Of Mice and Men – Misc Questions

OMAM Essays a) How does Steinbeck use details in this passage to present the bunkhouse and its inhabitants? Steinbeck uses many different ideas to present to present the bunkhouse and its inhabitants. Steinbeck emphasises that the inhabitants have little possessions by commenting about the â€Å"apple box† forming â€Å"two shelves for the personal belongings of the occupant of the bunk†. As all the occupants would be itinerant workers, which meant that they had a nomadic lifestyle, this also highlights that the occupants couldn’t afford possessions and probably didn’t have a lot of room for them anyway.This indicates that they had little home comfort and it was probably quite an uncomfortable, stark and hostile environment they lived in. This lack of familiarity or care is also further portrayed by the description of the bunkhouse itself. The walls are described as â€Å"whitewashed† and the floor is â€Å"unpainted†. This could indicate tha t the owners of the bunkhouse really don’t care about the inhabitants. However, this may not be because the owners dislike the inhabitants, more probably because the itinerant workers don’t stay around for long enough for the workers and the boss to have a proper relationship.This reiterates the point that â€Å"maybe everyone in the whole damn world is scared of each other† and the society they live in is truly a backstabbing and hostile environment. We even know that Crooks, who is a long time inhabitant of the bunkhouse, isn’t treated fairly and much more like an animal because he lives in the barn and he has little possessions. This also shows how cruel their environment is. Despite all of this – at least the inhabitants still have pride.This is demonstrated by George’s disgust when he finds a yellow can of pest killer next to his bed, indicating that the bed may be infested. Clearly, George was not expecting there to be pests in his bed which could indicate that he is a cynical man and has been hardened by his surroundings. This could also emphasise how out of place Lennie is. His docile approach just doesn’t fit in with his life. b) In the rest of the novel, how does Steinbeck present the lives of ranch workers at that time? Steinbeck has intentionally designed each character to represent a segregated group of society.Together, all the characters are presented in a microcosm and they all represent something much larger. For example, Crooks represents the prejudice that black people had to put up with and Crooks’ opinion of this treatment is evident throughout the book in an unbiased way, allowing the reader to digest the ideas of Steinbeck. George is one of the most pivotal characters in the book, as he represents the typical itinerant worker, trying to get money wherever they can after the backlash of the wall street crash.Unfortunately, like most workers at the time, they were all very lonely beca use of their nomadic lifestyle. Although George doesn’t appear to be lonely because of his friendship with Lennie, there are subtle indicators that being lonely is George’s fate. For example, George often plays Solitaire which is a game for one person. Steinbeck is highlighting how lonely it would be for the workers to always be working with no stable home. George also says that he â€Å"ain’t got no people† and that people like him who are alone â€Å"get wantin’ to fight all the time. This indicates that life for the workers was hard because all the workers were exactly like George – they were alone and they were a product of an inimical environment. Through George, Steinbeck is also describing how angry and nasty everyone becomes when they are alone. This is demonstrated by Crooks – who was cynical and nasty to Lennie when he showed weakness. Crooks has often proven to be an angry and bitter man, however this is only because of th e way he is treated. As he is black, people don’t talk to him or accept him for what he is.They even go as far as to not let him in the bunkhouse. Early on in the book, when Candy is describing Christmas on the ranch he says â€Å"they even let the nigger in† as if that was an unusual event – which of course for them it was. Through this, Steinbeck is addressing the segregation that blacks lived with in 1930s America. a) How do the details in this passage add to your understanding of George and his relationship with Lennie? In this passage it is clear that Lennie looks up to George

Understand Child Development and Young Person Development

CACHE Level 3 Diploma for the Children and Young People’s Workforce (QCF) Unit Ref: L/601/1693 CYP Core 3. 1: Understand Child Development and Young Person Development Rosanna King Learning Outcome 1: Understand the expected pattern of development for children and young people from birth – 19 years. Assessment Criteria 1. 1: Explain the sequence and rate of each aspect of development from birth – 19 years. Answer to 1. 1: Below I have explained the sequence and rate of each development from birth – 19 years old in great detail. 0-2 Years – Physical Development: The baby lies supine (1 month old) * The baby turns its head towards light and stares at bright and shiny objects (1 month old) * The baby can lift the head briefly from the prone position (1-4 months old) * Legs can kick vigorously both separately and together (1-4 months old) * The baby is beginning to use a palmer grasp and can transfer objects from hand to hand (4-6 months old) * The baby has good head control and is beginning to sit with support (4-6 months old) * The baby can roll from front to back (6-12 months old) The baby is very alert to people and objects (6-12 months old) * The baby will now be mobile, may be crawling, bear walking, bum shuffling and even walking (9-12 months old) * The baby may bounce is rhythm to the sound of music (9-12 months old) * The baby might be able to manage stairs and steps, but will need supervision (1-2 years old) * The baby can build afew bricks and arrange toys on the floor (1-2 years old) * The child can walk confidently and is able to walk without falling (1-2 years old) 0-2 Years – Communication Development: The baby responds to sounds, especially familiar voices (1 month old) * The baby makes eye contact (1 month old) The baby makes no-crying noises, such as cooing and gurgling (1-4 months old) * The baby cries with anger to show they are hungry, tired or need a nappy change (1-4 months old) * The baby begins to l augh and squeal with pleasure (4-6 months old) * The baby becomes more aware of others and start to communicate with them more and more (4-6 months old) * Babies begin to understand words like ‘up’ and ‘down’ raising their arms to be lifted up, using appropriate gestures (6-9 months old) * The baby can follow simple instructions e. . kiss teddy (9-12 months old) * The child begins to talk with words or sign language (1-2 years) * Child starts pointing and taking a real interest in books and enjoys looking at all the pictures and objects (1-2 Years) 0-2 Years – Intellectual and Cognitive: * The baby is sensitive to light (1 month old) The baby’s face, abdomen, hands and feet are very sensitive to touch (1 month old) * The baby recognises differing speech sounds (1-4 months old) * The baby can develop favourite tastes in food and recognise differences by five months (4-6 months old) * The baby prefers complicated things to look at from five to s ix months and enjoys bright lights (4-6 months) * The baby understands signs e. g. he bib means that the food is coming (6-9 months) * From 8 -9 months the baby shows that they know objects exist when they have gone out of sight (6-9 months) * The baby is beginning to develop images (9-12 months) * The baby gives some understanding of daily routine e. g. food, changing and then nap time (9-12 months old) * The child understands the names of objects and can follow a simple instruction (1-2 years old) * The child learns about things through trial and error (1-2 years) 0-2 Years – Social, Emotional and Behavioural: The baby often imitates certain facial expressions (1 month old) * The baby will smile is response to an adult (1-4 months old) * The baby stays awake for longer periods of time (1-4 months) * The baby shows trust and security (4-6 months old) * The baby has recognisable sleep patterns (4-6 months old) * The baby can manage to feed themselves using their fingers (6-9 months) * The baby is more aware of other people’s feelings, for example; they might cry and get sad if they see their brother or sister crying or sad. 6-9 months old) * The baby enjoys songs and action rhymes (9-12 months old) * The baby still likes to be near a familiar adult as appose to strangers (9-12 months old) * The child begins to have a longer memory (1-2 years old) * The child expresses their needs using words and gestures (1-2 years old) 0-2 Years – Moral: * Show joy by smiling, cooing and laughing when fed comfortable or safe. * No understanding of right or wrong starts to understand the word no. * Sensitive to adult approval and disapproval, despite tantrums and bursts of anger. -4 Years – Physical Development: * The child is very mobile and can run safely (2 years old) * The child can draw circles, lines and dots, using preferred hand (2 years old) * The child can jump from a low step (3 years old) * The child can build tall towers of bricks or bl ocks (3 years old) * The child has good spatial awareness (3 years old) 2-4 Years – Communication Development * Children are rapidly becoming competent speakers of the language they experience (2 years old) * The child can follow a simple instruction for example; â€Å"Could you bring me the spoon? ( 2 years old) * The child wants to share songs, dance and have conversations (2 years old) * The child might say â€Å"two times† instead of ‘twice’ and might say â€Å"I go there† instead of ‘I went there’ (3 years old) * The child loves to chat and ask alot of different questions (3 years old) 2-4 Years – Intellectual and Cognitive * The child can hold a crayon and move it up and down (2 years old) * The child talks about an absent object when reminded of it ( 2 years old) * The child pretend plays – often making up stories and characters ( 3 years ld) * The child represents events in drawings, models ect (3 years old) 2-4 Y ears – Social, Emotional and Behavioural: * The child begins to express how they are feeling (2 years old) * The child is learning how to dress themselves (2 years old) * The child is beginning to develop a gender role as they become aware of being male or female (3 years old) * The child makes friends and is interested in making new friends (3 years old) 2-4 Years – Moral: * Beginning to know right from wrong. Related article: Intervention When Development is Not Following the Expected PatternIs more self-controlled and less aggressive. Uses extreme verbal threats such as, â€Å"I'll kill you,† without understanding full implications, wants to be good, but is not yet mature enough to be able to carry out most promises. 4-7 Years – Physical Development: * A sense of balance is developing – the child may be able to walk in a straight line (4 years old) * The child can thread small breads on a lace (4 years old) * The child can play ball games (5 years old) The child has increased agility, muscle coordination and balance (6 years old) * The child can catch a ball thrown from one metre with one hand (7 years old) 4-7 Years – Communication Development: * The child begins to ask alot of where, when, how and why questions (4 years old) * The child talks confidently and with more fluency (5 years old) * The child begins to understand book language and that books have ch aracters (6 years old) * The child begins to realise that different situations require different ways of talking (7 years old) 4-7 Years – Intellectual and Cognitive: At age four, the child usually knows how to count up to 20 (4 years old) * The child can usually write their own name down on a piece of paper (5 years old) * The child includes alot more detail in their drawings (6 years old) * The child begins to establish what is real and what is a fantasy (7 years old) 4-7 Years – Social, Emotional and Behavioural: The child likes to be independent and is strongly self-willed (4 years old) * The child can wash their hands and brush their own teeth unassisted (4 years old) * The child has developed a stable self-concept (5 years old) * The child can begin to hide their feelings, once they learn to control them (6 years old) * The child can take responsibility e. g. in helping younger children (7 years old) 4-7 Years – Moral: * Is interested in being good, but ma y tell lies or blame others for wrongdoings because of intense desire to please and do right.Is very concerned with personal behaviour, particularly as it affects family and friends. 7-12 Years – Physical Development: * The child can ride a bike easily (7 years old) * The child plays energetic sports and games (8 years old) * The child is usually writing with an established style using joined up letters (9 years old) * Children differ is physical maturity. Girls experience puberty earlier than boys do and sometimes girls can be two years ahead of the boys with puberty (10 years old) * The child’s body proportions are becoming more similar to adults (12 years old) 7-12 Years – Communication Development: The child uses and understands complex sentences (7 years old) * The child is increasingly verbal and enjoys making up stories and telling jokes (8 years old) * The child uses reference books with increasing skill (9 years old) * The child can write fairly lengthy essays (11 years old) * The child starts to write stories that show alot of imagination (12 years old) 7-12 Years – Intellectual and Cognitive: The child has an increased ability to remember and pay attention, speak and express different ideas (7 years old) * The child is learning to plan ahead and evaluate what they do (8 years old) * The child enjoys tasks that are task-orientated, such as sewing and woodwork (9 years old) * The child begins to notice and understand the motives behind the actions of another (10 years old) * The child begins to devise memory strategies (11 years old) * The child starts thinking about different possibilities (12 years old) 7-12 Years – Social, Emotional and Behavioural: The child may become discouraged easily (7 years old) * The child takes pride in their competence (8 years old) * The child can become argumentative and bossy at times (9 years old) * The child is beginning to see things from another child’s point of view (10 ye ars old) * The child may be experiencing sudden, dramatic and emotional changes associated with puberty (11 years old) * The child succumbs to peer pressure more readily and wants to talk, dress and act just like their friends (12 years old) 7-12 Years – Moral: May experience guilt and shame. Has difficulty admitting mistakes but is becoming more capable of accepting failures and mistakes and taking responsibility for them. Is aware of right and wrong; wants to do right. 12-19 Years – Physical Development: * Physical development during adolescence is known as puberty. Age of puberty varies but is often between the ages of 9-13 years old for girls and 10-15 years old for boys. * Girls will experience the following during puberty; breasts develop, body size and shape will change and menstruation. Boys will experience the following during puberty; voice breaking, body size and shape will change, chest hair, penile errections and sperm. * Both girls and boys will experienc e the following during puberty; public hair, excess sweating and oil-secreting glands. 12-19 Years – Communication Development: * Become more independent and rely less on parents or carers * The young person has fast, legible style of handwriting * The young person communicates very well in an adult manner, with increasing maturity * The young person understands abstract language, such as idioms, figurative language and metaphors. 2-19 Years – Intellectual and Cognitive: * Around this time young people experience a shift in thinking from concrete to abstract – an adult way of thinking * They approach a problem is a systematic fashion and also use their imagination when solving problems 12-19 Years – Social, Emotional and Behavioural: * The young person may become self-conscious about physical changes their body is going through (e. g. too short, too tall, too fat, too thin) * The young person often feels misunderstood * The young person can experience a w ide range of emotions and sometimes have mood swings (e. . happy one minute and very down the next minute) * The young person wants to become accepted and liked 12-19 Years – Moral: * Knows right and wrong; tries to weigh alternatives and arrive at decisions alone. Is concerned about fair treatment of others; is usually reasonably thoughtful; is unlikely to lie. Experiences feelings of frustration, anger, sorrow, and isolation. Is confused and disappointed, state values and actual behaviours of family and friends; May be interested in exploring physical-emotional urges.

Apple Inc. Increasing Corporate Market Share Research Paper

Apple Inc. Increasing Corporate Market Share - Research Paper Example This is due to the increasing market competition of its business rivals. Within the global mobile market, Apple has only 1% market share while its computer market enjoys a 5% market share (Cusumano 23). Additionally, the company’s TV features are relatively unpopular. This paper presents a report of an analytical analysis of Apple Inc. with a view of illustrating how the company would achieve a greater corporate market share for its PC and non-PC products and services. The scope of the report will include analysis of the needs of the company’s customers, strategies through which the company will increase corporate market share and its competitiveness. Both Qualitative and Quantitative research methodologies are applied in collection of quantifiable and qualitative data for analysis and interpretation. The collection of data employs secondary research design in which findings for the company investigation are obtained from credible secondary information sources such as p eer reviewed journal articles, online databases and books. Data Analysis and Discussion The needs of Apple Inc.’s customers are quality of products and services. The company’s PC systems are expected to be of superior quality which includes creative and innovative design of hardware architecture and software platforms. Apple Inc. is faced with a challenge of maintaining its core success factors and competencies which include innovativeness, creativity, marketing, brand management and building relationships within the organization and with its customers (Casacchia 59). This scenario illustrates that the needs of the company’s customers are not being met effectively. Furthermore, the needs of the company’s customers include affordable prices for its OC and non-PC products. It has been revealed that Apple Inc. is experiencing difficulty in overcoming business rivals who provide low-priced products to the mobile and computer markets (Fontevecchia 6). The mobi le products from Google for example are posing a threat to the company’s success due to the low prices that the business rival provides for the customers. Additionally, the computer market is characterized by competitive prices from Dell and Microsoft both for hardware and software (Gelles, Chris and Richard 15). These findings demonstrate that Apple Inc. is faced with a challenge of satisfying the product and service cost effectiveness that its customers require. The strategies which Apple Inc. has employed in order to increase the market share for its products include marketing, strategic alliances, customer relations, creativity and innovativeness, provision of variety of products and services and the iPod platform. Within the digital music market, Apple’s iPod has enjoyed 70% of the market share as opposed to its closest competitor which has only 8% of the market share. Nonetheless, the company’s strategy for providing variety of products to its consumers ha s been faced by threats of new entrants to the market and substitutes for its computer and mobile products (Seitz 1). These threats have reduced the value that the customers have for the company. Additionally, the company’s customer base is increasingly becoming more diverse (Mallin and Finkle 52). New entrants in the market have also implemented new strategies and strengths in marketing which are posing new threats to the company’

Hot Potatos Essay Example | Topics and Well Written Essays - 500 words

Hot Potatos - Essay Example The chief analyst raised three main issues in the company’s management. A brief summary is given below: He said that the company is facing a tough competition and the main issue is that the competitors are using advanced technology now available in the market. This advanced technology enables the other companies to increase their sales through immediate advertisements and through the technology and receiving instant feedback enabling them to take prompt measures to enhance product quality and policy making of their companies. This advanced technology also ensures production at a comparatively low cost which results in their ability to introduce their products at fairly low prices in the market. This comparatively reduces the sales of the company as customers are always attracted towards low prices for same products in the market. Secondly, the competitors have employed some extra labor skilled in proper use of the company’s software and websites. These are IT experts properly trained in making quick decisions to meet demand in software changes and also quick production of its products. The company needs to ensure that the company makes sufficient advancements in its software in order to cope with the demands requirements of the customers. It is always very important for a company to meet the orders placed by customers in time as it is quite obvious that sales orders if maintained poorly and not responded to properly will make the company lose existing customers looking as well as new customers attracted by the quality of our products (Anthony Tarantino, 2006, p. 248). After the chief analysts’ report, the CEO decided to take some measures to overcome the problems that may be faced by the company in the future. He assigned the CFO of the company to determine the total costs of introducing new technology to the production sector of the company as well as the costs to hire skilled labor in order to operate that machinery. The decision to

Multiple Intelligences Essay Example | Topics and Well Written Essays - 2000 words

Multiple Intelligences - Essay Example Multiple intelligences developed by Howard Gardner have a great impact on education process explaining the ways of thinking, problem-solving and logic. Typically these skills cross the disciplines and include such things as communication, collaboration, information management, and higher-order thinking skills such as problem solving. These types of outcomes cannot be measured by written tests; they require performance measurement. Educators who assess by performance believe that being able "to do" is parallel to saying that a student has really learned something, rather than simply memorized it. The impact and role of intelligences in education was widely discussed in the literature during 1980s. The first attempts to define and explain this process made by Alfred Binet (1900) who tried to create a measure to predict which youngsters would do well in the primary grades of Parisian schools (Kagan and Kagan 1998). In the mid-1980s, Howard Gardner challenged the belief that IQ was fixed with his work at Harvard University, which was explained in his book Frames of the Mind (1985). He hoped to see society move from testing people to growing people, by focusing on the diverse ways people develop skills important to their lives. He redefined intelligence as the ability to solve problems and fashion products that are valued in a culture or community. His research showed intelligence as more complex, more diverse, and less fixed than originally thought. Garner (1985, 1997) and Sternberg (1985) have argued for specific, multiple domains of intelligence. Today, intelligence is being more broadly conceptualized and defined (Kagan and Kagan 1998). At the beginning of the 21st century, researchers applied Gardner's Theory to instructional technology and distance-based education, to different learning strategies and learning environments. For instance, Milheim and Osciak (2001) examine advantages and benefits of multiple intelligences within online learning environment and come to conclusion that it "can provide multiple avenues for learning based on an individual's preferred style regardless of the discipline or the geographic dispersion of the intended learners" (4). Another layer of literature examines practical application of multiple intelligences in different fields including leadership and employees training, physical education and gifted children. For instance, Kernodle and Mitchell (2004) analyze the benefits of multiple intelligences in teaching tennis at the secondary level or in a college. They find that "offering a variety of activities that enhance different intelligences also helps students who are weak in certain intelligences by giving them the opportunity to improve themselves in those areas" (32). Some researchers examine the role of multiple intelligences in development of gifted and talented children (Fasko 2001); identify sex difference in learning process and perception in children (Furnham and Ward 2001). They find that the role of the teacher is acknowledged in this perspective but only in the context of co-constructing meaning for content and skills. Thus, Kagan and Kagan (1998) admit that this is still the realm of procedural thinking. The

The Englishman's Boy by Guy Vanderhaeghe Essay Example | Topics and Well Written Essays - 1000 words

The Englishman's Boy by Guy Vanderhaeghe - Essay Example National Identity is a very important part of our personality. The formation of national identity is a process of comparing and contrasting the values of our own nation with the values, principles and beliefs of other nations. Certainly, not all the people have an opportunity to travel, thus we have got another reliable and valuable source of information that help us form our views and got different feelings. This is an art. We see films and beautiful pictures, read poems and novels created by our ancestors and also modern artists. Studying the masterpieces of art, we receive valuable information about our history and this helps us form our own identity. We can learn about what is good and what is bad from different examples provided by the pieces of art. Art also serves as a reflection of our own feelings and thoughts. However, not all the information we receive is true and it is important to understand what to absorb and what to ignore. When the Great War was over, a new important period started began in the life of America. Actually, this period predetermined the future of the United States and its reputation of the most developed country in the world. It was a period of thriving, the time of economic development. There were many new opportunities opened for Americans that time and it is natural that this made the United States attractive for immigrants. The myth of American Dream appeared and many people from different countries in the world left their motherland to come to the United States seeking for better life. The United States became multicultural that caused many problems for immigrants. Instead of American Dream they faced terrible racial intolerance. Guy Vanderhaeghe tells us the story of Harry Vincent, a young scenarist, and his boss, a rich man Damon Chance, who wanted to create a special movie. The main purpose of the Damon Chance is to create a real â€Å"American† movie, which would reflect n amely American spirit. Chance thinks that the main problem of Americans is the lack of national identity. Here we can trace the attitude of Chance towards immigrants. It seems that he accuses multiculturalism caused by mass immigration of the lack of national identity in The United States. He respects real American nation and is sorry that it does not have its own art. He wants to create a real American masterpiece: â€Å"The Germans gave the world their music. The Romans their architecture. The Greeks their tragedies. We recognize the soul of a people in their art" (Vanderhaeghe 108). Chance is irritated by European’s domination and wants to help form separate American national identity. He criticized Griffith: â€Å"It was pure genius on [Griffith’s] part to advertise [The Birth of a Nation] as fact. Americans are a practical people, they like facts.†¦ You mark my words, Harry, there’ll come a day when the public won’t swallow any of our stories unless they believe them to be real. Everybody wants the real thing, or thinks they do.†¦ Facts are the bread America wants to eat. The poetry of facts is the poetry of the American soul† (Vanderhaeghe 19). Damon paid Harry Vincent for creating a scenario about a history of a cowboy. The book is divided into two parts with parallel plots and this makes the novel unique. The second half tells us about a boy who is travelling to Cypress Hills. This was done by the author to demonstrate the different interpretation of the same story. We have a chance to trace the process of movie making and compare it with the story of a real boy. The reader can see how the both Harry and the Englishman boy try to resist the actions of their common opponent Damon Chance and how this forms the plot of the book. At first, Harry was impacted by Damon Chance’s aspiration to create a movie and was going to do his best to

Quantative methods report Essay Example | Topics and Well Written Essays - 3000 words

Quantative methods report - Essay Example of categorical variables 2. The measures of centre includes arithmetic mean, geometric mean, harmonic mean, median and mode where as the measures of spread are given by range, mean deviation, quartile deviation and standard deviation. The measures of shape are skewness and measures of position is kurtosis. 3. Event: Any activity subjected to experiment is called as an event. For example in tossing of an unbiased coin (experiment) the occurrence of head and tail are events. Since in any unbiased coin either head or tail can occur, they put together in a set is known as sample space. The sample space in a coin tossing experiment is S={H,T}. Similarly the sample space in throwing of a die is S={1,2,3,4,5,6}. Marginal probability is a measure of occurrence of an event keeping the occurrence of the other event as constant in jointly occurring events. The probability of joint occurrence of two events either independent or dependent is p(x,y)=pij where i=1 to m; j=1 to n; when x and y are d iscrete or else f(x,y)=fxy where x and y are both continuous. 4. The return is an expected value for an investment involving normal percentage values whereas the risk is the measure of uncertainity usually having a negative impact on return. The risk as per standard norms is 1 and if the value of risk is below 1 it is considered to be less risky and if the value of risk is above 1, it is considered to be highly risky. Suppose the return and risk involved in an investment is given in the following table as Table 2: Sample table indicating nature of investment Investment nature Stocks Bonds Real Estate Probability for investing 0.4 0.25 0.35 Return % 13% 8% 10% Risk 1.2 0.85 1.25 Note: The total investment is 250,000 (say), we can formulate a strategy to maximize the return based on the risk and return involved. Discrete distribution is concerned with the distribution of a variable which is countable or finite. For example in tossing of a die, the outcome is a discrete random variable and its distribution of the outcomes 1,2,3,4,5 and 6 can be described in the form P(X=x)=pi= where x takes any value 1,2,3,4,5 or 6 whereas a continuous random variable takes any value between a range of values (in an interval); for example if the frequency of arrival of a bus is 30 minutes and if we define the waiting time for a bus as a continuous random variable x, then the distribution of waiting time is given by f(x)= 0?x?30 =0 otherwise. 5. The sampling distribution is a distribution of the sample measures where the sample of size n is drawn out of a population of size N. If any random sample of size n is taken from a normal population of size N, then the sample mean is x and the distribution of sample mean is having expected value ? and variance ?2/n. ie. if the population is normal with mean ? and variance ?, then the sample will be having mean  with E()=? and SD is SE()= . The central limit theorem says that if a sample of size n having values x1, x2, x3....... ,xn follows normal distribution with mean ? and v

Compare and Contrast US Naval Innovation with that of Great Britain Essay

Compare and Contrast US Naval Innovation with that of Great Britain. What is the dichotomy and why - Essay Example From a close look at the navies of the treaties, both the United States and Great Britain navies were subjected to similar agreements that were restrictive of the innovations that they could pursue1. The United States response to the treaties and the agreements is however different from that of Great Britain and this was the result of all the differences observable to date. This paper is aimed at providing a comparison and the contrast between the US naval innovation and that of Great Britain. The paper will be guided by the thesis that United States naval innovations were dependent on logistic and reasoning while those of Great Britain was based on misguided sense of false security and elimination of imminent threat. From the Five Power Treaty that brought together five main powers into a naval agreement. The resolutions made U.S. Navy and the Great Britain navy to take different directions. For the Great Britain and Japan, the fortification clause had a major impact on the naval strategy and designs. For Great Britain, the influence on their innovation was shown by their arms limitations throughout the governments’ successions and policies formulation in relation to the treaties resolutions. Britain, therefore, appeared to focus directly on the benefits of the restrictions on the peaceful existence of the powers. However, United States was more concerned with logistics, their innovations after this treaty stayed relatively the same in the interwar periods. The Treaties of Versailles on its part enhanced further the restrictive force on naval innovation by prohibiting Germany from continued production of the U-Boats which were feared to have been used extensively in World War I2. The restriction on Germany provided a false sense of security to Great Britain making it believe that the treaties were important in eliminating threats. The treaties however failed to notice that if Germany had been

Written Play Reports Essay Example | Topics and Well Written Essays - 1000 words

Written Play Reports - Essay Example George Cuckor's direction is stunningly elaborate and his skilful handling ensures that none of the script's acerbic wit and irony is diminished under the fluff of the musical numbers. The execution of the musical is indeed praiseworthy and a truly befitting tribute to the George Bernard Shaw's classic 'Pygmalion'. Apart from the main protagonists, the actors playing Eliza Doolittle's father and Professor Higgins' mother, stand out for their acidic sharpness of dialogue and dialogue delivery. 2. Write a paragraph talking about the set design, the lighting, the costumes, the special effects, and the overall effect of the play. The film has sumptuous well lit sets of royal ballrooms where every pixel on screen appears buffed up and polished, with men and women draped in regal finery in sharp contrast to the threadbare and dirt encrusted faces of the poor folks at the shabby flower market. The set of Higgins' library stuffed full of hard bound books and recording instruments seemed quit e realistic for a professor of his stature. The background score backed by a full orchestra credibly supports and carries the play through its many moods. The costumes, scenery and production design-the handiwork of Cecil Beaton- was indeed successful in the challenge of intricately recreating London of a bygone era and ensuring that the characters and sets were not reduced to caricatures. It is known that an entire sound stage was converted into a hair, make-up and costume area for prepping for the famous Ascot sequence. 3. Write a paragraph describing the script of the play. Talk about which of the following aspects of the play are important to this script. You may not have to talk about all of these aspects in your paragraph, just the one's important to this play: subject matter, style, point-of-view, language, characterization, theme. While the story may be of a cockney girl's transformation into a lady on the back of a wager between an arrogant misogynistic phonetics professor and his friend- the genial interlocutor Col. Pickering, the subtexts run significantly deeper and are rife with satire and social commentary on how language and dialects create barriers and restrict social mobility; and a lowly flower girl's grit to break that barrier and to obtain a better station in life and be a respected lady as articulated by her in 'Isn't it loverly?' and an arrogant professor whose interest in the matter is entirely selfish. It is the style of presentation and articulation of these thoughts through intelligent verbal duelling and lyrical jousting like that of the pompous Prof. Higgins, the blatantly immoral Alfred DO little that entertains and enlightens the audience throughout. Despite perpetuating cultural stereotypes the musical remains non judgemental and largely fair and the watching the metamorphosis of Hepburn's Eliza Dolittle is quite enthralling. The film succeeds in showing the audience how accents and language reflects class and upbringing and how it can affect a person's life and lifestyle. 4. Who would you recommend see this play? This musical for all its attention to detail, its extravagant costumes and witty banter would appeal to the most discerning of audiences of all ages and watching the story unfold from a third person perspective is quite interesting, the story line itself being refreshing for a transatlantic audience. But even if these fail to impress, who can resist the charm and grace of Audrey Hepburn and the hope that the character of Eliza Dolittle holds out. Sound of Music 1) The movie in its appearance and content has more or less lived up to the source or origin, the autobiography of Maria Von Trapp (The Story of the Trapp Family

Antigone Essay Example for Free

Antigone Essay Remember those people who always thought they were right, and they always ended up in trouble for it? I Sophocles’ play Antigone, the main characters Antigone and Creon show how being so hubris can be tragic to your life. Set back in ancient Greece an epic battle takes place and brothers end up killing each other. One was allowed a proper burial, yet Polyinesis was not. Antigone felt disappointed by this and decided to bury them yet Creon the newly appointed king did not appreciate the rule breaking. Due to the fact that Antigone and creon exhibited excessive pride, their lives were ruined. Antigone’s arrogance and brashness ultimately led to her death. For example, on Creon’s first day as king he made a decree that no one should bury Polyneisis body. When Antigone heard this, she went to Ismene and asked for her help; yet Ismene refused and called her a criminal. Antigone still disagreed, â€Å"But I will bury him; and if I must die, I say that this crime is holy; I shall lie down with him in death†(673,55-57) This proved that Antigone was cocky and she was sort of stating that all her wrong doings are â€Å"holy†; she is referring to herself as always being right. This affected her though distorting her view on life. In addition to her argument with Ismene, Antigone then was caught in the act of burying Polyniesis and was brought in and questioned by Creon. â€Å"And you Antigone, with your head hanging – do you confess this thing†(679, 53) When Antigone didn’t deny her crime and boasted to the king and the elders, It proves how narcissistic that she is. If she would have not been so boastful I doubt that Croen would have been so harsh to Antigone. Furthermore, Creon then calls in Ismene to protest against her crime as well. Creon started o believe that she helped Antigone to plot against him. Ismene then lied and said she did help to Antigone’s surprise. Antigone become outraged and denied her hand in the deed. Ismene is displeased in antigone’s punishment, â€Å"Do you refuse me, Antigone? I want to die with you; I too have a duty that I must discharge to the dead†(681, 138). Antigone was then enraged further and rebelled against Ismene, â€Å"You shall not lessen my death by sharing it†(681, 139) Antigone is showing self centered and concided she was. She believed that she was so important because she did the right thing by the gods that no one should die except her because she was special. However, Antigone was not the only major character with this flaw. This proves how arrogance can really damage anyone’s life just like it did to Antigone and Creons’. Pride and the excess of it played a big role in this play, being the tragic flaw of both main characters; arrogance is not only a big part of this play but also our history, there will always be people who can never be wrong. Many people are just like Creon and Antigone, Always thinking they are right. Is your life headed for a fate just like Antigone’s plot?

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