the transport of electrical signals from the brain whereas the glial cells are thought to be the supporting cells of neurons by the uptake excess of neurotransmitter that are essential for signalling between neurons (Henn et al, 1971 and Purves et al, 2004) and plays a role in synaptogenesis of the neuron (Bacci et al, 1999). The glial cells are of three types: astrocytes, oligodentrocytes and the microglial cells. 1.2.1. Glial Cells Astrocytes are star shaped cells. The spatial arrangement of these cells between the capillaries and the neurons enables it in the modification of cellular responses, synaptic plasticity and survival of neurons (Abe et al, 2006 and Chen et al, 2003). Astrocytes important in glutamate transport, removal of free radical, controlling of haemostasis of brain and in maintaining a preferable environment for the active functioning of neurons by buffering K+ ions in their extracellular space (Chen et al, 2003; Gee et al, 2004 and Longuemare et al, 1999). Oligodentrocytes are type of glial cells that insulate the neuron with myelin sheath (Bear et al, 2001 and Lubetzki et al, 1993. The myelin sheath is a membrane which is made up of lipid and two proteins the proteolipoprotein (PLP) and the myelin basic protein (MBP). (Colman et al, 1982 and Boison et al, 1995). At regular intervals myelin sheath becomes thinner and is known as Nodes of Ranvier (Peter et al, 1966). These regions are the site for voltage gated sodium channels and a number of proteins. Microglial cells are the macrophages of the brain, which are formed in the bone marrow and are then transported to the brain by specialized protein called chemokines (Khoury et al, 2008) The study of chemokine receptors is one of the important research areas in the pathogenesis of Human Immuno Deficiency Virus. HIV can target microglial cells for their replication (Albright et al, 1999; Ghorpade et al, 1997 and Meer et al, 2000). Microglial cells are also studied for their inflammatory re sponses in the brain. The identification of role and mechanism by which microglial cells cause inflammation has paved path for finding targets and therapeutics for many diseases.(Bhatia, 2008; Huang et al 2008; Hwang et al, 2008 and Kim et al, 2008). 1.2.2. Neurons Neuron or the nerve cells are units of the nervous system involved in transfer of electrical signal between each other and to the effector cells. There are many types of nerve cells. Purkinje cells are one among them (Brown, 1991). The study of calcium ion channel of Purkinje cell is the subject of this project. The basic parts of neuron consist of a soma or cell body, axon, dendrites and neurites. All neurons are covered by the neuronal membrane. The soma or the cell body is similar to any other type of cell in the body. The axon is fibre that transport signal from the cell body to other neuron or to the target cell. The axons are covered by myelin sheath of the glial cells. The axon may be branched or unbranched. The main function of axon is to transfer the electrical signal from the axon hillock of soma throughout the axon known as the action potential and to transfer the signals to other cell in the form of chemical signal, the neurotransmitter (Purves et al, 2004 and Bear et al, 2001). The region of contact with other cells where release of neurotransmitter takes place is known as the synapse. The release of neurotransmitter is facilitated by synaptic vesicles of the presynaptic terminal (one which release chemical signal). The neurotransmitters are released by the synaptic vesicle in the space between pre synaptic and post synaptic terminal known as the synaptic cleft (Pu rves et al, 2004 and Brown et al, 1991). The neurotransmitters are then received by specific receptors of the post synaptic terminal which would generate an action potential in the cell. Apart from these receptors the ion channels of the cell membrane of the synaptic terminal also respond in the transfer of signal. Dendrites are branched fibres that arise from the cell. Their surface is lined with number of receptor to receive signals for the neuron (Brown,1991., Purves et al, 2004., Thompson,1993 and Bear et al, 2001). Purkinje cells are one of the largest types of neurons on the brain. They are found in the cerebellar region of the brain. The study of calcium ion channel of Purkinje cell is the subject of this project. Purkinje cells have a number of branches dendrites that receive synaptic inputs. As the dendrites receive signals it initiates a Ca2+ signal, which are important secondary messenger in the cells. The dendrites are the region for a calcium ion entry through the calcium ion channel. Similarly the soma contains K+ and Na+ channels(Schutter et al, 1994). These ions are of particular importance as their charge variation inside and outside the membrane trigger signalling in the cell. The transport of these ions is highly selective and they are maintained by the ion channel proteins of the Purkinje cell membrane and other neuronal membrane. These proteins form a pore for the transport of ions. Techniques such as the Patch clamp method have made the study of these ion channels easier (Bear et al, 2001). 1.3. Ion channel Ion channels are glycoprotein complex that allow specific ions through them. The proteins of ion channel are coded by different gene. More than 100 genes are known to code ion channels. The transportation of ion is important in generating action potential in the cell and is also important as the ions are second messengers in signalling. Diseases associated with the ion channel are known as channelopathies. Ion channels can be three major types voltage gated ion channel. Ligand gated ion channel and the stretch and heat activated ion channel (Purves et al.,2004). Voltage gated ion channels open and close on response to electrical potential. The voltage gated channels are made up of different protein sub unit. The subunits can move to open or close the channel (Horn, 2002). Depending on the type of ions they conduct they are further divided into Calcium channel, sodium channel and potassium channel. Ligand gated channels are those that respond to chemical signals. The ligand gated receptors are of five types nicotinic acetylcholine receptor (AChR), glutamate receptor, ÃŽ ³-aminobutyric acid (GABA), glycine-activated Channels and the ryanodine receptor(Stroud et al, 1990). Each of these receptors bind to specific ion and are found in different organs. The stretch and heat activated ion channel respond to heat or structural deformation of membrane (Purves et al, 2004). 1.4. Voltage Gated Calcium Channel (VGCC) Ca2+ ions are important secondary messenger in cells and play important role in biochemical pathways of cell. The level and entry of these Ca2+ ions in the cell is highly regulated. The regulations of these ions are controlled by the Voltage Gated Calcium Channel (Gribkoff et al, 2006). These VGCC are mainly found in excitatory cells such as the muscle cells and neurons. They exert their function by controlling muscle contraction, neurotransmitter release, neuronal plasticity, synapses, and neuronal excitability (Pietrobon, 2005 and Yang et al, 2005) . VGCC respond to membrane depolarization facilitating Ca2+ entry into the cell and thereby activating the signalling cascade of the cell (Yang et al, 2005). The normal functioning of the calcium channel protein is very important in a cell. Mutation in the gene coding channel protein, have been known to cause a number of diseases which include Timothy syndrome, Familial hemiplegic migraine type 2, episodic ataxia type 2, spinocerebellar ataxia type 6 and autism spectrum disorder which are grouped under â€Å"calcium channelopathies† (Bidaud et al, 2006 and Jen et al, 1999). Calcium channels also play a key role to mediate neuronal pain pathways (Gribkoff et al, 2006). A number of drugs have been known to block calcium channel and they are categorised as Calcium Channel Blockers. Verapamil was the first drug found to block Calcium Channel and later dihydropyridines (DHPs) class of drug was discovered to act as calcium channel blocker (Dolphin, 2006). DHPs are of much importance in studying the channel properties of the Dihydropyridine sensitive calcium channel. These DHP sensitive channels have dihydropyridine receptor for their bin ding (Campbell et al, 1988). Calcium Channel Blockers are now being found effective in the treatment of pain and hypertension (Atanassoff et al, 2000; Kize et al, 2001, and Thompson et al, 2001) but the question of safety in Coronary Heart Disease and the increased risk of cancer in patients remains unanswered (Eisenberg et al, 2004 and Fitzpatrick et al, 1997). 1.5. Calcium channel structure A calcium channel consists of five important subunits ÃŽ ±1. ÃŽ ±2, ÃŽ ², ÃŽ ´ and ÃŽ ³. The ÃŽ ±1 subunit is known as the pore forming complex (Yang et al, 2006). The ÃŽ ±1 subunit is a single polypeptide and its functions mainly include voltage sensing, gating and selective permeation (Horn et al, 2000). The structure of ÃŽ ±1 subunits consist of 24 segments (S1-S6) which constitute 4 domains, a C- terminal, N-terminal and Interlinkers. The linkers connecting domains are known as Loops and they are referred as loop I-II, loop II-III and loop III-IV depending on the domains they link (Dolphin, 2006). The intracellular loop of the ÃŽ ±1 subunit has interaction site for the binding ÃŽ ² subunit. The interaction can modulate the G- protein, an important second messenger in the cell (Dolphin, 1998). The specific binding of ÃŽ ² subunit to the tryptophan residue is important for controlling the gating of ÃŽ ±1 subunit of certain type of channels (Berrou, 2002). S4 is another important segment of the calcium channel. It is the voltage sensitive region of the calcium channel. S4 segment moves outward causing the channel to open by getting depolarised. S4 segment is positively charged due to the presence of arginine aminoacid making it voltage sensitive by translocation of the charges across the membrane (Sigworthl, 2003 and Horn et al, 2000). The S5, S6 and the linker connecting the S5 and S6 segment forms the boundaries ion conducting pore of the ÃŽ ±1 subunit. The ion conductance partly depends on the rotational movement of the S4 segment which either cause the S6 segment to open or c lose the pore (Horn et al, 2000). The ÃŽ ² subunits of the calcium channel are thought to be tissue specific and organ specific. Primarily they are of 4 different types, ÃŽ ²1, ÃŽ ²2, ÃŽ ²3 and ÃŽ ²4. Different isoforms of the ÃŽ ² subunits also do exist which include (CaB2a, CaB2b and CaB3) (Hullin et al, 1992 and Petegem et al, 2006). Their association with ÃŽ ± subunit is essential for modulation of VDI, CDI and CDF (Petegem et al, 2006). The ÃŽ ±2 subunit is also known as the ÃŽ ±2/ÃŽ ´ subunit as both the subunits are product of a single gene (Petegem et al, 2006). The ÃŽ ±2 and ÃŽ ´ subunits are linked together by disulphide bonds. Like other subunits ÃŽ ±2/ÃŽ ´ also exists as isoforms (Wang et al, 1999). They are known to play an important role in plasticity of neuron after a nerve injury and neuropathic pain processing (Luo et al, 2001). Gabapentin is a drug known to act on ÃŽ ±2/ÃŽ ´ subunit, but their binding affinity varies with different isoforms of the ÃŽ ´ subunit (Luo et al, 2001 and Luo et al, 2002).T he ÃŽ ³ subunit is found only in skeletal muscles. Their functional roles are yet to be discovered (Petegem et al, 2006). The C-terminus of calcium channel is a site for a number of protein- protein interactions in some channels. The expansion of the polyglutamine tract of the calcium channel is a major reason for the pathogenesis of the disease, Spino Cerebellar Ataxia 6 (SCA6). The cell death in SCA6 is thought to be caused by the poisoning of the nucleus by the localisation of C-terminal fragments (Kordasiewicz, 2006). 1.6. Calcium Channel Types Calcium channels account for the major amount of the calcium entry into the cell. The channel properties are tightly regulated to maintain Ca2+ concentration of the cell. The regulation was done through three well known processes. Voltage Dependent Inactivation (VDI) responsible for preventing entry of calcium into the cell. Calcium Dependent Inactivation (CD1) responsible for preventing entry of calcium into the cell whereas Calcium Dependent Facilitation (CDF) allows for the entry of calcium for signalling (Petegem et al, 2006). Based on the amount of current required to activate the channel the VDCC were termed either LVA channel (Low Voltage Activated) or HVA channel (High Voltage Activated). Later on due to the discovery of different current types, location of channel and sensitiveness to different types VDCC were broadly classified. Thus now 6 different types VDCC are known, in T type the current is transient, located in T-tubules and sensitive to dihydropyridine (DHP) (Dolphin, 2006). In L-Type the current is long lasting, found in neuron, heart and skeletal muscles and are sensitive to DHP. The N-Type stands for Non L Type or Neuronal and they are sensitive to ω-conotoxin GVIA (Petegem et al, 2006). The current found in Purkinje cells of the cerebral cortex were P-Type, they were sensitive to ω -agatoxin IVA. The Q-Type current are found in granular cells, however scientist consider P-Type and Q-Type to be same and are now term as P/Q- Type. The difference between the P Type and Q-Type is thoug ht to depend on the ÃŽ ² subunit to which it is associated(Dolphin., 2006). Another type of Residual current was also discovered which to date is not sensitive to any of the known toxin, this current is known as R-Type (Dolphin, 2006 and Petegem et al, 2006). 1.7. Calcium Channel Gene The alpha sub unit of the calcium channel are coded by 10 genes, therefore 10 different ÃŽ ±1 sub units are known. Of the ten types Cav 1.1 1.4 which is found in L-type, Cav 2.1 or the CavÃŽ ±1A is found in P/Q type channel, Cav2.2 is found in N type and Cav2.3 in R type channel. The Cav 3.1- 3.3 is found in T type channel. All these alpha subunit have one or more isoforms that would contribute to their functional diversity (Dolphin, 2006). The gene coding for the Cav 2.1, CACNA1A is found on the chromosome 19p13. This gene belongs to CACN family of gene that code for calcium channel. The gene characterised by the extension of CAG trinucleotide repeats. In humans the extension of the may vary from 4 to 18. Mutation of this gene cause diseases cause three major diseases FHM1 (Familial Hemiplegic Migraine 1), EA2 (Episodic Ataxia 2) and SCA6 (Spino Cerebellar Ataxia 6). Familial Hemiplegic Migraine is an autosomal dominant type of migraine caused by the missense mutation in CACNA1A. Three different mutations of CACNA1A cause FHM1 (Ducros et al, 1999). FHM1 affects the channel inactivation and the kinetics of the calcium channel (Kraus et al, 1997). The replacement of threonine with methionine is the mutation associated with FHM1. This mutation changes the channel structure causing more flow of calcium into cell. This ultimately results in the release of excess neurotransmitter (Ophoff et al, 1998). Episodic Ataxia 2 (EA2) is neurological disorder affecting the cerebellum and causing ataxia. The drug acetozolamide is known to be effective on EA2 (Ophoff et al, 1998). This disease has been found to have small but stable trinucleotide expansion but the role of the expansion is unknown for this disease (Jodice et al, 1997). The mutation in EA2 causes truncation of ÃŽ ±1A subunit which might cause a complete loss of the function of the channel (Wappl et al, 2002). 1.8. Spino Cerebellar Ataxia 6 Spino Cerebellar Ataxia 6 is also a neurodegenerative disease caused by the increase in number of CAG repeats in the CACNA1A gene (Tanaka et al, 2000). The number of trinucleotide repeat is between 22 and 28 in SCA6 (Riess, 1997). But it is not only the CAG repeats that are causing the disease. The ÃŽ ±1A have 6 isoforms and not all the isoforms are with the polyglutamine repeat. Therefore whether SCA6 is a channelopathy or Polyglutamine Disease remains a question among scientist (Frontali, 2006). The isoforms responsible for SCA6 is mainly limited to the C-Terminal. As the C-terminal is site for protein- protein interaction, changes in strength of interaction or changes in interacting partners tremendously affect the channel kinetics and other functional modification. As polyglutamine disease it cause toxic effect considered through aggregate formation (Pril et al, 2004). Comparison of number of repeats with other polyglutamine diseases where the repeat number is much high, the aggr egate formation alone cannot account for pathogenesis (Matsuyama et al, 1999). As a channelopathy the degeneration of Purkinje cell is caused by the poisoning of nucleus with the localised fragments of C-Terminal. The cleaved C terminal product is considered to have involved in signalling mechanism of the cell (Kordasiewcz et al, 2006). The isoforms of the C-Terminal of calcium channel are of considerable importance as the variation are found to be species specific (Kanumilli et al, 2005) and a few of them do not code for polyglutamine repeats. This invokes an interest in the C-terminal of the ÃŽ ±1A subunit of the calcium channel. The isoforms are formed by a process known as the pre-mRNA alternate splicing. 1.9. Splicing Transcription of messenger RNA (mRNA) from DNA and translation of proteins from mRNA forms the central dogma of molecular biology (Crick, 1970). These processes involves a series of important events, one among them is pre mRNA splicing. Before translation of protein, the mRNA needs to be processed by removing of non-coding introns. A human gene on an average consists of 8 introns. Splicing can lead to more than one type of mRNA from a single gene and consequently different protein isoforms (Faustino et al, 2003). Many different proteins are involved in splicing most importantly the spliceosome, a complex formed of small nuclear RNA (snRNA) and small nuclear ribonucleoproteins (Hagiwara et al, 2005 and Jurica et al, 2003). Small nuclear RNA can be of 5 important types U1, U2, U4, U5 and U6. All these in different combination target specific pre mRNA. The targeting is based on a number of factors which include phosphorylation of snRNAs, catalytic metal ions, enhancers, transcriptional coregulators and serine/arginine rich SR protein (Shi et al, 2006; Saba et al 2005; Auboeuf et al, 2007; Jurica et al, 2003; Hicks et al, 2005 and Manley et al, 2006). In general a spliceable introns has three regions splice donor, splice acceptor and a branch site. Most of the splice donor regions consist of AU nucleotide and the splice acceptor region consist of AG (Kenneth., 2005). Spliceosomes attach to these ends and by transesterification remove the introns, followed by the ligation of the exon (Rio,1993). Several mRNA have inherent splicing mechanism that does not require any spliceosome as they can splice themselves known as self splicing (Herrin et al, 1990 and Landthaler et al, 1999). Though most of the splicing is limited within the same mRNA, splicing also occurs between two different mRNAs by trans-splicing mechanism. The two mRNA exons called the mini exons were transcribed in different gene and were then combined to translate for a single protein (Bonen, 1993 and Bonen, 2008). Alternative splicing is a mechanism by which a few genes produce innumerable proteins that are diversified in structure and function. Nearly 75% of the human genes are involved in alternate splicing to give different protein isoforms (Hagiwara et al, 2005 and Stamm et al, 2004). The needs to understand alternate splicing have arised in almost all fields of biology. In evolutionary terms alternate splicing has a major role in the functional development of species right from the times of â€Å"RNA world†. The importance of isoforms has been understood through a number of studies. The Active and inactive forms of Sex lethal protein isoform are the determinants of sex of Drosophila (Herbert et al, 1999; Irimia et al, 2007 and Poole et al, 1998). Many different isoforms of normal proteins are discovered in cancer cells. These studies of these isoforms and their role have revealed some important diagnostic approach and cancer cell biomarkers (Brinkman, 2004; Skotheim et al, 2007 and Pampalakis et al, 2008). In the drug discovery process it is necessary to consider the mechanism of protein isoforms and pre mRNA splicing pathways and signalling molecules to identify new targets for drugs (Levanon et al, 2003 and Hagiwara et al, 2005). Alternative splicing in ion channels alter the conductance and functional properties of the channel. Splicing has been known in voltage gated sodium channel, voltage gated calcium channel, ligand gated ion channel and in calcium gated potassium channel. Although the ion channels differ in their properties, all share some basic function. These ion channels have multiple splicing site through which their channelling properties are regulated based on the organs where these channels are located (Copley, 2004; Raymond et al, 2004; Sarao et al, 1991 and Schaller et al, 1992). 1.10. CaV2.1 splice variants Variants in calcium channel protein, in particular the 47 exon of the c-terminal is the basis of this study. Splicing in calcium channel occurs at distinct region such as the loops between the II-III domains which is the major interacting site for ryodine receptor. Two isoforms BI and rbA are found in loop II-III of rat and rabbit. They differ in their interacting ability towards syntaxin and synaptotagmin proteins. These proteins can modulate the Ca+ influx of the neuron (Charvin et al, 1997 and Rettig et al, 1996). Site specific variations are found in exons 9, 31, 44, 46 and the extreme C terminus e47 (shown in Fig 3)(Kanumilli et al, 2005). The C- termini of calcium channels are involved in the modulation of G-proteins, molecular switching of calmodulin and are the site for protein-protein interaction. So a single amino acid change can potentially change the gating property and other function of the channel and its interacting partners (Chaudhuri et al, 2004; Gray et al, 2007; Krovetz et al, 2000 and Ligon et al, 1997) splice variants were known to occur in the C- terminal the calcium channel. A 5 base pair insertion (ggcag) was reported in pancreatic islets of rats a variant already known in human (Ligon et al, 1997). This 5 base pair insertion is expected to alter the length on the c terminal and hence channel property as it found before the stop codon, which means a change in the reading frame. The existence of variants with and without the 5 base pair (ggcag) insert before the stop codon of rat Purkinje cell is confirmed by Kanumilli et al (2005). Another independent study with mouse by Tsunemi et al (2001) also confirmed the 5 base pair insert. In addition, variants without the stop codon and a ggcag insert, 150 nucleotide deletions in the 5- end of the C- terminal is reported in mouse (Kanumilli et al, 2005). The absence of stop codon was also observed in the study by Tsunemi et al (2001) in mouse. Richards et al (2007) obtained similar results with rat Purkinje cell, the sequence of exon 47 were same as the rat pancreatic cells except for variations in other exons. However variation in the number of amino acid (156 residues, 153 residues and 115 residues) coded by exon 47 were observed in different clones. The 156 amino acid length was also reported by Ligon et al (1998). These finding and most other results describe the calcium channel properties in terms of activation or inactivation kinetics. However no protein- protein interaction study is available till date for the exon 47 with five base pair (ggcag) inclusion before the stop codon. The need for studies at the protein-protein interaction level is necessary which is evident from the studies of Dolphin(2006), Richards et al (2007), Sandoz et al (2001) and Kanumilli et al (2005). This study was aimed at studying possible protein-protein interaction for exon 47 of rat Purkinje cell. Then linking the interacting the protein to already known biochemical pathway is expected to give more insight the channel and possibly a new perspective in the treatment of SCA6. 1.11. Protein protein interaction studies Protein-Protein interaction is an important part in all biological process. A protein- protein interaction can altogether change the binding characteristics, kinetic property and their catalytic ability (Eisenberg et al, 2000). A number of methods have been developed and used to study protein-protein interaction. These methods can be the detection and analysis of interaction or can be screening against a family of proteins. Detection methods are mostly used to confirm and study known interaction. These methods include Protein Affinity chromatography, Affinity Blotting, Coimmunoprecipitation and Cross- linking. The screening methods include protein probing, phage display and the Yeast Two Hybrid System (Y2H) (Phizicky et al, 1995). Bioinformatics tools such as protein docking are also important in predicting the protein interactions (Smith et al, 2002). 1.12. Yeast Two Hybrid System (Y2H) Yeast two hybrid system is the most widely used protein screening methods. The requirement of an interaction between two domains DNA Binding Domain (DNA-BD) and Activation Domain (AD) for the expression of a reporter gene (lac-z) in yeast is being exploited in Y2H. The lac-z gene expression gives our ÃŽ ²-galactosidase enzyme which can be observed by colour change confirming interaction (as shown in Fig 4) (Criekinge et al, 1999). The protein of interest (bait) is usually fused with the BD and the interacting protein or the library protein is fused with activation domain. The protein of interest is normally termed as bait and the interacting protein is called a prey. Bacterial plasmid can be easily constructed to express fusion protein of interest. The bacterial shuttle vector can be isolated and transfected into the yeast for their expression. On expression the DNA-BD fusion protein will bind to the upstream activation sequence of the reporter gene. Two types of Y2H are known one is the GAL4 based system and the other is the Lex A based system. In Lex yeast two hybrid system the prey is fused with the Lex A binding domain. The specifically interacts with the Lex A operator upstream sequence which is the part of the promoter for reporter gene. The prey will be fused with the GAL 4 protein. In the GAL 4 system instead of Lex A the GAL 4 promoter will be used. Both the systems have their advantages and their dis advantages (Criekinge et al, 1999 and Luban et al, 1995) The yeast strain L40 is compatible with LexA operator and the GAL 4 promoter system. Most Y2H methods are done more than one reporter gene for more selectivity. HIS3 gene is one such reporter that is used for the nutritional selection of the cells. HIS3 reporter expression needs the interaction of proteins. So cells would not grow in a media lacking histidine if no interactions take place. Similar nutritional selections are also used in cell containing only the baits or only the prey. The nutritional selection for bait is tryptophan and for the prey is leucine. It is therefore important to use a defined media. A positive interaction between bait and the prey will allow growth in the Triple Drop Out media (TDO/ -His/-Leu/-Trp) (Criekinge et al, 1999 and Luban et al, 1995) The use of histidine reporter gene can sometimes account for leaky expression. In which case 3-AT (3-amino-s-triole) a competitive inhibitor of histidine can be tried in various concentration to find a minimum concentration at which cells grow and the enzyme is inhibited. Cells growing concentration of 100mM concentration cannot be used as baits (Criekinge et al, 1999). Toxicity caused by bait can inhibit the growth of yeast (Zhong et al, 2003). Toxicity tests have to be carried out to after the baits are designed. Autoactivation of the baits should be checked before proceeding to the, library screening as nearly 5% of the protein can initiate transcription without an interactor (Criekinge et al, 1999). After the library screening the plasmids can be isolated and used to transform bacterial cells. The interaction also has to be confirmed and isolated by techniques such as coimmunoprecipitation. 2. Aim This study was undertaken as a part of the project by Dr. Claire Palmer in finding novel protein-protein interaction for 5-base pair insert in exon 47 of rat cerebellar Purkinje cell(AF051526). Yeast 2 hybrid system was employed to study interaction. Accordingly two protein baits 5inSER and NLSER were constructed by colleague Surya to screen against library protein. Baits 5inSER is a 472 base pair length protein with ggcag NLSER is a 397 base pair length protein without the Nuclear Localisation Sequence. It was constructed to find the significance of the nuclear localisation signal (Surya, 2008). The aims of the project are To test for toxicity and autoactivation of baits. To determine the concentration of 3-AT at which the expression of Histidine gene is inhibited. Control mating experiment. 3. Materials and Methods 3.1. Control Mating 3.1.1. Control strains The control mating experiments were done prior to the library screen. The positive control yeast strains AH109 with the bait [pGBKT7-53] and Y187 with the target [pGADT7-T] , glycerol stock were provided. For negative control the bait strain was L40 with bait pBTM116/GluR2 and the target was the same Y187[pGADT7-T] The negative control bait was obtained by the transformation of L40 with the plasmids isolated from provided E.Coli cultures. 3.1.2. Small Scale Yeast Transformation A colony of Saccharomyces cerevisiae L40 yeast was inoculated into 10ml of YPAD media. It was left overnight in a shaking incubator (200rpm) at 30à ¢Ã‚ Ã‚ ° C. The overnight culture was diluted in 50 ml of YPAD to an OD600 Toxicity and Autoactivation of Baits Experiment Toxicity and Autoactivation of Baits Experiment Abstract Alternate splicing in exon 47 of the Purkinje cell calcium channel generates a splice variant with a five base pair insert (ggcag) before the stop codon in rat. This five base pair change the open reading frame of the exon 47 for resulting in an extended C-Terminal. Novel protein interaction at this region was hypothesised. Yeast Two Hybrid System was employed to screen against cDNA library to check for any protein interaction with 5 base pair insert region of exon 47. This project aimed to test the toxicity/ autoactivation of the baits in the yeast and to find the minimum concentration of 3-AT (3-amino-s-triole) at which it inhibits the HIS3 gene. The experimental result shows that there was no leaky expression of the HIS3 gene. The autoactivation/toxicity test results showed that the baits are less toxic than the control bait. The growth of non-interacting colonies in the Triple Drop Out media revealed that a more defined media should be used, demanding the repetition of experiment to obtain more convincing results. 1. Introduction 1.1. Nervous System The human nervous system consists of the Peripheral Nervous System (PNS) and the Central Nervous System (CNS). The PNS is formed of the cranial nerves and the spinal nerves. The central nervous system consists of the brain and the spinal cord. The brain can be divided into three major parts cerebrum, cerebellum and the brain stem. The cerebrum is divided into frontal lobe, parietal lobe, occipital lobe and the temporal lobe. The main function of cerebrum includes controlling of sensory organ, motor function, consciousness and imagining. The cerebellum is a uniform structure and its function is essential in movement and co- ordination of organs. The brain stem is made up of the mid brain, the pons and the medulla. The main functions of brain stem are transmission of information to and from the brain (Bear et al, 2001; Purves et al, 2004 and Thompson,1993). 1.2. Cells of CNS The brain consist mainly two types of cells nerve cells or neuron cells and the glial cells. The neuron are involved in the transport of electrical signals from the brain whereas the glial cells are thought to be the supporting cells of neurons by the uptake excess of neurotransmitter that are essential for signalling between neurons (Henn et al, 1971 and Purves et al, 2004) and plays a role in synaptogenesis of the neuron (Bacci et al, 1999). The glial cells are of three types: astrocytes, oligodentrocytes and the microglial cells. 1.2.1. Glial Cells Astrocytes are star shaped cells. The spatial arrangement of these cells between the capillaries and the neurons enables it in the modification of cellular responses, synaptic plasticity and survival of neurons (Abe et al, 2006 and Chen et al, 2003). Astrocytes important in glutamate transport, removal of free radical, controlling of haemostasis of brain and in maintaining a preferable environment for the active functioning of neurons by buffering K+ ions in their extracellular space (Chen et al, 2003; Gee et al, 2004 and Longuemare et al, 1999). Oligodentrocytes are type of glial cells that insulate the neuron with myelin sheath (Bear et al, 2001 and Lubetzki et al, 1993. The myelin sheath is a membrane which is made up of lipid and two proteins the proteolipoprotein (PLP) and the myelin basic protein (MBP). (Colman et al, 1982 and Boison et al, 1995). At regular intervals myelin sheath becomes thinner and is known as Nodes of Ranvier (Peter et al, 1966). These regions are the site for voltage gated sodium channels and a number of proteins. Microglial cells are the macrophages of the brain, which are formed in the bone marrow and are then transported to the brain by specialized protein called chemokines (Khoury et al, 2008) The study of chemokine receptors is one of the important research areas in the pathogenesis of Human Immuno Deficiency Virus. HIV can target microglial cells for their replication (Albright et al, 1999; Ghorpade et al, 1997 and Meer et al, 2000). Microglial cells are also studied for their inflammatory re sponses in the brain. The identification of role and mechanism by which microglial cells cause inflammation has paved path for finding targets and therapeutics for many diseases.(Bhatia, 2008; Huang et al 2008; Hwang et al, 2008 and Kim et al, 2008). 1.2.2. Neurons Neuron or the nerve cells are units of the nervous system involved in transfer of electrical signal between each other and to the effector cells. There are many types of nerve cells. Purkinje cells are one among them (Brown, 1991). The study of calcium ion channel of Purkinje cell is the subject of this project. The basic parts of neuron consist of a soma or cell body, axon, dendrites and neurites. All neurons are covered by the neuronal membrane. The soma or the cell body is similar to any other type of cell in the body. The axon is fibre that transport signal from the cell body to other neuron or to the target cell. The axons are covered by myelin sheath of the glial cells. The axon may be branched or unbranched. The main function of axon is to transfer the electrical signal from the axon hillock of soma throughout the axon known as the action potential and to transfer the signals to other cell in the form of chemical signal, the neurotransmitter (Purves et al, 2004 and Bear et al, 2001). The region of contact with other cells where release of neurotransmitter takes place is known as the synapse. The release of neurotransmitter is facilitated by synaptic vesicles of the presynaptic terminal (one which release chemical signal). The neurotransmitters are released by the synaptic vesicle in the space between pre synaptic and post synaptic terminal known as the synaptic cleft (Pu rves et al, 2004 and Brown et al, 1991). The neurotransmitters are then received by specific receptors of the post synaptic terminal which would generate an action potential in the cell. Apart from these receptors the ion channels of the cell membrane of the synaptic terminal also respond in the transfer of signal. Dendrites are branched fibres that arise from the cell. Their surface is lined with number of receptor to receive signals for the neuron (Brown,1991., Purves et al, 2004., Thompson,1993 and Bear et al, 2001). Purkinje cells are one of the largest types of neurons on the brain. They are found in the cerebellar region of the brain. The study of calcium ion channel of Purkinje cell is the subject of this project. Purkinje cells have a number of branches dendrites that receive synaptic inputs. As the dendrites receive signals it initiates a Ca2+ signal, which are important secondary messenger in the cells. The dendrites are the region for a calcium ion entry through the calcium ion channel. Similarly the soma contains K+ and Na+ channels(Schutter et al, 1994). These ions are of particular importance as their charge variation inside and outside the membrane trigger signalling in the cell. The transport of these ions is highly selective and they are maintained by the ion channel proteins of the Purkinje cell membrane and other neuronal membrane. These proteins form a pore for the transport of ions. Techniques such as the Patch clamp method have made the study of these ion channels easier (Bear et al, 2001). 1.3. Ion channel Ion channels are glycoprotein complex that allow specific ions through them. The proteins of ion channel are coded by different gene. More than 100 genes are known to code ion channels. The transportation of ion is important in generating action potential in the cell and is also important as the ions are second messengers in signalling. Diseases associated with the ion channel are known as channelopathies. Ion channels can be three major types voltage gated ion channel. Ligand gated ion channel and the stretch and heat activated ion channel (Purves et al.,2004). Voltage gated ion channels open and close on response to electrical potential. The voltage gated channels are made up of different protein sub unit. The subunits can move to open or close the channel (Horn, 2002). Depending on the type of ions they conduct they are further divided into Calcium channel, sodium channel and potassium channel. Ligand gated channels are those that respond to chemical signals. The ligand gated receptors are of five types nicotinic acetylcholine receptor (AChR), glutamate receptor, ÃŽ ³-aminobutyric acid (GABA), glycine-activated Channels and the ryanodine receptor(Stroud et al, 1990). Each of these receptors bind to specific ion and are found in different organs. The stretch and heat activated ion channel respond to heat or structural deformation of membrane (Purves et al, 2004). 1.4. Voltage Gated Calcium Channel (VGCC) Ca2+ ions are important secondary messenger in cells and play important role in biochemical pathways of cell. The level and entry of these Ca2+ ions in the cell is highly regulated. The regulations of these ions are controlled by the Voltage Gated Calcium Channel (Gribkoff et al, 2006). These VGCC are mainly found in excitatory cells such as the muscle cells and neurons. They exert their function by controlling muscle contraction, neurotransmitter release, neuronal plasticity, synapses, and neuronal excitability (Pietrobon, 2005 and Yang et al, 2005) . VGCC respond to membrane depolarization facilitating Ca2+ entry into the cell and thereby activating the signalling cascade of the cell (Yang et al, 2005). The normal functioning of the calcium channel protein is very important in a cell. Mutation in the gene coding channel protein, have been known to cause a number of diseases which include Timothy syndrome, Familial hemiplegic migraine type 2, episodic ataxia type 2, spinocerebellar ataxia type 6 and autism spectrum disorder which are grouped under â€Å"calcium channelopathies† (Bidaud et al, 2006 and Jen et al, 1999). Calcium channels also play a key role to mediate neuronal pain pathways (Gribkoff et al, 2006). A number of drugs have been known to block calcium channel and they are categorised as Calcium Channel Blockers. Verapamil was the first drug found to block Calcium Channel and later dihydropyridines (DHPs) class of drug was discovered to act as calcium channel blocker (Dolphin, 2006). DHPs are of much importance in studying the channel properties of the Dihydropyridine sensitive calcium channel. These DHP sensitive channels have dihydropyridine receptor for their bin ding (Campbell et al, 1988). Calcium Channel Blockers are now being found effective in the treatment of pain and hypertension (Atanassoff et al, 2000; Kize et al, 2001, and Thompson et al, 2001) but the question of safety in Coronary Heart Disease and the increased risk of cancer in patients remains unanswered (Eisenberg et al, 2004 and Fitzpatrick et al, 1997). 1.5. Calcium channel structure A calcium channel consists of five important subunits ÃŽ ±1. ÃŽ ±2, ÃŽ ², ÃŽ ´ and ÃŽ ³. The ÃŽ ±1 subunit is known as the pore forming complex (Yang et al, 2006). The ÃŽ ±1 subunit is a single polypeptide and its functions mainly include voltage sensing, gating and selective permeation (Horn et al, 2000). The structure of ÃŽ ±1 subunits consist of 24 segments (S1-S6) which constitute 4 domains, a C- terminal, N-terminal and Interlinkers. The linkers connecting domains are known as Loops and they are referred as loop I-II, loop II-III and loop III-IV depending on the domains they link (Dolphin, 2006). The intracellular loop of the ÃŽ ±1 subunit has interaction site for the binding ÃŽ ² subunit. The interaction can modulate the G- protein, an important second messenger in the cell (Dolphin, 1998). The specific binding of ÃŽ ² subunit to the tryptophan residue is important for controlling the gating of ÃŽ ±1 subunit of certain type of channels (Berrou, 2002). S4 is another important segment of the calcium channel. It is the voltage sensitive region of the calcium channel. S4 segment moves outward causing the channel to open by getting depolarised. S4 segment is positively charged due to the presence of arginine aminoacid making it voltage sensitive by translocation of the charges across the membrane (Sigworthl, 2003 and Horn et al, 2000). The S5, S6 and the linker connecting the S5 and S6 segment forms the boundaries ion conducting pore of the ÃŽ ±1 subunit. The ion conductance partly depends on the rotational movement of the S4 segment which either cause the S6 segment to open or c lose the pore (Horn et al, 2000). The ÃŽ ² subunits of the calcium channel are thought to be tissue specific and organ specific. Primarily they are of 4 different types, ÃŽ ²1, ÃŽ ²2, ÃŽ ²3 and ÃŽ ²4. Different isoforms of the ÃŽ ² subunits also do exist which include (CaB2a, CaB2b and CaB3) (Hullin et al, 1992 and Petegem et al, 2006). Their association with ÃŽ ± subunit is essential for modulation of VDI, CDI and CDF (Petegem et al, 2006). The ÃŽ ±2 subunit is also known as the ÃŽ ±2/ÃŽ ´ subunit as both the subunits are product of a single gene (Petegem et al, 2006). The ÃŽ ±2 and ÃŽ ´ subunits are linked together by disulphide bonds. Like other subunits ÃŽ ±2/ÃŽ ´ also exists as isoforms (Wang et al, 1999). They are known to play an important role in plasticity of neuron after a nerve injury and neuropathic pain processing (Luo et al, 2001). Gabapentin is a drug known to act on ÃŽ ±2/ÃŽ ´ subunit, but their binding affinity varies with different isoforms of the ÃŽ ´ subunit (Luo et al, 2001 and Luo et al, 2002).T he ÃŽ ³ subunit is found only in skeletal muscles. Their functional roles are yet to be discovered (Petegem et al, 2006). The C-terminus of calcium channel is a site for a number of protein- protein interactions in some channels. The expansion of the polyglutamine tract of the calcium channel is a major reason for the pathogenesis of the disease, Spino Cerebellar Ataxia 6 (SCA6). The cell death in SCA6 is thought to be caused by the poisoning of the nucleus by the localisation of C-terminal fragments (Kordasiewicz, 2006). 1.6. Calcium Channel Types Calcium channels account for the major amount of the calcium entry into the cell. The channel properties are tightly regulated to maintain Ca2+ concentration of the cell. The regulation was done through three well known processes. Voltage Dependent Inactivation (VDI) responsible for preventing entry of calcium into the cell. Calcium Dependent Inactivation (CD1) responsible for preventing entry of calcium into the cell whereas Calcium Dependent Facilitation (CDF) allows for the entry of calcium for signalling (Petegem et al, 2006). Based on the amount of current required to activate the channel the VDCC were termed either LVA channel (Low Voltage Activated) or HVA channel (High Voltage Activated). Later on due to the discovery of different current types, location of channel and sensitiveness to different types VDCC were broadly classified. Thus now 6 different types VDCC are known, in T type the current is transient, located in T-tubules and sensitive to dihydropyridine (DHP) (Dolphin, 2006). In L-Type the current is long lasting, found in neuron, heart and skeletal muscles and are sensitive to DHP. The N-Type stands for Non L Type or Neuronal and they are sensitive to ω-conotoxin GVIA (Petegem et al, 2006). The current found in Purkinje cells of the cerebral cortex were P-Type, they were sensitive to ω -agatoxin IVA. The Q-Type current are found in granular cells, however scientist consider P-Type and Q-Type to be same and are now term as P/Q- Type. The difference between the P Type and Q-Type is thoug ht to depend on the ÃŽ ² subunit to which it is associated(Dolphin., 2006). Another type of Residual current was also discovered which to date is not sensitive to any of the known toxin, this current is known as R-Type (Dolphin, 2006 and Petegem et al, 2006). 1.7. Calcium Channel Gene The alpha sub unit of the calcium channel are coded by 10 genes, therefore 10 different ÃŽ ±1 sub units are known. Of the ten types Cav 1.1 1.4 which is found in L-type, Cav 2.1 or the CavÃŽ ±1A is found in P/Q type channel, Cav2.2 is found in N type and Cav2.3 in R type channel. The Cav 3.1- 3.3 is found in T type channel. All these alpha subunit have one or more isoforms that would contribute to their functional diversity (Dolphin, 2006). The gene coding for the Cav 2.1, CACNA1A is found on the chromosome 19p13. This gene belongs to CACN family of gene that code for calcium channel. The gene characterised by the extension of CAG trinucleotide repeats. In humans the extension of the may vary from 4 to 18. Mutation of this gene cause diseases cause three major diseases FHM1 (Familial Hemiplegic Migraine 1), EA2 (Episodic Ataxia 2) and SCA6 (Spino Cerebellar Ataxia 6). Familial Hemiplegic Migraine is an autosomal dominant type of migraine caused by the missense mutation in CACNA1A. Three different mutations of CACNA1A cause FHM1 (Ducros et al, 1999). FHM1 affects the channel inactivation and the kinetics of the calcium channel (Kraus et al, 1997). The replacement of threonine with methionine is the mutation associated with FHM1. This mutation changes the channel structure causing more flow of calcium into cell. This ultimately results in the release of excess neurotransmitter (Ophoff et al, 1998). Episodic Ataxia 2 (EA2) is neurological disorder affecting the cerebellum and causing ataxia. The drug acetozolamide is known to be effective on EA2 (Ophoff et al, 1998). This disease has been found to have small but stable trinucleotide expansion but the role of the expansion is unknown for this disease (Jodice et al, 1997). The mutation in EA2 causes truncation of ÃŽ ±1A subunit which might cause a complete loss of the function of the channel (Wappl et al, 2002). 1.8. Spino Cerebellar Ataxia 6 Spino Cerebellar Ataxia 6 is also a neurodegenerative disease caused by the increase in number of CAG repeats in the CACNA1A gene (Tanaka et al, 2000). The number of trinucleotide repeat is between 22 and 28 in SCA6 (Riess, 1997). But it is not only the CAG repeats that are causing the disease. The ÃŽ ±1A have 6 isoforms and not all the isoforms are with the polyglutamine repeat. Therefore whether SCA6 is a channelopathy or Polyglutamine Disease remains a question among scientist (Frontali, 2006). The isoforms responsible for SCA6 is mainly limited to the C-Terminal. As the C-terminal is site for protein- protein interaction, changes in strength of interaction or changes in interacting partners tremendously affect the channel kinetics and other functional modification. As polyglutamine disease it cause toxic effect considered through aggregate formation (Pril et al, 2004). Comparison of number of repeats with other polyglutamine diseases where the repeat number is much high, the aggr egate formation alone cannot account for pathogenesis (Matsuyama et al, 1999). As a channelopathy the degeneration of Purkinje cell is caused by the poisoning of nucleus with the localised fragments of C-Terminal. The cleaved C terminal product is considered to have involved in signalling mechanism of the cell (Kordasiewcz et al, 2006). The isoforms of the C-Terminal of calcium channel are of considerable importance as the variation are found to be species specific (Kanumilli et al, 2005) and a few of them do not code for polyglutamine repeats. This invokes an interest in the C-terminal of the ÃŽ ±1A subunit of the calcium channel. The isoforms are formed by a process known as the pre-mRNA alternate splicing. 1.9. Splicing Transcription of messenger RNA (mRNA) from DNA and translation of proteins from mRNA forms the central dogma of molecular biology (Crick, 1970). These processes involves a series of important events, one among them is pre mRNA splicing. Before translation of protein, the mRNA needs to be processed by removing of non-coding introns. A human gene on an average consists of 8 introns. Splicing can lead to more than one type of mRNA from a single gene and consequently different protein isoforms (Faustino et al, 2003). Many different proteins are involved in splicing most importantly the spliceosome, a complex formed of small nuclear RNA (snRNA) and small nuclear ribonucleoproteins (Hagiwara et al, 2005 and Jurica et al, 2003). Small nuclear RNA can be of 5 important types U1, U2, U4, U5 and U6. All these in different combination target specific pre mRNA. The targeting is based on a number of factors which include phosphorylation of snRNAs, catalytic metal ions, enhancers, transcriptional coregulators and serine/arginine rich SR protein (Shi et al, 2006; Saba et al 2005; Auboeuf et al, 2007; Jurica et al, 2003; Hicks et al, 2005 and Manley et al, 2006). In general a spliceable introns has three regions splice donor, splice acceptor and a branch site. Most of the splice donor regions consist of AU nucleotide and the splice acceptor region consist of AG (Kenneth., 2005). Spliceosomes attach to these ends and by transesterification remove the introns, followed by the ligation of the exon (Rio,1993). Several mRNA have inherent splicing mechanism that does not require any spliceosome as they can splice themselves known as self splicing (Herrin et al, 1990 and Landthaler et al, 1999). Though most of the splicing is limited within the same mRNA, splicing also occurs between two different mRNAs by trans-splicing mechanism. The two mRNA exons called the mini exons were transcribed in different gene and were then combined to translate for a single protein (Bonen, 1993 and Bonen, 2008). Alternative splicing is a mechanism by which a few genes produce innumerable proteins that are diversified in structure and function. Nearly 75% of the human genes are involved in alternate splicing to give different protein isoforms (Hagiwara et al, 2005 and Stamm et al, 2004). The needs to understand alternate splicing have arised in almost all fields of biology. In evolutionary terms alternate splicing has a major role in the functional development of species right from the times of â€Å"RNA world†. The importance of isoforms has been understood through a number of studies. The Active and inactive forms of Sex lethal protein isoform are the determinants of sex of Drosophila (Herbert et al, 1999; Irimia et al, 2007 and Poole et al, 1998). Many different isoforms of normal proteins are discovered in cancer cells. These studies of these isoforms and their role have revealed some important diagnostic approach and cancer cell biomarkers (Brinkman, 2004; Skotheim et al, 2007 and Pampalakis et al, 2008). In the drug discovery process it is necessary to consider the mechanism of protein isoforms and pre mRNA splicing pathways and signalling molecules to identify new targets for drugs (Levanon et al, 2003 and Hagiwara et al, 2005). Alternative splicing in ion channels alter the conductance and functional properties of the channel. Splicing has been known in voltage gated sodium channel, voltage gated calcium channel, ligand gated ion channel and in calcium gated potassium channel. Although the ion channels differ in their properties, all share some basic function. These ion channels have multiple splicing site through which their channelling properties are regulated based on the organs where these channels are located (Copley, 2004; Raymond et al, 2004; Sarao et al, 1991 and Schaller et al, 1992). 1.10. CaV2.1 splice variants Variants in calcium channel protein, in particular the 47 exon of the c-terminal is the basis of this study. Splicing in calcium channel occurs at distinct region such as the loops between the II-III domains which is the major interacting site for ryodine receptor. Two isoforms BI and rbA are found in loop II-III of rat and rabbit. They differ in their interacting ability towards syntaxin and synaptotagmin proteins. These proteins can modulate the Ca+ influx of the neuron (Charvin et al, 1997 and Rettig et al, 1996). Site specific variations are found in exons 9, 31, 44, 46 and the extreme C terminus e47 (shown in Fig 3)(Kanumilli et al, 2005). The C- termini of calcium channels are involved in the modulation of G-proteins, molecular switching of calmodulin and are the site for protein-protein interaction. So a single amino acid change can potentially change the gating property and other function of the channel and its interacting partners (Chaudhuri et al, 2004; Gray et al, 2007; Krovetz et al, 2000 and Ligon et al, 1997) splice variants were known to occur in the C- terminal the calcium channel. A 5 base pair insertion (ggcag) was reported in pancreatic islets of rats a variant already known in human (Ligon et al, 1997). This 5 base pair insertion is expected to alter the length on the c terminal and hence channel property as it found before the stop codon, which means a change in the reading frame. The existence of variants with and without the 5 base pair (ggcag) insert before the stop codon of rat Purkinje cell is confirmed by Kanumilli et al (2005). Another independent study with mouse by Tsunemi et al (2001) also confirmed the 5 base pair insert. In addition, variants without the stop codon and a ggcag insert, 150 nucleotide deletions in the 5- end of the C- terminal is reported in mouse (Kanumilli et al, 2005). The absence of stop codon was also observed in the study by Tsunemi et al (2001) in mouse. Richards et al (2007) obtained similar results with rat Purkinje cell, the sequence of exon 47 were same as the rat pancreatic cells except for variations in other exons. However variation in the number of amino acid (156 residues, 153 residues and 115 residues) coded by exon 47 were observed in different clones. The 156 amino acid length was also reported by Ligon et al (1998). These finding and most other results describe the calcium channel properties in terms of activation or inactivation kinetics. However no protein- protein interaction study is available till date for the exon 47 with five base pair (ggcag) inclusion before the stop codon. The need for studies at the protein-protein interaction level is necessary which is evident from the studies of Dolphin(2006), Richards et al (2007), Sandoz et al (2001) and Kanumilli et al (2005). This study was aimed at studying possible protein-protein interaction for exon 47 of rat Purkinje cell. Then linking the interacting the protein to already known biochemical pathway is expected to give more insight the channel and possibly a new perspective in the treatment of SCA6. 1.11. Protein protein interaction studies Protein-Protein interaction is an important part in all biological process. A protein- protein interaction can altogether change the binding characteristics, kinetic property and their catalytic ability (Eisenberg et al, 2000). A number of methods have been developed and used to study protein-protein interaction. These methods can be the detection and analysis of interaction or can be screening against a family of proteins. Detection methods are mostly used to confirm and study known interaction. These methods include Protein Affinity chromatography, Affinity Blotting, Coimmunoprecipitation and Cross- linking. The screening methods include protein probing, phage display and the Yeast Two Hybrid System (Y2H) (Phizicky et al, 1995). Bioinformatics tools such as protein docking are also important in predicting the protein interactions (Smith et al, 2002). 1.12. Yeast Two Hybrid System (Y2H) Yeast two hybrid system is the most widely used protein screening methods. The requirement of an interaction between two domains DNA Binding Domain (DNA-BD) and Activation Domain (AD) for the expression of a reporter gene (lac-z) in yeast is being exploited in Y2H. The lac-z gene expression gives our ÃŽ ²-galactosidase enzyme which can be observed by colour change confirming interaction (as shown in Fig 4) (Criekinge et al, 1999). The protein of interest (bait) is usually fused with the BD and the interacting protein or the library protein is fused with activation domain. The protein of interest is normally termed as bait and the interacting protein is called a prey. Bacterial plasmid can be easily constructed to express fusion protein of interest. The bacterial shuttle vector can be isolated and transfected into the yeast for their expression. On expression the DNA-BD fusion protein will bind to the upstream activation sequence of the reporter gene. Two types of Y2H are known one is the GAL4 based system and the other is the Lex A based system. In Lex yeast two hybrid system the prey is fused with the Lex A binding domain. The specifically interacts with the Lex A operator upstream sequence which is the part of the promoter for reporter gene. The prey will be fused with the GAL 4 protein. In the GAL 4 system instead of Lex A the GAL 4 promoter will be used. Both the systems have their advantages and their dis advantages (Criekinge et al, 1999 and Luban et al, 1995) The yeast strain L40 is compatible with LexA operator and the GAL 4 promoter system. Most Y2H methods are done more than one reporter gene for more selectivity. HIS3 gene is one such reporter that is used for the nutritional selection of the cells. HIS3 reporter expression needs the interaction of proteins. So cells would not grow in a media lacking histidine if no interactions take place. Similar nutritional selections are also used in cell containing only the baits or only the prey. The nutritional selection for bait is tryptophan and for the prey is leucine. It is therefore important to use a defined media. A positive interaction between bait and the prey will allow growth in the Triple Drop Out media (TDO/ -His/-Leu/-Trp) (Criekinge et al, 1999 and Luban et al, 1995) The use of histidine reporter gene can sometimes account for leaky expression. In which case 3-AT (3-amino-s-triole) a competitive inhibitor of histidine can be tried in various concentration to find a minimum concentration at which cells grow and the enzyme is inhibited. Cells growing concentration of 100mM concentration cannot be used as baits (Criekinge et al, 1999). Toxicity caused by bait can inhibit the growth of yeast (Zhong et al, 2003). Toxicity tests have to be carried out to after the baits are designed. Autoactivation of the baits should be checked before proceeding to the, library screening as nearly 5% of the protein can initiate transcription without an interactor (Criekinge et al, 1999). After the library screening the plasmids can be isolated and used to transform bacterial cells. The interaction also has to be confirmed and isolated by techniques such as coimmunoprecipitation. 2. Aim This study was undertaken as a part of the project by Dr. Claire Palmer in finding novel protein-protein interaction for 5-base pair insert in exon 47 of rat cerebellar Purkinje cell(AF051526). Yeast 2 hybrid system was employed to study interaction. Accordingly two protein baits 5inSER and NLSER were constructed by colleague Surya to screen against library protein. Baits 5inSER is a 472 base pair length protein with ggcag NLSER is a 397 base pair length protein without the Nuclear Localisation Sequence. It was constructed to find the significance of the nuclear localisation signal (Surya, 2008). The aims of the project are To test for toxicity and autoactivation of baits. To determine the concentration of 3-AT at which the expression of Histidine gene is inhibited. Control mating experiment. 3. Materials and Methods 3.1. Control Mating 3.1.1. Control strains The control mating experiments were done prior to the library screen. The positive control yeast strains AH109 with the bait [pGBKT7-53] and Y187 with the target [pGADT7-T] , glycerol stock were provided. For negative control the bait strain was L40 with bait pBTM116/GluR2 and the target was the same Y187[pGADT7-T] The negative control bait was obtained by the transformation of L40 with the plasmids isolated from provided E.Coli cultures. 3.1.2. Small Scale Yeast Transformation A colony of Saccharomyces cerevisiae L40 yeast was inoculated into 10ml of YPAD media. It was left overnight in a shaking incubator (200rpm) at 30à ¢Ã‚ Ã‚ ° C. The overnight culture was diluted in 50 ml of YPAD to an OD600