Unit 9 - Gpo Planning Scenario Assignment - 801 Words

Hello I am the IT Administrator, I will be help you answer your questions dealing with software deployment. 1. What is Group Policy? Group Policy can be used to install, upgrade, patch, or remove software applications when a computer is started, when a user logs on to the network, or when a user accesses a file associated with a program that is not currently on the user’s computer. 2. What is Repackaging Software? It is taking a snapshot of a clean computer system before the application is installed, installing the application as desired, and taking a snapshot of the computer after the application is installed. A clean computer system is defined as a system with no other applications installed and only those service packs, hot†¦show more content†¦Software restriction policies are designed to identify software and control its execution. In addition, administrators can control who will be affected by the policies. 5. What about Configuring Software Restriction Rules? The functionality of software restriction policies depends on the rules that identify software, followed by the rules that govern its usage. When a new software restriction policy is created, the Additional Rules subfolder is displayed. This folder allows you to create rules that specify the conditions under which programs can be executed or denied. The rules that are applied can override the Default Security Level setting when necessary. 6. What does Network zone rules do? It can control from a specified zone, such as a local computer, a local intranet, trusted sites, restricted sites, or the Internet. This type of rule can be applied to allow only Windows Installer packages to be installed if they come from a trusted area of the network. For example, an Internet zone rule could restrict Windows Installer packages from being downloaded and installed from the Internet or other network locations. 7. What about application install, how to set this? The package is set to Assigned, the Install This Application At Logon option is available. This option allows the application to be installed immediately, rather than advertised on the Start menu. If users have slow links between theirShow MoreRelatedBritish Telecom Case Study.5136 Words   |  21 Pagesrequirements of people increases, British Telecommunication (BT) has a leading corresponding growth in the size and number of information processed. Therefore, the genuine analyses on BT are required for such an important and strategic company. In that assignment, we followed the Systems Design approach which includes analysing the components of the system, brainstorming the alternatives to improve the processes and implementation of the preferred solution. First of all, we presented some information onRead MoreProject Management9882 Words   |  40 PagesProject with all its parts in Unit 10. You will deliver drafts of some parts of the Capstone Project at different times throughout the course so you can receive feedback from your instructor. Please see the Syllabus for a full schedule of due dates. Briefly, the parts of the Capstone Project are due as follows: ï‚ § With Unit 6: o o Network Schematic o Server Configuration o ï‚ § Active Directory Network Infrastructure Configuration With Unit 8: o Client Configuration Read MoreProject Mgmt296381 Words   |  1186 PagesIntegration of project management processes [3.1] 6.5.2 Setting a schedule baseline [8.1.4] 6.5.3.1 Setting a resource schedule 6.5.2.4 Resource leveling 7.2 Setting a cost and time baseline schedule (1.3.5) [8.1.3] 6.5.2.3 Critical chain method Chapter 9 Chapter 10 Reducing Project Duration Leadership Chapter 2 Organization Strategy and Project Selection 1.4 Projects and programs (.2) 1.4.1 Managing the portfolio 1.4.3 Strategy and projects 2.3 Stakeholders and review boards 12.1 RFP’s and vendorRead MoreAnz Bank142091 Words   |  569 Pageseconomies of scale, increase our speed to market and strengthen the operating risk control environment for the business. The  Group’s regional delivery centres provide full service regional coverage across our operating time zones helping to drive lower unit costs, improve quality and lower  risk. Our business risk profile improved, with a continuing shift to investment-grade clients and shorter tenor Trade Finance, and greater earnings diversification across products and geographies. Combined withRead MoreTransforming Total Sales into Net Profits51271 Words   |  206 PagesTransforming Total Sales into Net Profits V iable ision Transforming Total Sales into Net Profits Gerald I. Kendall, PMP Copyright  ©2005 by Gerald I. Kendall ISBN 1-932159-38-X Printed and bound in the U.S.A. Printed on acid-free paper 10 9 8 7 6 5 4 3 2 1 Library of Congress Cataloging-in-Publication Data Kendall, Gerald I. Viable vision : transforming total sales into net profits / by Gerald I. Kendall. p. cm. Includes bibliographical references and index. ISBN 1-932159-38-X (pbk.Read MoreAnnotated Bibliography: Plagiarism39529 Words   |  158 Pagesthought (see Lewis et al., 2011). Moreover, as these and other authors report, academic plagiarism even when discovered, often goes unsanctioned (Bartlett and Smallwood, 2004; Kock, 1999). This situation Corresponding author: Belinda Luke, QUT, GPO Box 2434, Brisbane, 4001, Australia Email: b.luke@qut.edu.au 448857ORG0010.1177/1350508412448857Luke and KearinsOrganization 2012 Article Downloaded from org.sagepub.com at FLORIDA INTERNATIONAL UNIV on January 20, 2013 2 Organization 0(0) implicates

Challenges Of The European Immigrant - 1794 Words

Taryn Ernst Professor Franks Intersections 19 November 2015 Challenges of the European Immigrant For the last year, people from the various regions of the globe have been fleeing their home countries and migrating toward several nations across Europe. What pushes them away and what draws them to their final destinations? Ascertaining a single motivation of immigration remains too complex to determine because a variety of factors and goals have lead to this grand movement. Several issues arise from increased numbers of immigrants who may or may not be finding what they seek. The most common reason for this sudden migration involves increasing violence in the home countries of the immigrants. Civil unrest in Syria has continued with†¦show more content†¦Whether they seek education, employment, or housing, all of these immigrants must first be granted asylum. Ahmed Umar took the long voyage from Mogadishu, Somalia in hopes of arriving in Germany to find education and employment opportunities (Five Migrant Stories). Immigrants have come from within Europe as well. Poverty in the nation of Kosovo has driven its members to seek economic refuge inland. Kosovo has a nearly forty percent unemployment rate that affects women and children in a disproportionate manner. Kosovo remains Europe’s poorest country with a per capita GDP of  £2,700, or about 2,900 US dollars. Approximately one-third of the population lives below the poverty line (Poverty Spurs). Some people leave their home countries in search of better social services. Ali Fellah left Iraq because there was a breakdown of services such as electricity, clean drinking water, and fuel. â€Å"I’m not thinking about me. It’s about the future for my son† he says. Diseases spread quickly and sufficient services were not available. He also felt threatened by the government of his home country. Fellah could not see a future for his son if their family stayed in Iraq (Five Migrant Stories). Sara Arbini decided to leave her husband in Syria in order to gain access to proper medical care. She says, â€Å"It’s like we went back 200 years.† Finding work, clean water, and electricity becameShow MoreRelatedNative Immigrants And Asian Immigrants914 Words   |  4 Pages The opportunities of racial minorities such as the Chinese or African Americans different from those of European immigrants because diversity played a big role in the quality of urban setting. When the industrial revolution happen a lot of immigrant were in search for better economic opportunity, so as Chinese left their home countries due to poverty and famine, cities were the first place they settle down in, making their way to the US they had great opportunity, from owning their own businessRead MorePuerto Rican Obituary By Pedro Pietri1185 Words   |  5 PagesRican immigrant. 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Thirty years ago, it was considered by many Europeans that multiculturalism (society being diverse and united at the same time), is the answer to social problems in Europe. Today, however, many believe that the raising number of immigrants is the cause of those problems. This perception prompted many people to speak out against multiculturalism and talk about the risks. That perception hasRead MoreEurope, The Catholic Church, And The Refugees Of Refugees Essay1675 Words   |  7 Pageswell-developed European countries. The majority of Europe is Christian, with a large portion of that being Catholic, and so in the midst of this situation it is crucial to examine how European responses to the migrant crisis have compared to the Catholic Church’s position on migration, to see if people are truly putting their religion into practice. 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On the Necessity of Rationalism Free Essays

In the process of considering the various means of justification, a relativistic conception of reality assumes that the truth and hence the validity of a statement may only be assessed in relation to the perspective of the discipline which holds a particular belief. In this sense, truth is dependent upon the internal coherence of beliefs within a system of thought. In his The Last Word, Nagel claims that such is not the case. We will write a custom essay sample on On the Necessity of Rationalism or any similar topic only for you Order Now He argues that the truth and hence the validity of statements are dependent upon an unqualified notion of reason. He claims that the truth of a statement is independent upon any particular perspective. If such is the case, it follows that the truth of any statement is independent from the schema [truth schema] presented by any system of thought. In relation to scientific claims, it thereby follows that the truth of scientific claims ought to be assessed through the unqualified notion of reason as opposed to merely their internal coherence within the scientific conception of reality [scientific framework]. The aforementioned argument is based upon the critique of the intrinsic limits to subjectivist doubt since challenges to the independent validity of reason must themselves assume the independent validity of reason. Any explanation of reason deriving from outside the mind can itself be explained only from inside the mind, as having its own independent validity. In the case of scientific knowledge, he argues that it is mistaken to assume that the scientific discipline has freed itself from the limits of the Cartesian problem through the replacement of judgments about rules of practice from objective judgments. Nagel argues that if science will continually adhere to a subjectivistic and hence relativistic framework, the discipline will fail to provide an objective account of reality. He claims, â€Å"the general aim of such reasoning [scientific reasoning] is to make sense of the world in which we find ourselves and how it appears to us and others† (81). If such is the case, it is necessary to conceive of the conception of the world which is not based upon an a priori conception of reality dependent upon a preconceived and limited conception of the word. According to Nagel, such an account is not provided by science. The reasons for this lies in the subjectivism of science (Nagel 84). Subjectivism within science [scientific methods] is apparent if one considers that the scientific â€Å"demand for order cannot itself be rationally justified nor does it correspond to a self-evident necessity† (Nagel 84). He notes that scientific subjectivism can only end if it adheres to rational means of knowledge acquisition. It is only through the defense of rationalism that an objectivist account of evidence is possible. Nagel further argues that the appeal of subjectivism arises out of a certain reductionist impulse in modern explanation as this reductionist impulse enables the explanation of things to hinge on their reduction to local and finite terms thereby ensuring subjectivist conclusions. Although this enables the assurance against rationalist explanations that refuse to make reason into something irrational or that conceive of reason as a capacity for grasping the universal and infinite principle, the reductionist explanation is in itself dependent on an irreducibly nonlocal and objective understanding of reason. Nagel argues that doubt about reason presupposes reason’s independent validity hence reason’s independent validity cannot be coherently doubted. He rightly argues that to object to reason on the grounds we cannot strictly explain it in naturalistic terms is to misunderstand the irreducible nature of the concept since reason cannot be so explained without losing its meaning or validity and that, as such, it is justified in a different way, by showing it to be necessary to intelligible thought and action. Science thereby must opt for a rationalistic as opposed to a subjectivistic account of reality for it to maintain its value as a discipline. Work Cited Nagel, Thomas. The Last Word. New York: Oxford University Press, 1997. Essay Number Two Edmund Gettier’s Counterargument Against the Platonic Tripartite Account of Propositional Knowledge The Platonic tripartite definition of propositional and fallibilist knowledge found in the last section of the Theaetetus states that knowledge of P occurs when an epistemic agent S knows that P if and only if (1) P is true, (2) S believes that P, and (3) S is justified in believing that P (90). A well-known opposition to such an account of propositional knowledge questions the sufficiency of the aforementioned conditions. It is argued that although the aforementioned conditions are necessary in the definition of propositional knowledge such conditions are insufficient due to their failure to ensure S against conditions wherein knowledge of P occurs as a result of mere epistemic luck (Gettier 123). This critique is best known as the Gettier type counter examples towards the tripartite definition of propositional knowledge mentioned above. A logical problem is posited by the Gettier type counter examples. This logical problem is evident in the lack of successful coordination between the truth of P and the reasons that justify S in holding P. Floridi notes that Gettier type counter examples arise â€Å"because the truth and the justification of P happen to be not only independent but also opaquely unrelated that they happen to fail to converge or agree on the same propositional content P†¦without S realizing it† (64). In order to understand this, it is important to lay down the main assumptions of Gettier’s counter argument that seeks to explicate the aforementioned logical problem. Gettier’s argument against the tripartite account of propositional knowledge, which involves the conception of knowledge as justified true belief arose as a result of the following claim: knowledge [propositional knowledge] does not merely involve justified true belief. Such a claim is based upon the following assumptions. First, there are instances wherein the warrant is not a sufficient condition for a belief in P. This is evident if one considers that instances of belief and knowledge of P are in some respects epistemically different [other than in terms of truth] from belief of P without knowledge of P. Second, there are instances wherein warrant is fallible. This is due to the insufficiency of truth and justification as warrants for knowledge. The evidence of such, according to Gettier is apparent if one considers that it is possible for P to be false even if S believes that P possesses epistemically significant properties such that whenever a belief possesses such properties and is true the belief may thereby qualify as knowledge. Lastly, there is the closure of knowledge under obvious and known entailments. The last assumption argues that if S is justified in believing P and a deductively valid inference is drawn from P to another belief Q then S is justified in believing Q. This is a result of the entailment of Q from P. From what was stated above, it is possible to present the usual form of Gettier’s attack against the tripartite account of knowledge. Gettier’s counter argument is based upon the critique of warrant, fallibility, and closure. Note that combination of the three claims mentioned above leads to a contradiction. From what was mentioned above it follows that it is possible to believe in an obvious deductive consequence of P, which is Q, while in the process retaining the epistemically significant properties of the belief in P. If such is the case, it is possible to have a justified true belief of any property which has led S to have a belief in Q or any other type of belief which has Q’s epistemic characteristics. Note that this contradicts the assumed necessity that P and Q differ from each other since one qualifies as knowledge [S believes and has knowledge of P] whereas Q merely qualifies as a belief [S believes but does not have knowledge of Q]. Works Cited Floridi, L. â€Å"On the Logical Unsolvability of the Gettier Problem.† Synthese 142(2004): 61-79. Gettier, E. â€Å"Is Justified True Belief Knowledge?† Analysis 23(1963): 121-23. Plato. Theaetetus. Trans. M.J. Levett. Indiana: Hackett Publishing Co., 1992. How to cite On the Necessity of Rationalism, Essay examples

Proposed CCS Alumni Tracing Software free essay sample

Nowadays, they have used various forms of market research to identify their key value to their alumni and reinforce that value consistently throughout all forms of immunization, either on print, online or through personal visits, events, and presentations. The College of Computer Studies (CSS) of the University of Southern Philippines Foundation currently retrieves records of graduates from the Office of Alumni Relation. The office will records and updates the alumni directory with the use of spreadsheet.Information is gathered through various sources such as social networks, emails, yearbook or by filling out the alumni tracer as a clearance requirement. USPS Alumni Relations also maintains a bloodspot where photos and articles of latest news and activities related o alumni association are posted. Thus, the researchers strongly feel the need to develop a website and establish an Alumni association for the CSS Department. The main purpose of this study is to trace historical student and graduate information. We will write a custom essay sample on Proposed CCS Alumni Tracing Software or any similar topic specifically for you Do Not WasteYour Time HIRE WRITER Only 13.90 / page It also aims to provide hubs of information offering college news, events, issues, along with association activities and organize seminar/workshop and training programs to build and foster CIT growth and development trends among CSS alumni, faculty and students. With these objectives, the researchers composed of CSS faculty, induct a software development study for tracing CSS Alumni. It will serve as communication portal of CSS students and alumni professionals and their alma mater.The proposed CSS Alumni Tracing Software will: Provide a lifelong link between the alumni and their alma mater. Maintain a dynamic Alumni database. Engage the current students with the Alumni and can get useful career guidance from the alumni. Create common interest groups and provide a forum for discussion. Inform the alumni about the ongoing and future activities and help organize reunion with the help of Alumni association.

How to Write Business Emails that Sound Friendly - The Writers For Hire

HOW TO WRITE BUSINESS EMAILS THAT SOUND FRIENDLY In today’s technologically advancing world, email has become the official choice for communication within businesses. The lack of face-to-face communication can cause a lot of issues and misunderstandings, though. Because of this, it is important that business emails convey a clear message, elicit the desired response, and, above all, not offend in any way. But how do you write emails that are both professional and friendly? Identifying Good vs Bad Email Etiquette Before we tackle how to write friendly business emails, we must first identify what a good friendly email looks like, vs. a not so friendly email. Take these emails, for example: See the difference? In the first example, the author seems to take a bit of a hostile, negative tone. Reading this email may leave the recipient feeling like they have been attacked, and will, therefore, immediately put them on the defensive. In the second example, though, the author was able to communicate exactly the same information, but in a much more positive and empathetic way. Emails that are friendly and positive are much more likely to be received well and will illicit the response that you are hoping to get. So, now that we have established what a friendly business email looks like, let’s discuss how to write one. The Basics When you are writing business emails, keep in mind that the recipient(s) probably already has a lot on their plate, so they are not going to want to have to read through a bunch of fluff. Keeping your emails clear and concise will ensure that they are not only read in their entirety, but that your intended message is understood. Effective, yet friendly, business emails, should be brief and punctilious while conveying professionalism and affability. While emails will vary, depending on who they are being sent to and the intended message, ideally, business emails should follow this format: A subject line of no more than 6 to 10 words. Your subject line should be direct and spam-proof. ‘Workshop Date Changed’ will immediately alert your recipient that there is information in your email that they need to read. You want to avoid things like ‘Urgent’ or ‘Reply Needed,’ though, as these subject lines might send your email straight to spam. You want the recipient to get and read your message. Check out this handy free tool that highlights phrases or words in your email that might trigger a recipient’s spam filter. An appropriate greeting/salutation. Every business email should start with an appropriate greeting. When you are writing to another professional—even to someone you may already know—be friendly and respectful, while not overly casual or laid back. Starting the email with a simple ‘Hi,’ followed by the person’s name sets a friendly tone, but does not sound stiff or too formal. When you are addressing someone by their first name, be sure that you have the correct spelling and are not using any nick-names (unless you have previously been directed to do so). The last thing you want to do is start your email off by offending someone. The Body. Try to keep the text of your email short and to the point. When possible, one or two paragraphs of one to two sentences each is best. Your email should have a clear introduction that states the purpose of the email and a conclusion that is forward leaning. Also, checking that your text is spam proof is important here as well. Your signature. At a minimum, your signature block should include your full name, title, the company name, and your contact information, including a phone number. This will make it easy for your recipient to identify who you are, what your position is, and how they can best contact you if needed. The Friendly Part So, how do you write business emails that are friendly but not too casual? Recall the tips above on appropriate greetings and tone, and then consider the real world experience of Brittany Cooper, Project Coordinator for The Writers For Hire (TWFH). Brittany, who has years of sales and customer relations to her credit, has mastered the knack of communicating in a professional but friendly way. She deals directly with clients at the beginning and end of each project, and checks in with them along the way. She is often the person clients feel most comfortable talking to if problems crop up. Brittany describes her communication style as â€Å"upbeat† – always upbeat. She confesses to using lots of exclamation points to express â€Å"lots of gratitude;† it’s her way of communicating to clients that TWFH is â€Å"excited to work with them† and â€Å"values the relationship.† Another way to convey a friendly and â€Å"upbeat† tone is to add a personal touch to the beginning or end of your email. Starting out by saying â€Å"I hope you are having a great week,† or signing off with â€Å"Have a fantastic day!† immediately gives your email a positive tone. Brittany also makes it a practice to seek feedback –usually via email on every project, following up to see if everything went smoothly and ask if anything could be done to make the process better. Of course, things don’t always go smoothly. There are times when clients need to be gently reminded of hard and fast approaching deadlines. In those situations, it is important to keep a friendly and non-accusatory tone. Simply reminding them of the approaching deadline, and asking if you can assist in any way, will help ensure that the deadline is made (without making the client feel like they are being scolded or blamed). Even in the situations where a deadline is actually missed, it is important that you avoid using any kind of language that comes off as negative and harsh. For example, instead of saying â€Å"When you missed the deadline, you caused our project to be pushed back,† use something more neutral, such as â€Å"With the missed deadline, the project was pushed back†¦Ã¢â‚¬  and then follow it up with a positive suggestion for how to rectify the situation. And as a rule of thumb, everything you write in an email should be read and re-read before you hit â€Å"send.† Try to read each email from the perspective of your recipient. If anything sounds like it could be possibly taken in an adverse way, then it is safe to assume that it should probably be re-written with a more positive spin. Other Helpful Tips to Keep in Mind Use traditional fonts such as Arial, Calibri, and Times New Roman; these fonts are not only classic, they are easy to read. Stick to the color black, use 10-12 pt., and keep the font and size consistent throughout the email, including in the signature block. As much as you may enjoy playing around with different fonts and colors, business emails are just not the place to get creative. Never write when upset. An angry screed defeats the purpose of conveying important information or soliciting a desired response. Chances are high that your anger will come across in the words you choose, and the recipient of your email will be able to sense your agitated tone. Be cautious when using language such as â€Å"but† and â€Å"unfortunately.† Adding those words to your sentence tends to negate what is said in the first place, and can also come off as being condescending. â€Å"I apologize for the delay. I will have the completed document to you by the end of the day† will be received much better than â€Å"I apologize for the delay, but it couldn’t be avoided. Unfortunately, I can’t get the document to you until the end of the day.† Spell check but also proofread. Spell checkers are our friends, but they can be fickle at times. Pay close attention to each suggestion you click on and re-read the entire text after you spell check. It’s hard to be taken seriously when you send an email full of spelling and punctuation errors. Think twice before hitting ‘reply all.’ The sender may have wanted a dozen or more people to see their email to you, but all recipients do not necessarily need to see your reply. Consider carefully. Is your reply important for everyone to read? Is the content of your reply appropriate for everyone to see? Don’t use humor unless you know the recipient well – make that very well. What may be funny in a social setting among business acquaintances might not come across as funny in writing. Written communication is easy to mis-construe, so it’s always best to just focus on the point and leave humor for in-person conversations. Don’t overuse the word â€Å"please.† This does not mean that you shouldn’t be polite and write please when it is appropriate. You should just make sure to reserve it for places where it sounds natural. Saying â€Å"Please find the attached document and let me know if you have any questions† does not sound natural, and frankly makes for an awkward sentence. Instead, consider saying â€Å"I have attached the document. Please let me know if you have any questions.† Be sensitive to cultural differences. In some cultures, it may be considered rude to address someone by their first name. In others, discussing certain topics over email may be offensive. Before sending an email, do some homework on what may and may not be appropriate for your recipient. Finally, remember that every email leaves a trace. Don’t write anything that will reflect badly on you or others should your exchange become public.

Decolonization Abandonment essays

Decolonization Abandonment essays De-colonization began with the British colonists in the United States who declared independence in 1776. Most of Latin America gained independence a few decades later. De-colonization continued through the mid-1970s, mostly in Asia and Africa, until almost no European colonies remained. Most of the newly independent states have faced tremendous challenges and difficulties in the post-colonial era. The stability and harmony of de-colonized countries are not guaranteed once the countries are left to the hands of indigenous people. Colonies were flourishing under the colonial administrative government which creates bureaucratic, legislative and educative filters that guarantees indigenous interest. Through law, politics, policy and culture brought by colonizers, the indigenous reproduce themselves while legitimizing and rectifying their origins. Along with an adequate and appropriate government system, both colonial and indigenous realities can contribute to the future shape and nature of the nation state. Economic investment by the administrating power directly benefits the inhabitants of a given region (Guarini n.p.). Suitable structures in the field of credit and savings have been set up successfully in colonies and this has managed to organize and support the production of goods necessary to the economic equilibrium of the region. By meeting the demands of the people, a higher standard of living is achieved. All citizens benefit from security arrangements when the territories host colonial security forces (Plunkett n.p.). It is guaranteed that the colony will be protected by the armed forces available if there ever is a case of war. This is possible as every state has a right and obligation to defend its colonies (Cunningham n.p.). The people of a majority of the territories no longer view, if they ever did, the activities of foreign econom...

Is the nature of crime in our society accurately presented by the Essay

Is the nature of crime in our society accurately presented by the media Discuss - Essay Example This has meant that media has been unable to showcase success within the crime regimes and has not been able to showcase its true face at exposing crime and the criminals who are at large on most of the occasions (Weeks, 2011). The most interesting aspect of properly understanding the nuances related with crime and its propagation within any society of the world and especially within Australia is made when people are trying to get the hang of the criminal elements which are plaguing the people and the society in essence. What is required is a vision to set the good and the bad apart from one another so that both virtue and vice have their respective positions within the societal domains. The people who make media such a huge phenomenon are indeed the ones who have to decide what is deemed as right and what can be judged as wrong without any doubt. These include the anchors, the media persons, the talk show hosts, the reporters, the news analysts and so on. The need is to realize that this huge phenomenon must not be blown out of proportion rather the emphasis should be kept on bringing sanity within the related ranks (Thorn, 2008). It will assure that the media stays on track and the criminal element prese nt within the Australian society is checked at all times. One of the most significant reasons why crime is being propagated lies solely in the fact that the criminals are being allowed to do just that. The media is playing its negative role on most of the occasions, and this is earmarked as something that is proving to be a serious problem if seen within the related settings. How crime gets the back seat is important for the society’s different dimensions and this must be brought to the fore none other than the media alone. The element of being responsive in terms of reporting, finding out facts from just about everywhere and directly reporting with

Media Advertising Criticism Essay Example | Topics and Well Written Essays - 2500 words

Media Advertising Criticism - Essay Example The major role of advertising is to increase sale of products and services, in addition to creating and maintaining the brand identity and image. It also entails communicating the change that exists in the product line and introducing a new product or service (Bartels 46). It is also viewed as a favorable representation of products to make customers, consumers and the public aware of the existent products. In other words, it lets potential users, buyers, and the public as a whole become familiar with the various brands of products, goods and services found in the market. Advertising has faced various criticisms regarding the content, ethics, privacy and the cost of the adverts. In this essay, we are going to discuss the reasons why advertising is essential irrespective of the criticisms that have been lodged against it (Beckman 70). Additionally, we shall examine in details some of the issues that bring about the criticisms of advertising. For instance, that it does not protect other people’s privacy and that it lays emphasis on inaccurate or inappropriate content. Beckman brings out the fact that media organizations at times misinterpret and withhold relevant facts because they are subverted by the advertiser’s demands. ... They believe that tobacco adverts may convince the younger generation that smoking is cool, yet is not. In some countries such as Canada, Europe, South Africa and New Zealand, the advertising structure operates in a system of self-regulation where advertisers, media and advertising agencies agree on the code of advertising standards that all stakeholders try to uphold. This aims at ensuring that the advertising is decent, legal, truthful and honest (Beckman 89). Thousands of policy researchers, opinion makers and politicians often wish to transmit information to the larger public. In order to do that successfully, they need a medium of communication. Therefore, media organizations always serve as the intermediaries of conveying such messages. Transmission of information and news to the public is extremely expensive, and this has prompted media organizations to significantly depend on advertising in order to cover some of their costs. There are few organizations and corporations that spend heavily on adverts, and this has compelled media agencies to accept advert orders from them irrespective of whether they violate the media ethics or not (Bartels 100). Advertising in its essential nature boldly appeals to the self-interest of customers for the patent and selfish gain of the capitalists. Therefore, criticizing advertising is to criticize capitalism and ethical egoism (Albion 48). Anderson makes us understand that, in the psychological point of view, people are in control of their mind; thus, they cannot be manipulated unless they allow it. There are three facts that uphold this statement. First, reason is volitional, a fact that negates determinism; thus, removes the support for the view that advertising possesses

ABBREVIATION ERRORS Assignment Example | Topics and Well Written Essays - 500 words

ABBREVIATION ERRORS - Assignment Example d that the Institute of Medicine reported that more than 7,000 deaths a year are due to medication errors which was published in their 1999 review To Err is Human (Landers ). Due to this disturbing report, a campaign was launched in 2006 by FDA and IMP to reduce errors, wherein one of the main thrusts of that campaign was to eliminate some error-prone notations like U and IU ( FDA press release 2006 ) The following three abbreviations chosen that were prone to error are the following : BT ( bedtime ),q1d ( once a daily), and u ( for units ). BT is commonly mistaken as BID ( twice daily ) which can be lethal when the medication can cause extreme reactions to the patient when an over dose is given. Another mistake would be q1d which can be thought of as 4 times daily since it closely resembles qid .;thus, administering a dosage four times the original dosage can have produce other unwanted symptoms such as allergy or even shock that can complete the doctor’s treatment. The third abbreviation is u ( units ) which is commonly mistaken 0 or 4 thereby increasing the dosage as much as ten times. Consequently, the result would be an overdose that would stress out the liver or kidneys of the patient which can cause serious complications or even death. The Purpose of the safety goals is to promote safety when using abbreviations in prescribing. The Goals main focus is clarity in written communication that will result to correct administration of

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