Business Plan: Anti-bullying dance event Essay

Business concept Discussions initially took place within the team to find the best possible project we would enjoy planning and eventually to execute. After a number of meetings we decided on a dance event and need to identify how that could benefit the student community. We want to promote health, and upon further discussions we thought of the idea to link anti-bullying. Once we were decided on our event we decided the best place to start was with the Student Union and Student Services to identify what is currently done at the university to provide awareness/information to students and lecturers. Whilst there is a place for students to seek advice when they are being bullied none of us where initially aware of this resource, and this seemed the perfect opportunity to raise awareness to benefit the university community. Discussions with Anwar Azari (Support Services Advice and Representation Manager) and Yasmin Bastow (Vice President of Academic Support and Campaigns) took place who were both keen to assist us with the event. This also led us to Peter Lovatt (Psychologist, Dancer and Lecturer at the University of Hertfordshire) and Marcella Wright (Head of Equality) Objectives 1. Raise awareness of anti-bullying 2. To provide students the opportunity to learn about anti-bullying 3. Create awareness of the University of Hertfordshire’s zero tolerance of harassment and bullying 4. A dance event with different tutorials and a short presentation providing students and lecturers from the University to increase their knowledge on bullying, but do something fun whilst learning through linking dancing and anti-bullying. Market Research Approximately 70% of young people experience bulling (Cyber Mentors, 2011). According to Anti-bulling network (2011) bullying can occur at any time regardless of age. At the University of Hertfordshire there is a zero tolerance of harassment and bullying policy (University of Hertfordshire, 2011). Bullying UK (2011) identify that examples of bullying include name calling, damaging someone else’s possession with intent, spreading rumours, threats and intimidation. At least 20 children each year commit suicide because they are being bullied (Cyber Mentors, 2011). Primary research has been carried out to examine the demographics of students and lecturers at the University of Hertfordshire in terms of age and gender, their awareness of anti-bullying and if they would participate in our event and how much they would be capable of paying to join in. (See appendix 1 for the questionnaire and results of the research). Furthermore Dr Peter Lovatt; Psychologist, Dancer and Lecturer at the University of Hertfordshire, uses scientific research to investigate the relationship between dance and health, dance & self-esteem, dance & thinking, dance & hormones, dance & emotion recognition. He works with choreographers to create new dance works which have a psychological basis (Lovatt, 2011) In honour of International Anti-Bullying Day, two schools, David Lloyd George Elementary and Churchill’s Secondary came together to create a message about acceptance and challenge through a flashmob dance in January 2011 (MrDarrenj88, 2011) The customer groups we will be targeting are: 1. University of Hertfordshire students at Hatfield campus 2. University of Hertfordshire lecturers at Hatfield campus Risks we have identified: 1. Breakages to equipment loaned to us for the event such as the projector or sound equipment 2. Health and safety of students, lecturers and the event organisers leading up to the event and during the event 3. Weather impacting on people travelling to the event 4. No one showing up/limited interest from students and lecturers 5. Unavailable venue or equipment 6. Dance societies not being available to assist with our event Reward we have identified from the event: 1. Community awareness of bullying issues and the effects that it can have on other people 2. Creating an atmosphere for conducive learning Measure success of the event 1. Individuals from the University of Hertfordshire take part in our event 2. To ensure that they are not just taking part for ‘free’ we will also ask for feedback to assess their knowledge of bullying straight after the event. Promotion and advertising The key target market for this event is university community; students and lecturers. We plan to promote the event through a number of marketing vehicles which include: Within the university; TV screens in restaurants, notice boards, social media sites linked to the University, Universe newspaper, support from Yasmin and her team in the Student Union, support from Anwar and his team in Students Support Services, and work with the various Schools at the University to provide awareness of the event. Additionally to ensure we provide direct awareness and opportunity for students to ask questions about the event we will distribute flyers to promote the event two weeks prior to our event. Financial cost of anti-bulling dance event The cost for the event has remained zero following discussions and negotiations with the many people we have met with. A summary of the resources required for the event and how they have effectively been funded is specified below: Resource How fund ed 1. Dancers 1. Student Society and in-kind Zumba teacher 1. Audio equipment 1. Media Students 1. Projector 1. Student Union 1. Posters 1. Student Support Services 1. Printing 1. Student Support Services 1. Speakers about bullying 1. Head of Equality 1. Venue 1. Open space in the Forum 1. 1. 1. 1. ________________ References Anti-bulling network (2011) Information. Available from: http://www.antibullying.net/communitymoreinformation.htm [Accessed 1st November 2011] Bullying UK (2011) Anti-bullying Advice. Available from: http://www.bullying.co.uk/advice/anti-bullying-advice [Accessed 1st November 2011] Cyber Mentors (2011) How many people are affected by bullying? Available from: http://cybermentors.org.uk/index.php?option=com_content&task=view&id=19&Itemid=40 [Accessed 1st November 2011] MrDarrenj88, 2011. Anti-Bullying Flashmob January 2011. Available from: http://www.youtube.com/watch?v=MhYyAa0VnyY [Accessed 22nd October 2011] Lovatt, P. (2011) Psychologist & Dancer. Available from http://dancedrdance.com/default.aspx [Accessed 22nd October 2011] University of Hertfordshire (2011) Student Guide to A Safe and Secure Environment. University of Hertfordshire.

Monopoly and American Dream

Monopoly: Reinforcement of the American Dream Many board games are used to bring in family, friends, and even strangers to come together and socialize. What many people do not know is that sometimes these games teaches our society the values, skills, and social statuses in each individual’s life. Video games such as Medal of Honor or Call of Duty teach young teens (even children), the American pride of being a soldier. Board games such as Life teaches individuals about life in general or what is expected by society when children move on to be adults (go to college, have a job, have kids, get married).I’ve decided to examine the Monopoly board game, where it teaches a variety of values, skills, and social inequalities. Some good aspects about the Monopoly game are the teachings of real life accounting. A player learns how to budget their money and makes decisions on what to spend. There are even taxes, such as luxury tax and income tax. Mastilak (2012) states that  "Monopoly involves investing money into a financial enterprise, developing a strategy, making investment decisions, paying expenses, collecting revenues, and competing with other similar enterprises. † Monopoly teaches individuals the value of the American Dream.It is supposedly said that everyone starts off in the same social conditions and everyone has equal chances to climb the social classes. In the game, every player starts off with the same amount of money. In life, everyone is born with the same opportunities among your peers. For example, individuals born in a low social class have the same amount of chances to reach the higher social statuses. To reach a high social class, individuals have to invest themselves in the American dream, so that one-day individuals will own a house, have money, have luxurious items, and â€Å"live happily. The Monopoly game incorporates the American dream elements into the game. The paper money obviously represents money, the houses and h otels represents real estate; luxuries are included in the game as well, such as a jet plane, a limo, a yacht, and a bullet train. Even household luxuries such as water and electricity are included. The game is based on competition; the winner is clearly the wealthiest. The game represents corporate culture, where the game is about winners and losers, it’s about greed and it’s about being heartless. Players are suppose to use every way to get their wants, even if it means hurting their family and friends.For example, if a player lands on another player’s spot, the player has to find a way to pay for landing in the spot, even if it means that the player doesn’t have the money for it. Monopoly also teaches players the rules of social engagement. Taking turns, following the rules, and fair play are general norms of social engagement (Glasberg, Nangle, Maatita, and Schauer 1998). Glasberg, Nangle, Maatita, and Schauer also bring out a good observation when pl ayers noticed the political socialization. They stated that since unknown players made up these games, the players did not debate or negotiate the rules.What I’ve noticed about the game is that the square that says â€Å"In Jail†, â€Å"Just Visiting†, the price of the estates are relatively cheap. This reinforces the idea that people who are in jail are most likely people from bad neighborhoods. It can also mean that the estates are cheap because it’s next to a jail and it brings down the value of the estate. While on the other hand, the estates near the â€Å"Go To Jail† square are significantly higher on prices. The way I interpreted this is that higher security is placed among valuable estates. It’s like in life; people with the money can afford to buy security cameras or high security equipments.Or it can mean that higher securities are placed around rich neighborhoods. Another square, called the â€Å"Free Parking† represents lu ck. The way I interpreted the â€Å"Free Parking† spot is the chances of winning the lottery, or in the case of the game it is to take all the money piled in the middle of the board game. There are many Americans who play the lottery, hoping to beat against the odds to win a vast amount of money. If the game were to be played different, for example some players start of wealthy while other players start off poor, the real life application may be accurate.A professor from Pennsylvania State University tested 50 students with the poor and rich elements. As suspected, the rich gets richer and the poor gets poorer. According to the article Classroom Monopoly Game Shows Rich Often Get Richer, 20% of the people control 40% of the wealth and 20% splits 1%. The remainder divides the middle 59% in the United States. It’s just really interesting that how easy it is for players to adjust to the power of money and how accurate it is sometimes. For example, it was very interesting that a student stole $100 from a neighbor because the student was poor.That’s how it is in some places; people have no other choice but to steal for survival. Monopoly does have many useful skills such as accounting and money investments. However, the board game does reinforce American values, it incorporates the lemets of the American Dream and the corporate culture as well. Even if the game is played differently, these values remain the same and some players even become greedy and heartless (do anything to win, even if it means hurting friends or family). It’s very interesting, it taught me new ideas, and made me think of games that they are not always as it seems.Works Cited 1. Classroom monopoly game shows rich often get richer. (1992, Feb 22). Journal Record. Retrieved from www. search. proquest. com 2. Glasberg, D. , Nangle, B. , Maatita, F. , & Schauer, T. (1998, Apr). Games children play: an exercise illustrating agents of socialization. Teaching Sociology, 26( 2), 130-139. Retrieved from www. jstor. org 3. Mastilak, C. (2012): First-Day Strategies for Millennial Students in Introductory Accounting Courses: It's All Fun and Games Until Something Gets Learned, Journal of Education for Business, 87(1), 48-51. Retrieved from www. ebscohost. com

Baldrige Criterion

To many people, strategic planning is something meant only for big businesses, but it is equally applicable to any type of business entity or organization. Strategic planning is matching the strengths of an organization to available opportunities. To do this effectively, an organization need to collect, screen, and analyze information about its environment. The organization also needs to have a clear understanding of its strengths and weaknesses and develop a clear mission, goals, and objectives (Wikipedia, n. . ). Acquiring this understanding often involves more work than expected. The organization must realistically assess its current state and device a plan of action to successfully make it better. So how does an organization gauge how well they are doing in term of matching their strengths to available opportunities? A self-assessment using the Baldrige Criteria for Performance Excellence can help an organization achieve high performance and move toward performance excellence (Balbridge. om). Even if the organization isn’t ready to apply for the Malcolm Baldrige National Quality Award, the Baldrige criteria are a framework for evaluating any organization’s processes, their impact on results, and its progress toward goals and objectives. The Baldrige criterion consists of seven key categories/indicators of success. One of these categories is strategic planning and it contains ten questions that are not routinely asked on how an organization can function more efficiently. In the case of University California, Berkeley’s and University of Colorado, Boulder’s campus-wide IT strategic plan, this paper will address some of these questions as they relate to Balbridge’s criteria for assessing strategic planning. The questions I will cover are: describe how the organization sets its strategic objectives into action plans, what the organization’s action plans are, and how the organization is able to project future performance on these key performance indicators or measures. In addition, this paper will also describe the following: how each university used the strategic planning process to address their needs, what are the university’s current strategic objectives, and the goals for each objective and the timetable for achieving these objectives. First of all, one of Baldridge’s criteria in strategic planning is answering the question of the organization’s current strategic objectives, the goals for each, and the timetable for achieving them. The following are the objectives for UC Basic IT resources that are adequately supported and refreshed in order to carry out their research, teaching and learning, and administrative work. 2. Seamless, integrated, immediate, and continuous self-service access to information and services. 3. Robust technology tools to support collaboration. 4. Access to tools and data/information that enable community members to develop their own integrated solutions. The UC-Bolder defined their strategic objectives as the following: 1. Universally available wireless network including all campus buildings and strategic open common spaces as well as access to a campus VPN. 2. Faculty purchase and renewal program allowing all faculties a significant subsidy for a new computer every several years. 3. Free antivirus and encryption to protect data as well as access to a variety of major software licenses. 4. Integrated email, calendaring, and scheduling (Exchange). 5. Accessible and multi-layered IT support including both centralized and dedicated IT personnel. 6. Classroom and online IT training. Although I did not go in depth about their objectives and timetables, clearly in each report, both universities exclusively defined what their IT strategic plans and objectives are for their future success. Charles McNamara (n. d. ), a leading strategic planning advisor, stated that goals should be designed and worded as much as possible to be specific, measurable, acceptable to those working to achieve the goals, realistic, timely, extending the capabilities of those working to achieve the goals, and rewarding to them, as well. By clearly defining what their goals and needs are, both universities mirrored the Baldrige criteria for strategic planning. Secondly, one of the ten questions asked in Baldridge criteria for strategic planning is how do you ensure that financial, human, and other resources are available to support the accomplishment of your action plans? In other words, how do the universities convert their strategic objectives into action plans through resource allocations? UC Boulder satisfied that question by having approximately 300 employees in the Information Technology Services. In addition, UC Boulder has also clearly defined how they will allocate their IT resources to ensure they meet their strategic goals. According to the website UC Boulder’s IT allocation is as follows: 1. Campus programs and projects (28%) 2. Academic technologies and spaces (25%) 3. Support, operations, and services (including network and telephony) (42%) 4. Administration amp; support (5%) With UC Berkeley, however, it paints a different picture. UC Berkeley did have an IT allocation but they used the generic term â€Å"resources† throughout their strategic plan and were not at all defined as compared to UC Boulder. Their means of resource support simply stated: â€Å"Researchers and research support staff across disciplines require a minimum level of research support with technical compatibility to facilitate research and the sharing of data, and to avoid significant reinvestment and training for each new research initiative. † By far UC Berkeley does not meet Baldridge criteria by not specifically allocating their resources. With goals and objectives clearly defined by both universities, a plan of action must take place. According to McNamara (n. d. ), action planning is carefully laying out how the strategic goals will be accomplished. Action planning often includes specifying objectives, or specific results, with each strategic goal. Therefore, reaching a strategic goal typically involves accomplishing a set of objectives along the way in that sense, an objective is still a goal, but on a smaller scale. Often, each objective is associated with a tactic, which is one of the methods needed to reach an objective. Therefore, implementing a strategy typically involves implementing a set of tactics along the way in that sense, a tactic is still a strategy, but on a smaller scale. He added that action planning also includes specifying responsibilities and timelines with each objective, or who needs to do what and by when. It should also include methods to monitor and evaluate the plan, which includes knowing how the organization will know who has done what and by when. With that said each university’s plan needs to address adapting and evolving with new and emerging technology to stay in accordance with the Baldrige criteria. UC Berkeley acknowledges how new technology will affect their strategic plan with the following statement in their plan, â€Å"New and emerging technology solution-building capabilities. They attempt to address new technologies in their 2030 plan, but unlike UC Berkeley, they do not address it specifically. UC Boulder’s attempt at planning for emerging technologies: â€Å"Flagship 2030; not only will advancements in research computing across campus help facilitate growth and excellence in research, the open, collaborative, and flexible spirit in which such advancements are pursued will help ensure research computing resources allow for new approaches to research, scholarly, and creative work, and bolster structural support for research and creative programs across campus. Last but not least, the Baldrige criterion asks the question. â€Å"What are your key performance measures or indicators for tracking the achievement and effectiveness of your action plans? † I have read both the universities report in its entirety but I did not find any mention of how each university would measure the successfulness of their strategic plan. It is kind of odd to me that they didn’t mention any indicators or measurement. Is this a common practice among the IT community to not take into account the importance of measuring the effectiveness of their IT strategies? When discussing measuring for effectiveness of the IT plan at UC Berkeley, Mr. Jack McCredie explains, â€Å"It is much more of a description of an end state that we are working for. We are more goal oriented, not number oriented, in our process. One UC Berkeley goal was to wire the campus, not count the number of nodes that are actually installed. Our board doesn’t seem to require particular dashboard numbers that say we are 38 percent of the way to accomplishing our goal.    Clearly in my opinion, both of the universities failed one area of the Baldridge criteria and are not showing any efforts and thoughts into establishing proper measures of effectiveness into their plans. In conclusion, in comparing the strategic IT plans of UC Berkeley and the University of Colorado at Boulder, similarities and differences become quickly evident. When Baldridge criteria for strategic planning are taken into account, in conjunction with comparing each strategic plan, the variation in d epth of commitment clearly shows. In my opinion, each universities IT strategic plan is not superior over the other as both have faults and missing some key ingredients in successfully attaining IT strategic planning superiority. As Charles McNamara stated, â€Å"A frequent complaint about strategic plans is that they are merely to-do lists of what to accomplish over the next few years. Or, others complain that strategic planning never seems to come in handy when the organization is faced with having to make a difficult, major decision. Or, other complains that strategic planning really doesnt help the organization face the future. These complaints arise because organizations fail to conduct a thorough strategic analysis as part of their strategic planning process. Instead, planners decide to plan only from what they know now. This makes the planning process much less strategic and a lot more guesswork. Strategic analysis is the heart of the strategic planning process and should not be ignored.

Ideology in modern politics Essay Example | Topics and Well Written Essays - 1500 words

Ideology in modern politics - Essay Example Morgenthau (1978) states that collective moral values are not applicable to the actions of a political party and an effort to do so is unrealistic. Realist theory recognises that morality must be filtered through legitimate situations and circumstances occurring within a nation state when attempting to ensure national security and longevity. This has been witnessed in the United States where the collective ideology of liberty and freedom, as mandated by social sentiment, was oppressed by political actors in an effort to depose domestic terrorism from the country after the terrorist attacks in New York’s World Trade Centre. In 2001, the American government launched the Patriot Act which gave political actors more authority to conduct wire-tapping on domestic citizens and engage in observation of citizen activities as a justification for ensuring national security and guaranteeing better safety of patriotic citizens. Circumstances, in this situation, forbade reaching decisions o n how to curb terrorist activities based on fundamental and universal social attitudes related to liberty and freedom. The aforementioned example of the U.S. Patriot Act underpins a sense of nationalism. The United States maintains a strong sense of nationalism and patriotism toward the view that this nation is a hegemonic authority and founded on a significant sense of ethnocentrism. Prior to this recession, in the UK free market-oriented society, if government had provided capital to businesses in this fashion, citizen dissent would likely have been significant.

Language as a Political Instrument Essay Example | Topics and Well Written Essays - 1000 words

Language as a Political Instrument - Essay Example This research will begin with the statement that while the use of language has been in existence since time immemorial, people normally convey various messages using selective language, with some qualifying for political language. Certainly, language can play a pivotal role in relaying political messages especially in situations and environments where the impending messages may spark strong reactions. More often than not, euphemism has been at the center stage of driving political messages whereby one uses a language that appears to be more controversial or less serious to disguise the reality of the situation. In this regard, it is important to note that language the manner in which people use language determines the positivity or negativity of the language especially when it comes to political statements. Similarly, in James Baldwin’s essay, â€Å"If Black English Isn’t a Language, Then Tell Me, What Is?† Baldwin claims that Black English result from political alienation. He frequently compares and contrasts Black English from the ‘right’ English. While some political acts appear so gruesome to be put in plain language, most people who make political statements tend to employ euphemism in their speeches in order to negate the veracity or intensity of the situation they are trying to explain. In this regard, James Baldwin claims that Black English result from political alienation essay in the essay If Black English Isn’t a Language, Then Tell Me, What Is?... Owing to the fact that in most cases, the language a person uses defines who they are, people have the tendency of evolving the language they use in order to evade being submerged in situations that they are not in a position to articulate (Baldwin 653). This is especially so because different people speaking the same language can have different connotations for the same language based on their background and geographical location. Although everyone uses language in order to control and articulate various realities in life, it is worthy to note that language can sometimes transform into a political tool owing to its ability to bring out the identities of various persons and communities. This is especially true when it comes to the French and Britons who have heavy accents and various political undertones attached to their language and therefore identifying a foreigner in such countries is quite an easy task (Baldwin 654). According to Orwell, most people who use political speech do s o in defense of various ugly scenarios, although this often backfires in the end (5). In order for a political language to be effective in its context, euphemism and vagueness need to be injected through various language articulations. A classic example of cases where political languages have used euphemism are the ‘continuance of British rule in India’ and the ‘the dropping of the atom bombs on Japan’. These statements appear less critical in their current context though the situation on the ground at the time of the two major events was quite astonishing, at least according to history books. In essence, the language used by the political class in describing past or present events normally portray a major understatement for example a country

Alan Mathison Turing Essay Example | Topics and Well Written Essays - 2500 words

Alan Mathison Turing - Essay Example Alan Mathison Turing was an English mathematician, logician, and cryptographer. He is often considered to be the father of modern computer science as he has contributed immensely towards the field of computer science through his Turing test. Though not considered a philosopher, he is cited by many modern day philosophers. The Turing Test which is named after him is the most significant contribution he has made in the world of modern computers. Though there are many who has opposed this test, it has undoubtedly a turning point in today's world. In a very short span of life, Alan Turing has provided significantly. This paper describes briefly Alan Turing's Life and his achievement through the Turing Test. This paper also gives a brief overview of the future that this test holds and concludes that the Turing Test has been, and will continue to be, an influential and controversial topic.Alan Mathison Turing was an English mathematician, logician, and cryptographer and he was an original thinker. He is often considered to be the father of modern computer science as he has contributed immensely towards the field of computer science through his Turing Test. Even though Turing never designated himself as a philosopher, his 1950 paper "Computing Machinery and Intelligence" is most frequently cited in modern philosophical literature as it gave a new approach to the traditional mind-body problem, by connecting it to the mathematical concept of computability (Hodges, 2002). Today, it is well known fact that the Turing Test is one of the most discussed topics in artificial intelligence, philosophy of mind, cognitive science, and mechanical science. This paper describes in detail about the life of Alan Turing and also how his Turing test has an influence in the modern science. Besides, in this paper Turing's ideas are discussed in detail and also present the important comments made by many philosophers and others. Turing's test and criticism by different people are also discussed. Finally, the paper concludes looking at the future applications of Turing's test and looks at the current situation and analyzes programs that have been developed with the aim of passing the Turing Test. Alan Turing Life Alan Mathison Turing was born to an upper middle class British family in London, 23 June 1912. He was educated at Sherborne School. While in his school, he was criticized for his handwriting by his teachers. He also struggled at English and mathematics as he was too involved with developing his own ideas to produce solutions to problems using the methods taught by his teachers. From his early years of schooling he has amazed many of his teachers. He tried to always find his own solutions and in spite of producing unconventional answers, Turing did win almost every possible mathematics prize while at Sherborne. This has amazed many of his teachers. The one subject he liked was chemistry. However from a very early age, he carried out experiments following his own plan which did not please his chemistry teacher. As a result of these Turing's headmaster once wrote:- "If he is to stay at Public School, he must aim at becoming educated. If he is to be solely a Scientific Specialist, he is wasting his time at a Public School". In spite of the difficult schooling years, Turing entered King's College, Cambridge, in 1931 to study

The Evolution of the Eukaryotic Cell Essay Example | Topics and Well Written Essays - 2500 words

The Evolution of the Eukaryotic Cell - Essay Example Proponents propose that flagella derived from the symbiotic relationship of a host cell with a parasitic spirochete. A parasitic spirochete attached to surface of the host cell to gain food through the cell membrane, and the host cell gained motility from its whip-like motions. The beneficial relationship between the organisms evolved in the same manner as that of mitochondria and chloroplasts. Serial Endosymbiotic Theory (SET) The endosymbiotic theory related to the primitive origins of the organelles: mitochondria and chloroplasts. According to the endosymbiotic theory, these originated as prokaryotic organisms, which were engulfed by a larger prokaryote through phagocytosis. This larger prokaryote was probably a rickettsia bacterium, which is an anaerobic proteobacteria that was a precursor to the mitochondria organelle. Similarly, chloroplasts come from an autotrophic prokaryote called endosymbiotic cyanobacteria. The theory has in origins in 1905. Konstantin Mereschkowsky with chloroplasts and Ivan Wallin in the 20s advanced a similar idea for mitochondria. Later on, Henry Ris found that they contain DNA. The modern attribution goes to Lynn Margulis for her work in 1981, Symbiosis in Cell Evolution. She contended that eukaryotic cells started as communities of networking bodies such as endosymbiotic spirochetes that developed cilia and flagella. The problem with this is that cilia and flagella do not c ontain DNA. Another organelle, the peroxisome, is thought to have emerged this way. They, too, do not contain DNA, however. Christian de Duve's peroxisome idea did not last long. (Cooper, 2005) Modern evidence that suggests the endosymbiotic theory is viable: Mitochondria and chloroplasts contain DNA, which is... The endosymbiotic theory related to the primitive origins of the organelles: mitochondria and chloroplasts.   According to the endosymbiotic theory, these originated as prokaryotic organisms, which were engulfed by a larger prokaryote through phagocytosis.   This larger prokaryote was probably a rickettsia bacterium, which is an anaerobic proteobacteria that was a precursor to the mitochondria organelle.   Similarly, chloroplasts come from an autotrophic prokaryote called endosymbiotic cyanobacteria.   The theory has in origins in 1905.   Konstantin Mereschkowsky with chloroplasts and Ivan Wallin in the 20s advanced a similar idea for mitochondria.   Later on, Henry Ris found that they contain DNA.   The modern attribution goes to Lynn Margulis for her work in 1981, Symbiosis in Cell Evolution.   She contended that eukaryotic cells started as communities of networking bodies such as endosymbiotic spirochetes that developed cilia and flagella.   Another organelle, th e peroxisome, is thought to have emerged this way.   They, too, do not contain DNA, however.   Modern evidence that suggests the endosymbiotic theory is viable:†¢ Mitochondria and chloroplasts contain DNA, which is different from that of the cell nucleus, and that is similar to that of bacteria (in being circular and in its size).  Ã¢â‚¬ ¢ They are surrounded by two or more membranes, and the innermost of these shows differences in composition compared to the other membranes in the cell.

Personal Skills Assignment Example | Topics and Well Written Essays - 1500 words

Personal Skills - Assignment Example Transferable Skills of Time Management: Transferable skills of time management reflect the way in which an individual can plan all his due projects to be submitted within time. Certain tasks are related to specific objectives and thus need specific scheduling. Using the right technology, materials and facilities for the tasks to be completed at the right time is essential. It is also needed for an individual to be self-dependent doing his own work on time as well as making scheduled plans for his team reflecting effective leadership and management. The progress of the tasks needs to be monitored as part of the scheduling process and one need to be persistent in his performance (Ellis 2012). Thus these are the transferable skills that enable individuals to manage their time effectively in order to complete and deliver all necessary tasks and projects on time. Working With Others: While completing higher education and later on carrying on with the career life, one needs to have certain interpersonal skills, particularly when they have to work along with others either as team or at the workplaces. Skills of communication are extremely essential for this purpose. It is essential to interact effectively, listen to others such that one can be accepted well in the workplace benefitting the timely completion of tasks along with healthy relationships in the workplace with all. Powers of goof communication and negotiation are certain interpersonal skills that one needs to develop in order to be successful within an organization, and such skills are initially developed at the higher education institutes (What are Interpersonal Skills? 2013). Use of ICT and Managing Learning: Information and Communications Technologies (ICT) has become extremely vital in present day learning and education. With ICTs the modern system of learning and education has developed significantly, thereby developing the ways in which individuals can learn and manage their learning. With information technologies, the traditional forms of teaching are available in the classrooms but along with that the benefits include making notes and study structure available online such that students can access them even when they are not in classrooms. Also, since internet has its widespread use in the present day world, with ICTs involved in learning management, students get the facilities and opportunities to learn and utilize the use of internet technology before they enter the professional life. Thus it helps to manage the learning process of the students without affecting the classroom based teaching (Punie, Zinnbauer and Cabrera 2008). Academic Skills: Different academic skills also develop students. Individuals need to learn how to identify and choose the correct information and sources for their learning to be more effective. For this students need to make use of internet sources as well and carry out detailed research to identify the right sources for their learning. This is also a part of the transferable and interpersonal ski

Financial analysis in healthcare Coursework Example | Topics and Well Written Essays - 2500 words

Financial analysis in healthcare - Coursework Example First, there is likely to a shortage of health care facilities. This is because the central government controls the health facilities. With such a low gross domestic income, the country may not be able to set up adequate health facilities to serve the public. WHO recommends that the health facilities should be at least five kilometres from where people live, in this case, this may not be possible making the health care centres inaccessible (Torrance, 2009, p. 34). As a result, the health care in this country can be described as poor Secondly, there is a shortage of health workforce team. Since the government is the sole employer, with the meager resources it is earning, it may fail to employ adequate staff to attend to people. As a result, there will be pooling of people at the hospitals without adequate staff to attend to them. Besides, with this shortage the workers can prefer to work in the private sector where the salary is promising. This will make the few workers at the hospital to be overworked and unable to deliver the services. With the majority of the workers without proper orientation on how to manage the health care finances, they may fail to meet the demands of the government and that of the citizens. Citizens as the consumers of health care suffer from the poor health care. First, they do not have adequate finance to pay for the health care services. This is because first they do not have access to insurance that would have made it easy to pay for the health care bills. For the few who can pay from their pocket in the private market or hospitals, the services are given based on the user fees. This will make the users not to get adequate health. Secondly, the health insurance that is provided by the private sector is not accessible to all. This is because only a few can afford the said insurance either because it is expensive or inaccessible. Besides, there is no mandatory insurance scheme. Without any cost sharing, there is likely to be a

Business Environment Essay Example | Topics and Well Written Essays - 500 words - 3

Business Environment - Essay Example The firm has developed its brand through the high quality food and service that it offers to all its customers. The firm has been able to capture a desirable percentage of market shares in terms of food market and on the other hand Waitrose has also gained its importance in the organic food market and fish market. This British supermarket chain which is partnership firm has an employee base of 91,000 (Randall and Seth, 2011).The organization operates on the concept of centralized decision making. Albam a small high street retailer was founded by Alastair Rae and James Shaw who have proved to be great partners through their decision making approach and strategy formulation. Both of them worked together with all its workers so as to deliver the best services to all its customers and position themselves as a strong brand in the market (Pride, Hughes and Kapoor, 2011).The retail business of Albam is focused towards developing sophisticated and practical menswear that is aligned with the ever changing fashion scenario. The small retailer has extended its branches into two other locations in London. The business ownership structure of this small retail firm is that the owners and staff aim at providing personal service to all its customers. The staffs get feedback from the customers and on that basis they develop their designs so that they are able to retain their customers and create more of timeless fashion (Menipaz and Menipaz, 2011).Thus it can be stated that ownership structu re of Waitrose which is a big firm, is completely different with that of a small firm such as Albam. In case of Albam the focus towards customer loyalty and dealing individually with customers but on the other hand the owners of Waitrose are more aligned towards smooth running of the subsidiary by enhancing their level of service through providing online facilities to all its customers (Heskett, Sasser

Education Essay Example for Free

Education Essay When we think of someone who is well-educated, we typically think of doctors, lawyers and people in those categories. This does not always means a person has to have a fancy degree or title behind their name to be well-educated. Being well-educated can come in many different ways. So what does it mean to be well-educated? In this essay, I will give some examples as to what I feel well-educated means to me, other than having a degree. As I read thru the article by Alfie Kohn about principle leadership on what does it mean to be well-educated; I became very intrigued. Over my 21 years with the US Army, I have had several officers over me who have their degree but still not educated on life in the military. Yes, they went to college and got educated in the books, but when it comes to military life or combat, it’s still a lot to be educated on. There were times when an officer came to me for advice or what to do because he knew I had many years of experience on the job which therefore made me more educated than he. It’s no different from when I first enlisted in the military; there were people over me I had to go to because I wasn’t educated in the job. In Alfie Kohn’s article, he stated â€Å"my wife has a doctoral degree from Harvard but if you ask her what 8 times 7 is, she would freeze up. † The same concept applies with some officers I worked under in my military career. You could go to them about a lot things, and they could give you answer without thinking but it could be something as simple as putting up a frame tent and they wouldn’t have a clue. But if you take a person that has set up a frame tent several times without any hesitation and not ask the question, â€Å"how? † I consider this person well-educated because he has done this task many times. I do believe a person becomes well-educated with time and practice. Just because you go to college and graduated doesn’t necessarily mean you well-educated on the job. For me, being well-educated can mean different things for different people. For instance, take my dad. My dad is 65 years old and retired from his job for as long as I can remember a mechanic. After his retirement, my dad sits in his chair and watches cartoons, old television shows and westerns and tells me stories of his past. My dad never even finished high school but I do consider him to be a well-educated man because of things he has taught me and the way he raised me. My father never had much growing up, so he had to work in the fields and didn’t have much opportunity for education. He had to start work at a very young age as a mechanic once he turn 16; a job he done till he retired at age of 62. By those many years in one profession; even though he doesn’t have a high school diploma nor college degree, I still consider him a well-educated man from his years of experience. You may be able to walk up to my dad and ask him an algebra problem but if you ask him anything dealing with mechanical on a car, there would be no problem. He didn’t have and school education, but he knew how to count and manage his earnings. He also married my mom when he was 18 and done a really good job of raising three kids. I remember growing up and watching my father work on people car out in the yard and I use to wonder what he was doing? I didn’t have a clue of what he was doing but I know the person he whose car he was working on was very satisfied after he was done. He taught me how to be a man and what mistakes not to make from his mistakes. I know he wasn’t good with math but he could really save money and make his money work for us. One thing I never forgot he said, â€Å"always put some money away from every check for that rainy day. † Once I got my first job, I have always done just that; even still to this day. He also taught me the responsibility to always take care of your family first before anything else. It didn’t quite understand then what he was referring to then, but as I got older, I knew he was just educating me on how to be a man that takes care of his family and home. For that I consider my father to be well-educated. I believe if a person just takes the time and be patient at whatever there pursuing, he or she will become well-educated. For some, going to college and receiving a degree and be very difficult, whether is financial or just didn’t score high enough to get into college. I do believe that if we got to college and graduated with a degree, we can get better jobs and be well-educated in the books but maybe not hands on in the field of their choosing. I know once I graduated from high school, going to college was the last thing on my mind and joining the military was always my desire. I’ve always felt like school just wasn’t for me because I had hard time maintaining grades and learning the books. I have always been more of hands on person more than school. I do consider myself well-educated because I spent 21 years in the military and have accomplished all tasks set before me with high standards. In conclusion, I believe that a person doesn’t necessarily have to go to college and get a degree to be considered well-educated. We as people can determine who we feel is well-educated by knowledge and experience as well as schooling; whether it be at junior college, university or technical college. So what does it mean to be well-educated is a question that can be argued for years to come.

The Green Movement Essay Example for Free

The Green Movement Essay Purpose: The purpose of this lab report is to demonstrate how changes in abiotic factor such as changes in precipitation patterns can influence plant populations. Introduction: During this observation I was observe ring the plant species called Kentucky Blue Grass. In this observation I will show how changes in the weather can affect the population of the grass. I will also explain how this may have some effect on Global Warming as well. Hypothesis: My hypothesis of this lab was that if you have more rain the population of the grass will greatly increase. Along with decent to great weather to help the grass grows and become healthy. Methods: There were three different times that I changed the rainy season to last The first experiment the rainy season lasted 150 days for five years, the next season lasted 100 days for five years and the last rainy season as set to last 50 days for five years. Results: My hypothesis was correct to a certain extent. I would think that the cooler the temperature the better the population but it seems that the warner the weather is when the biggest population occur. Discussion/Analysis: During the first observation I changed the rainy season to last 150 days. When the experiment started the temperature outside was 30 degrees and the population of the grass started at 500,000. As the first year went on the population slowly raised but for the most part stayed the same. When you look at the last days of year three, there seems to be a dramatic low point in the population. While looking at the data when the temperature raises to 42.1 degrees the population went down to 54,748. Going into the fourth year the population made another drastic turn and the population rose to 609,747. Getting close to the end of the fifth year the population changed back to its steady pace of 600,000. In the next observation when the rainy season lasted 100 days there was a little bit of difference but not that much. In the first observation the temperature rose to a maximum of 70 degrees and that lasted just one day and during that day the population was at 820,624. When the season lasted 100 years in the experiment the temperature constantly change with much difference. During the first year the temperature stated at 30.2 degrees and the population was at 500,000. In the middle of that year the temperature rose to 71.3 and the population grew to 821,915.If we look at the end of the third year the population took a drastic fall. The population was steady at first but feel from 824,489 to 3,168. The fourth year seemed to be the year that the temperature rose and feel the most but the population was the most consistent

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