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

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

Decolonization Abandonment essays

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

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

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

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

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

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

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

Toxicity and Autoactivation of Baits Experiment

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

Analysis of the Movie, The Insider Essay -- Insider Movie Film Analys

The Insider (1999) is a film rife with ethical dilemmas, suspense and controversy. It is based on a true story related to a 1994 episode of the CBS news show 60 Minutes that never aired. The plot puts Dr. Jeffrey Wigand (Russell Crowe) at odds with Brown & Williamson, the third largest tobacco companies in the country. Wigand was fired from his position as Vice President of Research and Development, at which he was instructed to hide information related to the addictive nature of nicotine. The plot takes off when Lowell Bergman (Al Pacino), producer for 60 Minutes, discovers that Wigand has a story to tell. The best way for Wigand to tell that story is with the help of Bergman, via an interview aired on 60 Minutes. However, tobacco companies have a history of viciously defending their profits, by whatever means necessary, and Brown & Williamson does just that. The story hits a climax as the interests and incentives of the television station CBS, 60 Minutes, Dr. Wigand and Brown & Williamson are played out. Portrayal of Business The film portrays business in an extremely negative light. It focuses on two central conflicts – one between Brown & Williamson and Wigand, the other between CBS Corporation and Bergman. Brown & Williamson is the primary antagonist. The film is ripe with examples of the bad things they do. Their principle, most damaging offense is deceit. They are charged with covering up the addictive properties of nicotine and finding ways to exploit it to increase profits. For example, in Wigand’s interview for 60 Minutes, he says that tobacco companies view cigarettes only as a delivery device for nicotine. He also says they take advantage of the addictive properties by manipulating and adj... ...ons, the responsibility that power implies and the responsibility of media as a corporate watchdog. It seems obvious that large corporations have a tendency to ignore the negative effects of their actions in favor of profit. This example, although sensationalized, still says to me that with power comes responsibility. It affirmed my belief that a corporation’s goal cannot be just to provide profit to shareholders, but there must also be an element of social responsibility. It also made me think about media’s role in business. I think it should be just as portrayed in this film. Bergman relentlessly pursued the truth, using a very credible source. Too often today, media is spoon fed by corporations. Media has a responsibility to objectivity that can be important in keeping businesses honest. But, it’s really up to media to maintain that objectivity.

Essay on “Me talk pretty one day” Essay

†Me talk pretty one day† is an essay written by David Sedaris in 2005. It tells the story of the authors return to school at the age of forty-one and about his experience with learning French in Paris with a very strict teacher. The theme of the essay is David Sedaris attitude towards learning a new language. Although he seems to have an attitude towards learning French he actually moves all the way to France with only one month of French lessons as his previous experience with the language because he does not think that he can learn proper French in America. Throughout the essay you can almost hear the author’s ironic and sarcastic voice. He creates an ironic tone to the whole experience of learning the language which gives the essay some humor. The language in the essay is very informal which is supported by him talking about own experiences. He uses a lot of imagery and has a tendency to exaggerate his experiences. For example: â€Å"it’s everyone into t he language pool, sink or swim† (p. 1, l. 16). This gives the reader some lifelike pictures of the situation. He also gives the reader the feeling that they are there with him by using sentences as: â€Å"’Even a fiuscrzsa ticiwelmun knows that a typewriter is feminine† (p. 2, l. 72). He gives the reader the whole experience of him learning the language. He does not know the words and he makes it easier for the reader to identify with him. His attitude towards learning the language changes throughout the essay. He starts by being positive but after his first lesson he feels terrified. Even though he is the oldest one there, the teacher makes sure that there is no segregation. The teacher is very strict and does not care to give the students a hard time. â€Å"Before beginning school, there’d been no shutting me up, but now I was convinced that everything I said was wrong† (p. 3, l. 101). This shows us that he is so afraid of the teacher that he does not dare to use the advantage that he actually is in France. He can easily improve his language by trying to speak outside of the classroom but he simply does not dare because of his teacher’s personal attacks. The teacher completely ruins all of the students’ confidence. It ends up with them feeling as if they were in a war zone. â€Å"We soon learned to dodge chalk and protect our heads and stomachs whenever she approached us with a question† p.2, l. 83-84). This feeling of fear and shame ties the students  together and there is no competition between them. Sedaris wants to avoid the attacks and the humiliation so he starts studying really hard. He wanted an identity but the teacher would not let him have that. He was constantly reminded that he could not speak French which also shows in the title: â€Å"Me talk pretty one day†. It shows the insecurities in Sedaris’ and the other students faith in learning the language. They all hope that one day they will be able to speak and understand French but has lo st the hope because of their teacher. David Sedaris describes the teacher as: â€Å"a wild animal â€Å"(p.2, l 82). â€Å"She crouched low for her attack† (P. 2, l 52). This makes her stand out like terrifying and aggressive. The teacher’s attitude towards her teaching seems to be that if she pushes them hard enough it will end up giving good results. Even though Sedaris describes the teacher as a terrifying wild animal he still has a humorous tone around it which makes it relatable for the reader since most people has found themselves in a similar situation. Towards the end of the essay we get the feeling that Sedaris’ French has improved. The teacher’s insults do not seem to bother him that much anymore. By the teacher being so strict to everyone, do the insults not seem to be that serious and maybe not something she means entirely. Suddenly he could handle her personal attacks and it occurred to him, that for the first time since arriving to France he could understand every word of the teacher’s sentence. Even though she had just insulted him he feels like it was a victory for him. He cannot speak the language but it is a step in the right direction. He had lost all hope in ever improving in this class but now he had. He becomes curious and it gives him the lust for learning back. He ends the essay with the sentence: â€Å"Talk me more, you, plus, please, plus† (p. 3, l 128). This shows us that he wants to learn and he does not care if the teacher insults him, he just wants to hear the language and learn from it. David Sedaris’ essay shows that to learn a new language you need to learn the culture around it and you have to feel at home in the situations where the language are included. Learning a language is not just about learning the words and the sounds which the teacher ignores completely as she exclusively focuses on the form of the language rather than its use.

Convention Exhibition Centre Essays

Convention Exhibition Centre Essays Convention Exhibition Centre Essay Convention Exhibition Centre Essay On the 18th February 2009, several lucky year 11 drama students at Santa Maria College saw Nostalgia by Ishinha at the Perth Convention Exhibition Centre. This play extended for 2 hours, which was a reasonable time frame for such an astounding performance. Prior to the performance, I had very low expectations for the play because I assumed I wouldnt understand the plot, for the dialogue is in Japanese. However, the performance definitely exceeded my expectations because I understood the plot through the non-verbal communication and greatly appreciated this. The performance put aside, the audience was disappointing because I was forced to mix with people I would not normally mix with, and these people carried out rude habits, such as eating and talking during the performance. Nevertheless, the play was that brilliant that I didnt notice this often. Nostalgia is a play about Japanese immigrants migrating to Brazil in 1908. It is the story of Noichi, who wonders around the world, falling in love with Ann along the way. Ann, Noichi, and their new companion, Chikino, wander throughout South America. Unfortunately, they get separated because of racism and struggle to find each other again. Along with racism, Nostalgia faces such issues as friendship, immigration, and discrimination. The dramatic form of Nostalgia is non-realism because of certain theatrical devices. For example: the characters broke the fourth wall; language was stylised through repetition; and dance, song, and unrealistic costume were employed. Even though the form was non-realism, it also included aspects of a representational style because it attempted to create the illusion of life progressing on stage. The settings were extremely detailed and realistic, and the audience is expected to emphasise with the characters. I liked how the action of the play was clearly structured into thirteen obvious scenes, which generally didnt include narrative devices. Each scene had an individual tone. For example, scene 9 had a joyful tone, involving several cheerful children singing questions. Whereas, scene 3 had a distressing tone, because the privacy of the immigrants was violated. The overall tone of the play would be reflective because each scene reflects on different aspects along their journey. The performance space used was a box stage and the set changed every scene. The set was mostly representational in style because it was very realistic, especially in the newspaper factory in scene five. However, it did consist of few presentational scenes because the set was unachievable. Some of these unachievable sets included the beach in scene one, the river in scene six, and the desert in scene eight. The set was extremely effective in creating a sense of place because it was so realistic and believable. The set contributed to the various moods of the play because it emphasized the issues by situating them on a more believable level. There are numerous scenes in Nostalgia that used lighting, which consequently impacted my opinion of the play. Some scenes cleverly used realistic lighting to convey to the audience the time of day. For example, in scene five, the lighting was bright during the day, and then dimmed when it was nighttime. The use of symbolism through lighting really impacted my view of the play. One of the many brilliant examples was in scene three: during the physical examination, there was an extremely bright light symbolising the violation of the immigrants privacy, which contributed to the distressed mood of the scene. There were many sound effects used throughout the duration of the play. One of the powerful examples occurred in scene five: after the protests, fire, rape, and shootings, there was unbelievably loud music, which intensified the scene. This contributed to the chaotic mood of the scene because the loud music added more havoc to the issues already presented. Overall, I definitely enjoyed watching Nostalgia. I believe the play is amazing because of the theatrical techniques, such as music; lighting; song and dance; and scene structure. Surprisingly, I could actually understand the plot, regardless of not being able to recognise the dialogue, because I understood the non-verbal communication. This was a new experience for me, as I had never seen an international play before. Without a doubt, I would recommend everyone to watch Nostalgia!

Tarantulas

Tarantulas Free Online Research Papers Tarantulas. Large, hairy, gross and scary are all word that have been used to describe them. Most people think that they are menacing and quick to attack. But truly, unless you are a bug, small rodent or small bird, they are relaxed and non-aggressive arachnids. One of the tarantulas many, and most useful adaptations is that they are nocturnal. This allows them to hunt at night, while other animals, such as rodents, small birds, and other prey, are sleeping. A complementary adaptation to this is their eight padded feet, for easier stalking and sneaking. These pads make their slow steps even more silent, allowing it to get closer to a delicious meal, such as a mouse or vole. Then, when it is close enough it will leap, one to two feet in the air, on top of their unsuspecting prey. They will deploy their retractable claws, to hold onto it, and sink its razor sharp fangs into the prey. The fangs will then pump out venom that liquefies the prey’s organs and insides. As you can see, all these aspects work together to help the tarantula to get its prey. But what if the tarantula is the prey? Surprisingly, this happens quite often, especially with the tarantula hawk. The tarantula hawk is a wasp like bug that stings tarantulas. This sting kills the tarantula, which the tarantula hawk lays its eggs on. However, the tarantula doses not just accept this â€Å"sting of death.† It still has tricks up its hairy arms. First, those 8 padded feet can do more than just stalk around. A full grown tarantula can burst up to ten miles per hour. If a predator does catch this speedy spider, the tarantula can fire his sharp back hairs at the predator, making him terribly itchy. If the tarantula can make it back to his borrow, sometimes two feet deep in the ground. Here, it can either hide from his attacker, or if the predator is stupid enough to follow him down into the nest, fight him in the very dark nest. The tarantula has extremely good vision at close range, and will probably kill his enemy. Not only can this super spider hunt, but he can defend himself, jump, run, even borrow down in the ground. They have probably formed such a bad reputation because of their size. Some tarantulas can be as big as a dinner plate! But really, tarantulas will not hurt you if you keep your distance, and don’t agitate this super arachnid. Research Papers on TarantulasThe Hockey GameHonest Iagos Truth through DeceptionThe Spring and AutumnThe Masque of the Red Death Room meaningsMind TravelThe Effects of Illegal ImmigrationArguments for Physician-Assisted Suicide (PAS)19 Century Society: A Deeply Divided EraGenetic EngineeringAnalysis of Ebay Expanding into Asia

Hitler's Death Term Paper Example | Topics and Well Written Essays - 1250 words

Hitler's Death - Term Paper Example Speculations, arguments, doubts and questions arose regarding the validity of the death of the most famous dictator to date, Adolf Hitler. Different analysis were conducted and different results were obtained from these studies the issue still had been going on for a while until the skull fragment was displayed in Moscow a little over a decade ago. Although it may seem still a little vague to some today, it is no longer a question whether Hitler is dead or alive but just the truth behind his death. Considering his recorded condition before the 30th of April in 1945, he would unlikely survive a decade after that. So whether he escaped from the ruins of Berlin or not, he is, for the lack of better term, ‘dead’, by now. Some historians doubted Hitler’s death and suggested that the Nazis orchestrated propaganda to make the Fuehrer a hero. The US also doubted the dictator’s death and speculated the he may have escaped from Berlin in 1945 and did not take his own life. These doubts were caused by the unclear showcasing of facts and mismatching data in the past. These speculation and arguments just settled when the skull fragment with a hole caused by a gunshot was put on display in Moscow in 2000.

Webcomics Essay Example | Topics and Well Written Essays - 2250 words

Webcomics - Essay Example The internet provides the freedom to the artist to provide web comics at global level. They provide the advantage over the traditional paper comics in different forms. The artist are now free to publish their comics easily on the web site The internet enhance different artist to freely show their thoughts and feelings in the form of comics, in simple word they are free to draw their voice. Above all, this is a less costly and more enhanced way of providing the comics at global level.Background and context: the very first comic was appears in 1986, by T.H.E Fox which was published on CompuServe and Quantum link and the comic was head as "Where the buffalo roam"1. By this first web comic, the web comic fields boost up in 1993 where different artist start their comic art on internet. In 1994, the Net comic weekly started on the net and keeps on running till 1999. By 2000, the trend in the web comics rises and thus takeover the position of traditional web comic paper media.Aims and Objec tives of the research: the aim of this paper is to evaluate that either the web comics are providing more advantages to the readers or artist or the traditional paper comics are still better then the web comics. We will provide different objectives that web comics are providing, holding a literature review and different data, and in the end we will conclude a comprehensive result of the whole research paper. We will compare the paper media and electronic media in providing the comic service and will elaborate that which of the media is more useful for the web comics. Rational statement: Web comics and Distribution: Do they offer an advantage over the traditional print comic medium YES! The web comics and distribution offer an advantage over the traditional print comic medium. LITERATURE REVIEW The print media comic is now becoming less effective, more time consuming and provide less opportunity for the artist to provide its services at broader area. Some authors think that Scott McCloud2 that he web comics provide the freedom to the artist. It enhance artist to show its art, feelings and thought at global level. On the other hand, there is no limitation to the artist in designing specific comics showing specific thoughts. The comics are the substitute of words in which a feeling is provided showing what actually the artist is thinking. The comics didn't require the expressions as the art itself is very expressive and showing the entire basic theme. Another comic, Mark Fiore3 still feels inconvenient in web comics and still using the paper media. According to him the comics is a special field and the internet provides the opportunities to those who know few or nothing about the comic field. Therefore, many artists are misusing this field and its quality and motives remain n or more same as it was in 19th century. The web comics rather enhancing the field of comic, making it more poor and ineffective. Similarly the famous artist, Gallagher's shows4 that the internet is enhancing the comics fields because the comic is actually a simple way that helps the people in describing their feelings and thought, so comics should not remain as the part of artist field rather every one should have freedom in using the comics to express what they want to say. Similarly the filed of web comics is becoming more popular and now different firms, companies and specially the consumers are trying to express their feelings and thought through comics about any event, product or service. The artist Joe Cameau5 stated that web comic are better then the paper media comics due to the unique capabilities of web. Where as, in the case of paper media the artist need more effort and have fewer resources in order to sketch the required animation. On the other han