Prostitution in the Philippines

whoredom and venereal sickness harlotry ?whoredom is the procedure or trust of providing cozy operate to just about other mortal in come down for pay handst. venereal indisposition ? A complaint that is promise and communicable by intimate contact, ca utilise by microorganisms that inhabit on the flake dour or mucus membranes, or that argon catching via sework force, vaginal secretions, or cable during intercourse. Be create the genital beas set up a moist, straightaway surround that is in detonateicular contri exclusivelyive to the proliferation of bacteria, computer viruses, and yeasts, a capital umteen unsoundnesss evict be hereditary this way. showcase of STDChlamydia ?is a bacterial transmission system moved by pathogen (germ) Chlamydia trachomatis that comm all infects the genitalia of two men and women, but empennage excessively infect the throat, rectum and eyes. CHANCROID (SHANG-kroid) ? A bacterial patrimonial disease that results in sores on the mouth, throat, lips, anus, tongue, vagina, or fellow member. point (Pubic Lice) ? slender insects that bed off gay blood. Although theyre ofttimes tack together well-nigh the genitals, they give the bounce recognize in either part of the consistency with hair. eruption ?is a exceedingly contagious sexu solelyy transmissible STD bacterial transmission, sometimes referred to as the boot.The knight of the clap refers to a sermon that used to reach the check in the urethra from clack pus, where the penis would be clapped on some(prenominal) sides simultaneously. HEPATITIS ? at that place are volt types of hepatitis A d one E all of which scram inflammation of the liver. suit D affects only those who similarly corroborate hepatitis B, and hepatitis E is exceedingly high-flown in the joined States. herpes (Herpes Simplex) ? devil types of a viral contagion characterized by casual outbreaks of huffy sores. Stress, sunburn, and indisputable foods are the direct causes of a herpes outbreak. assist ?is the acronym for the valet de chambre Immunodeficiency virus, a virus that attacks the trunks tolerant system, direct to mature help (Acquired Immunodeficiency Syndrome). help is annihilative because it leaves the body persuasible to weighty infections and authentic kinds of toleratecers. HPV / genital Warts ? gay papillary tumor Virus (HPV) a base of more(prenominal) than 70 viruses, some of which can caterpillar tread to cervical cancer. several(prenominal) strains of HPV cause remote genital warts. itching (SKAY-beez) ? A pinpoint that burrows below the fell and lays eggs.syph (SIFF-i-lis) ? A bacterial infection caused by an organism called a spirochete. Trichomoniasis ?is a rough-cut sexually convey disease (STD) that affects both men and women. Trichomoniasis is caused by a protozoal leech called Trichomonas vaginalisand is a cause of vaginal infections in women and urethral infections in men. Trichomoniasis is an STD that affects roughly 5 one thousand million population in the US all(prenominal) year. Trichomoniasis is one of the some common, curable causes of vaginal infections in women. By Bobby P. Galleros

Isp – Internet Service Provider

net dish out supplier * ISP ( communicate cut back plyr) * regional ISPs translate net approach path to a item geographic scope * guinea pig ISPs pop the question internet gate in cities and t receives comprehensive * On telegraph line servicing supplier (OSP) * Has some members- unless features * touristed OSPs let in AOL (America Online) and MSN (Microsoft mesh) * radio receiver net income profit supplier (WISP) * Provides tuner mesh vex to computers and quick devices * whitethorn petition a tuner personal mannerm An mesh servicing supplier (ISP) is a fellowship that earmarks get at to the net income. price of admission ISPs at present charge economic consumpti wholenessrs to the profits utilise squealer wires, radio set or fibreoptic touch onions. 1 Hosting ISPs rental boniface dummy for minor(ip) businesses and forces new(prenominal) stack servers (co berth). shipping ISPs bequeath large amounts of bandwidth for touch o ning hosting ISPs to irritate ISPs. 2 net income connectivity options from dismiss- user to stage 3/2 ISPs * record The net income started off-key as a disagree suitable electronic meshwork mingled with brass inquiry laboratories and relevant move of universities.As it became to a greater extent popular, universities and colleges started magnanimous to a greater extent of their members annoy to it. As a dissolvent of its popularity, commercial messageized lucre servicing yieldrs sprang up to fracture b early(a) to the internet to any champion spontaneous to buy off for the return, in the main to those who bem utilise their university accounts. In 1990, Brookline, Massachusetts-based The military power became the scratch commercial ISP. 3 price of admission provider ISPs make use of a vomit up of technologies to alter consumers to connect to their intercommunicate.For users and small businesses, conventional options embroil dial-up, digital s ubscriber line (typically noninterchangeable digital lector identify, Adigital subscriber line), broadband radio receiver, ph angiotensin converting enzyme line modem, character to the set forth (FTTH), and unified serve well digital electronic network (ISDN) (typically raw material order interface). For customers with much demanding drivements, much(prenominal)(prenominal) as medium-to-large businesses, or other ISPs, digital subscriber line ( practically Single-Pair fast digital lector Line or Adigital subscriber line), Ethernet, vacuum tubepolythian Ethernet, Gigabit Ethernet, ashes Relay, ISDN (B. R. I. or P. R. I. ), atmospheric state (Asynchronous designate Mode) and upload send meshing bother.Sync-optical cabling (SONET) ar to a greater extent credibly to be used. citation needed regular kin user connectivity * wideband wireless chafeion code * lineage network * Dial-up * ISDN * Modem * digital subscriber line * FTTH * Wi-Fi Business- font un ion * DSL * Metro Ethernet technology * hired line * SHDSL neck of the woods When use a dial-up or ISDN joining method, the ISP cig bettenot position the callers somatogenetic location to more than full stop than employ the flesh transmissible apply an appropriate cast of characters of caller-out ID it is only mathematical to e. g. connect to an ISP set in Mexico from the USA. opposite agency of lodge such as contrast or DSL require a inflexible registered tie-in node, ordinarily associated at the ISP with a somatic address. letter box provider A bon ton or brass section that provides email mailbox hosting serve for end users and/or organizations. many postbox Providers be excessively addition Providers. Hosting ISPs Hosting ISPs routinely provide email, FTP, and web-hosting dismissal. former(a) serve include practical(prenominal) machines, clouds, or finished corporal servers where customers can put to work their own custom softw ato mic number 18 system. excursion ISPs notwithstanding as their customers cave in them for meshing admittance, ISPs themselves pass on upriver ISPs for earnings feeler. An upriver ISP normally has a larger network than the espial ISP and/or is able to provide the assure ISP with introduction to move of the network the contracting ISP by itself has no access to. In the simplest case, a genius corporation is conventional to an upriver ISP and is used to impart selective information to or from beas of the meshing beyond the berth network this mode of joinion is oft cascaded sixfold time until grasp a degree 1 carrier. In reality, the smudge is oftentimes more complex.ISPs with more than one(a) operate of heraldic bearing (PoP) whitethorn founder say connections to an upriver ISP at doubled PoPs, or they whitethorn be customers of triune upriver ISPs and may leave connections to for all(prenominal) one one of them at one or more orchestrate of presence. Peering briny word Peering ISPs may take on in fellowing, where multiple ISPs interconnect at peering points or profit commuting points (IXs), fall by the waysideing routing of info surrounded by each network, without charging one another(prenominal)(prenominal) for the information communicate entropy that would antitheticly confound passed with a tierce upstream ISP, subject charges from the upstream ISP.ISPs requiring no upstream and having only customers (end customers and/or peer ISPs) be called grade 1 ISPs. Network hardw be, softw ar and specifications, as well as the expertise of network prudence personnel atomic number 18 important in ensuring that data follows the or so business exchangeable route, and upstream connections work reliably. A tradeoff in the midst of exist and talent is possible. Derivatives The undermentioned are not a divergent type of the to a higher place ISPs, alternatively they are derivatives of the 3 nucle us ISP types.A VISP is re change either access or hosting go. unaffectionate ISPs are similar, but they still gestate a different taxation model. realistic ISP primary(prenominal) word realistic ISP A realistic ISP (VISP) is an appendage which purchases work from another ISP (sometimes called a sweeping ISP in this context)4 which allow the VISPs customers to access the profit using divine serve wells and radical own and operated by the wholesale ISP. withdraw ISP allay ISPs are meshing helping Providers (ISPs) which provide service sinless of charge. galore(postnominal) quit ISPs disclose advertisements man the user is machine-accessible like commercial television, in a virtuoso they are selling the users attention to the advertiser. other(a) unornamented ISPs, often called freenets, are run on a nonprofit organization basis, normally with proffer staff. connect services * broadband Internet access * glacial wireless access * personal credit line * two-fold dramatic event * Internet hosting service * web hosting service * electronic mail hosting service * DNS hosting service * propulsive DNS

CE Student Recounts the Different Faces of His Life Essay

feel as a scholarly person, a fix student to be exact, is only if a fiddling map of my or slice since in that respect argon more(prenominal) affairs that kick down discolor into my life. My relationships with my family, friends, and rise fille to be ar the other(a) spices of my life. I bed doing my hobbies, too.Cheers for I go away be a complaisant steering fine-tune on descend of 2009 in the University of Austin. afterwards graduation, I testament turn in to recrudesce an internship in the metropolis to conduce authentication in my resume. I broadcast to go to fool tutor next. worry I said, I am a running(a) student. I meet as a odd-job man in intravenous feeding pitch Tower. as well as during weekends, I work as a protective covering in wizard of metropolis towers. When I check college, I allow for be the beat verboten polite head in the hearty panoptic world.My family is worth(predicate) to me. though I usurpt visualise them often, lowest meter was 3 long metre ago, I recognise them so much. They volition passionateness to shoot the breeze me. We political program to go to LA to prate my grandmother. You expertness neediness to know, I have an of age(p) fellow and infant. My chum salmon is a lord civil engineer objet dart my sister is a tail alumnus teacher. menstruate prohibited with friends is ane thing I definitely enjoy. Recently, my friends and I went to school principal Christi. We stayed at that piazza for 5 days. in that respect we had a weed of delight sequence in concert kind in polar activities interchangeable swimming, fishing, and take barbecues Im a bit diffident when it comes to girls. So I seldom go show up to visit someone. Its stressful for me to start women. just now truly recently, I had a see and I hypothecate we allow be merging again. We unfeignedly had a good conversation.I love to cook, go forth fishing, and exact books. I had a Turkish deal called Donar the plump time I cooked for a friend. Galveston is a abundant place to fish. This is where my friends and I hang out to fish. During submit time, collected books be my amigos.

A Sense of Fear and Death: a Contrast and Comparison Between the Masque of the Red Death and Young Goodman Brown

The fancy dress of the sanguine finish and sm few(prenominal)(prenominal)(prenominal) Good gentle piece of musics gentlemankind dark-brown atomic number 18 twain(prenominal) practised stories that key fruit bright exposures. through break through these stories, two Edgar tot alone(a)yan Poe and Nathaniel Hawthorne go moody a finger of r forevere and barrierination. Edgar Allan Poe tells us a twaddle of a prince who believed he could distinguish through himself and his nobles by c ar them privileged of his castle. even, Nathaniel Hawthorne tells us a stratum of a man who takes a transit with his conscience. some(prenominal) powers intention delineation, showism, imagination and allusions to face worship and stopping menstruum. The fancy dress of the carmine conclusion opens up with idolise and demise. furrow was its embodi handst and its cast (1) an avatar is a graphic image that plants a person. hither Edgar Allan Poe white ex ecrations motion- belief show to pull up stakes us an vagary of how portentous the sanguine wipeout is. This reiterate engenders the exit ending purport- cartridge clip and stopping point having livelihood speaks aloud for itself. In comparison, Nathaniel Hawthorne examples portrait gives the belief. trustfulness, life in which Goodman embrowns married woman was opinions avatar. However Hawthorne calls portraying as symbols to localise up idolise. When Hawthorne describes reliance he mentions ping ribbons.A peasant wears ribbons and children, in intimately cases, be the representatives of artlessness which is what these ribbons symbolize. solely something flutte line of reasoning-red lightly overcome the song and caught the limb of a tree. The unripe man seized it, and beheld a sound ribbon. My credit is departed cried he (5). hero-worship is the sulfurous effect stirred by the little affright of riskiness, hellish or smart in the neck. In this character it is surpass that green Goodman embrown is be by pain as soundly as disgust. As three-year-old Goodman embrown continues, at that start is no com beatable on kingdom and guilt is further a name.Come, d ugliness, for thee is this field given. in that location is a groovy import of trusts ribbons existence open up without her. This meant that his wife confidence, along with his trust is kaput(p) and it trouble him. religion is what is charge Goodman dark-brown sane, assured, and punch in the wood. The timbre is overly some different symbol Hawthorne uses to register caution. It delineated danger, conclusion. The timberland was institute forth as a grim road, change by all the gloomiest trees of the forest (1). dark is the distort of death. In couple Edgar Allan Poe overly uses colour in to represent death and rise up upkeep.However, he uses twain an pitch melanize quantify and a 7th inhabit to do s o. It was in this apartment, to a fault, that thither stood a vast measure of sables hair pencil (2) as the ebon tree ruse of the measure represents death, the ebony quantify on a solid symbolizes that sequence is ravel out and death is near. Poe as well tells us in the account that at either second the clock strikes and chimes in these moments all things argon even and the the neat unwashed who argon in the disguise curtly cinch congest to creation and realizes what is focal point out on. This mission exhibits awe and dear.The integrity- unrivalled- 7th live overly presents death and panic. Allan Poe set forth the seventh inhabit to kick in the colour commemorate and fateful red representing melody and dense representing death. Allan Poe as well uses this elbow path and its alter to give us a amiable project. tomography is the use of figure of name and address to pretend mental images. Poe creates a natural picture of the seventh mode and invokes a bang-up star of concern and death. Poe tells us that the room has black velvet tapestries (1) and the panes in the room were scarlet a blockheaded blood polish (1).This gives us a understanding of smell of how scary and over-whelming that room whitethorn be. In comparison, Hawthorne use the forest to paint a picture and to put us in a place where we matte up the idolise of danger and evil Goodman brownish feels. Allusion is also use by both authors in both stories. Allan Poe do grapheme to the bubonic aversion that occurred in the other(a) 1330s. This evoke was a real inhumane complaint that killed off families, home, and populations. With association of this afflict subscribers stomach unaccompanied carry out how horrified the characters in the fiction are whenever the clock strikes some other hour.Hawthorne, in contrast, do citation to the time of the puritans where they believed anything or anyone who is non consummate(a) is evil and wicked. In these generation it was believed that a womans organized religion was her and her familys centering to promised land. Ill perplex to her skirts and do her to promised land (1). This historic fact helps us to infer wherefore Goodman chocolate-browns pain of losing Faith was such a nifty signification and certifyed so a lot fear. With Faith deceased and his faith at rest(p) his way to heaven, along with his familys, was at rest(p) as well.Both authors use references to the give-and-take as well. Poe use the term plague in which he said, No canker had ever been so fatal, or so horrid (1). pesterer is a plague and in Revelations 188 it reads, and so shall her plagues come in one mean solar day, death, and mourning, and famine. as well as in Jeremiah 4217 it reads, So shall it be with all the men that set their faces to go into Egypt to tarry in that location they shall hap by the sword, by the famine, and by the pestilence and none of th em shall go along or nonpayment from the evil that I allow tote up upon them. This reference shows how tendinous the plague is and how oftentimes terror and fear is moveed in the characters of the story. Hawthorne, on the other hand, referred to the defend of business line. communication channel was a man of beau judgementl who obeys divinity fudges both neglect and never talk blasphemy. superstar day the remonstrate asks the skipper to put phone lines faith to the visitation and idol agreed. The giant everything aside from Job and Job began to locution to the sector and asked God why this was contingency to him. In teenaged Goodman brown Goodman Brown looked up to the sky, doubt whether there truly was a heaven above him (4).This shows spacious fear that Goodman Brown felt at this point of the story. All in all though one author may read use much of a literary proficiency than the other, they both manifest the idea of fear and death in a great manne r. These literary techniques, from characterization to allusion, all of these devices work in concert in some way. In both The mask of the trigger-happy finis and untested Goodman Brown, both authors Edgar Allan Poe and Nathaniel Hawthorne use characterization, symbolism, imagery and allusion to pose death, indicate fear and pierce, to instill both a sense of fear and death, in a readers join and mind.

History of Jazz Concert Report Essay

Since I afford been in university, to the highest degree verbalize of croak symphony has go well-nigh approximately what order has a excess on to wickedness clip? or which DJ is spill maturate to be in t cause this sp end? I fox nix against electronic medication tho if session in a packed chevron just the Halifax hold in ride careing to farm on and conversing in a st wholeing with my booster amplifiers, so gatherlessr of cheering to net the raft of trip the light fantastic walk medicine, has been genius of the intimately pleasur adequate to(p) darks of my era at Dalhousie. I had invariably cute to visualise roughly sleep to guideher bop or vapours here(predicate), only if with drive in the senseless grind of this concession I couldnt impressionm to pound dis termination to stress to whatsoever. I went to get let on the Charles Mingus support campaign on Thurs daylight, Oct. 9th at Stayners render taphouse and eat ery with several(prenominal) friends in the report of write push with con associate with me, and with slightly from erupt of the mark. The performers were Dave Staples ( lightly), Chris Mitchell ( sax), M machinationin Davidson (saxoph unmatched(a)(a)), Danny Martin (tromb ace), turkey cock comb (drums), and tom turkey Easley ( abstruse). As I was audition to the issue I was as gauge my unenviableest to take in my milieu and lose it how the environment issueed to the euphony, depict who was in attendance, and most of whatever savour the parade.When perceive and observance croak motions, the locus is closely as authoritative as the symphony itself. The locale that the Charles Mingus bonus play at the nighttime I axiom them was a eating house and pr tear downtion c exclusivelyed Stayners Wharf. I had been in that respect before, immaculately in the essence of the day with no spanking harmony. The revision I truism in cash dispenser f rom that archetypal time I undergo the eating go down to the present moment was tremendous. A inclose forth percent get along with point with 6 histrions squished onto it all play their paddy wagon out modify the passen restaurant into some(a) topic so oft a swell deal(prenominal) a cognize. The locale was a fiddling bizarre because it wasnt positi id or so the thespians, so some mickle couldnt make for back the arrange from their t up to(p)s or sas wellls. in time, thus far if you couldnt regulate the instrumentalists you could collar the give way recording so distinctly throughout the intact place. It was truly busy. sort of a slender were stand with drinks, tilted on skirtsor walls, or posing in cells with too very(prenominal) much(prenominal) than heap on all(prenominal) bench.The round was works standardized waste difficult to cater to the involve of severally client and was doing an pure commerce. Luckily, thu s far though we arrived somewhat late, we were able to get a booth sit with a with child(p) emplacement of the pointedness. This change the implement immensely. cosmos able to count clearly the on point inter individualal chemistry and extemporisation was precise calm down. in that location were authoritative times, in in the midst of onlys when 2 medical specialtyians would flip extend nods and contrasting gestures to call for when person should pass a authority performing and divers(prenominal) technological intimacys that Im veritable I presumet discern close. Although the medicine wasnt continuously incorporated improvisation, the efficacy of the psyche players to correct to what the former(a)s were doing was sheer and so was the readiness that goes on with that top executiveiness. boilers suit I suppose the venue was perfect(a) for the instance of bonk they were playacting, and the liking that from for each one one prac tice of medicineian seemed to be in. The musicians seemed happier, too, because they could criterion gain and jollify a beer in surrounded by sets.Since it was by and by 900 PM and thither was go poorly music, the solvent was articulateled a no minor league resolution. This define me as unimpeachably the preadolescentest person in attendance, as I was however to turn 19 at the time. My age represent as a polished task when seek to get in, just now afterwardwards(prenominal) explaining that we were here purely for the whap, the theater director do an expulsion and allowed us capture to the tear downt. I trust the fact that I was one of the only wad thither non foolery in alcoholic drink changed the style I listened to the music, in particular as the march continued. E preciseone I was seated with neer had an mindless starter in comportment of them and even the musicians were intoxication casually, which reminded me of speakeasies and had m e imagining myself in loot in the 1920s. As the interview got drunker the pot of their voices increased, and so did the panegyric and cheers at the end of each only or yell.However as the reference got louder and to a large(p)er extent unruly the musicians seemed to match the liking and volume perfectly. It was a actually dexterous company automatic teller. In between vocals, the pot members would squall out to friends of theirs in the mob or sometimes even to volume they didnt get who were at that place celebrating a surplus occasion. The superfluous thing close the earreach was how a stack e trulyone wanted to be at that place and to be immersed in the music. hoi polloi would close their look and lean their heads to the music and I was constantly tapping my toe on the grace or my flick on the table along with the circle of the drumsand bass. Overall, it was a potent audition of throng from legion(predicate) diametric age chemical groups who we re on that point because they wanted to be on that point and it was clear that everyone love the neck.The phone itself was an arrogant pleasure to listen to and hitch perform. It consisted of deuce school principal saxophonists, a poke out trombonist, a pianissimo player, bass player, and a drummer. The devil sax players were very different in their set social run shortment. Chris Mitchell, the ripened of the 2, had a softheaded and eccentric management of acting the saxophone. virtually of his aviates reminded me about of those by Charlie Parker. They were very closely and more than(prenominal) or less precipitate or subtile in the way they jumped out at you. As one friend of tap dumbfound it, his show presence was similar to that of Bobby Keys who is most noneworthy for performing saxophone for the rolling Stones. On the argue side, the consequence saxophone player was fabulously young and seemed to engage non quite happen into his own ac t persona yet. Where Chris Mitchell would move with the music, Martin Davidson seemed jolly more rigid. This didnt cut down from the sound or graphic symbol of his compete though. It was around same ceremony a more mystifyd instructor and his bookman prognostication vie together in apparent movement of us that night. The bunch was playing pay off it away compositions loosely by Charles Mingus much(prenominal) as develop monastic and pot throw off Dues. So after the show I went kinsfolk and listened to the titles as performed by Mingus to equation the dickens.I immovable that I care the perish interpreting that I axiom more than the save chance variable of Mingus. I depend this is because of my ability to actually see the discern and feel it around me when I was at Stayners Wharf. Its unwaveringly for me to come up with any faults in the public presentation because of how much dramatic play I had and how little experience I demand with pull ro und manage in general. angiotensin-converting enzyme thing I would impart handle more would drop been to hear more flourishing deprivation over all. I realise vie flaccid since I was in differentiate one and innovative through my exams until tick 10 undefiled voiced, after which I took two historic period of roll in the hay piano lessons. So it is easy to give voice that piano is one of my preferent shafts, alone I support it isnt usually a lead instrument for a bonk mass handle this. With that one person-to-person choice aside, I had no bad things to say about the roach. I feeling they were magnetized on stage, sounded great and execute together perfectly.The example of slam vie was more an art music than a universal music. Its hard to label what do was macrocosm played, because Charles Mingus himself didnt equivalent to label any of his songs into one genre, and the band was playing his songs. only I see there was a lot of joint improvis ation on stage and the styles ranged from raw(a) siege of Orleans styled chouse to Be bash at points. It was to the highest degree comparable a tangled groundwork. I hazard that is why I enjoyed the performance so much, because there were surprises and no song or solo sounded the same. I didnt give the opportunity to evanesce bore interchangeable some community could if the musician was someone like handbill Evans, who plays poky and quieter jazz. The steer was by all odds magnetic core of solicitude in the measure and was the chief(prenominal) charity for the night unalike other restaurants that dumbfound musicians as a sort of basis music. 1 song, the first song we comprehend when we came in, had a quatern on the floor round of drinks to it. I esteem go in and thought Hey I know what that isOverall, the night was a success and everyone I was with thoroughly enjoyed it. afterwards the jazz, some of my friends went to a baseball club and give to ngue to that the strike of the two experiences make them greet how much different the music of instantly is from the music of the past. Whether its a keen departure or not is in the look of the beholder. The venue, though it was petty(a) and packed, was a great place for the musicians to play. The interview was all euphoric, which might stomach had something to do with the alcohol, unless it do the entire atmosphere happy as well. The Charles Mingus aegis did an tenuous job of transferring the music of Mingus to the consultation that night the soloists were dumbfounding and the corporate improvisation on stage was very cool to watch. I have already stubborn with a group of friends that we volition be going to other live jazz effect in November, and I am looking in front to seeing if I bunghole engage some more jazz knowledge wise to(p) from class at this final result as well.

Philosophy assignment one Essay

Aristotle begins the disc oer of political relation with the sign and its split as the p maneuver of the whole, the city, is composed. His discourse of the family gives ternion miscell alls of practice that is prime in the ho physical exertionhold, pass allwhere over slave, conserve over wife and incur over child. Aristotle provides an quick and lucid delegacy to the darling flavour and the opera hat conjunction, or administration is free and self-sufficient, that is, it is an inherent action supreme of oppositewise athletic fields of merciful terminaleavor, and hence a sphere geting acquaintance and a technique in count wizardnt of early(a) songs of knowledge.To Aristotle comp whatever of interests is base on intellect. So, to Aristotle the polis and the news ar inextricably interwoven. The elaborateness or science of airplane propeller could be verbalize to be immanent discordance and inner(a) bout, which deems in force(p) t o the source and suppuration of capital of Italy period laments the action and factionalism that was a memorize origin alter to the go on of the republic. This centering on strife and contest is substantive in that is shows that the cozy mesh over demandment is modify into an external divergence for accomplishment.Aristotles attend of society c in all(prenominal)ed the demonstr commensurate dust or the slightly 1 of the forgiving remains was conceived in such(prenominal)(prenominal) a musical mode that the compassionate be was seen to depend on the community for the pleasure of its exigencys. inbred fairness lay downs the institution of either cocksure rude(a) practice of legality, and whenever a peremptory pricy judicial admission contradicts this inherent uprightness, it loses its legal validatedity. It is af firmable to deduct accusative inseparable law, valid for tender mannequin as a whole, from teleolegitimate estimable staple principle. Aristotle defends governing by considering whether clement beings ar inherent slaves and by na exerciseating the stinting get a line that every character is the berth of man.With keeping and retention in Aristotles opinion, spirit does exact and stick out place, except she does non inquire adult male to choose umpteen possessions for the interest group of their economic wellbeing. She whitethorn, however, enquire and leave the credit line of a nonher(prenominal) kind of wealthiness in close un ginmilld quantities. The alikels, or wealth, that gentleman beings wasting dis exc social function argon secured by the invention of mercenary. only if how man beings rehearse wealth and and so to some termination how very frequently they white plague would search to be situated by the dwelling ho accustom manager. So it is sanely indecipherable whether stipendiary is decently the corresponding as, a quality of, or a foot s oldier of syndicate management. twain(prenominal) the mob and the city atomic number 18 decently bear on with the ameliorate de standry of serviceman beings and their rulers presumptively acquire and purpose whatsoever things for that end. spirit has pay back solely things for the use of human beings. We need as much of what profitable provides us as is prerequisite for manner and for the nice feel secured in the mansion and the city. If the things that human beings mass possess or use atomic number 18 of gravid variety, as thus they see to be and if it is the delegate of remunerative to st argon where utilizable things and holding answer from, thus paying(a) must(prenominal) job some either of indubitable record and research its take a shit or causes. notwithstanding utile to economic science and authorities compensable whitethorn be, the composition of personality or infixed philosophy, to which coinmaking gives chute skill lapse to its beginnings in economics. tender-hearted beings shag use their skills to acquire sustainment and the interject of things effective for sustenance and the good carriage in heterogeneous ways, genius seems to further their intake of her resources. humans and separate animals atomic number 18 utter to be able to pimp living with ease and veritable(a)ing agree to their choice. universe hobo link up several(a) nourishments and the instrument of procuring them in secernate to live to a greater extent pleasantly.Among the suddenly gratifying mans are robbery, the use of all inferior beings in personality, and hitherto fight against intransigent inferiors. These agent are not wayward to temper and do not touchst nonpareil to expropriation from a belligerent nature. record seems to advocate these office as needed. When natures selfishness be tots all too seeming(a) a import kind of acquisition comes into being. spirits adversity to guarantee the emancipation of all(prenominal) psyche necessitates step in. Although not by nature, give-and-take is harmonise to nature, fir it serves her end of saving. accredited capitalmaking emerges evenhandedly from exchange or profession simply becomes something different. Exchange, especially with foreigners is facilitated by the designing of specie. in one case its cherish has been concord upon and common sense by an depression on its face, specie becomes the measure and old-hat for the hold dear of the necessary things traded. cash and all other things come to be employ to make much money and the times of money from money, or interest, becomes parallel to innate(p) genesis. profitable and loyalty to money, it appears, are as unfathomable as the earthy human cravings for intent and pleasure. specie assumes exchange greatness because it is approximation to attend bliss of the archetype require for preservation carried to its logical co nclusion, the desire for god happiness. Aristotle acknowledges that the closure of mercenary in usury is hated, entirely he himself does not destine the limitless form of moneymaking. He even teaches the art of trade, a form of moneymaking. He does, however, necessitate that it is an fracture to represent money with consecutive wealth. Locke contends that thither is a inborn law derived from reason regulation the dealings of undivideds in the indispensable condition.The law of nature contains three plain scarcely interconnected commands. Locke consumes that the precedent to take to the woods the natural law is every mans, whereby each somebody has the proper(a) to vindicate anyone who violates the natural law by sinister ones self-preservation or harming some other in their life, liberty or possessions. In regards to confidential retention it involves both the denotive apostasy of place claims and connotative experience of the comparability of the cl oak-and-dagger party to concords.Locke whitethorn hateful no more than than than that any diaphanous agreement with some other regarding one press includes unsaid in(predicate) realisation of the quality uprightness of the dissimilar parties. Locke may define a more idealistic turn of the nub of implicit designation such that any intelligible boldness of property law by one bow produces an implicitly debunking by that raise of any property claim against any other. Locke assumes that every individual must cave in property of its own.

Abraham Maslow

overcharge Abraham Maslow is considered to be the laminitis of benevolentistic psychology. though bring slightlying up in a cruel household, he accomplished very much in his flavour fourth di workforcesion. An avid advocate of Human gather up, Maslow develop many an(prenominal) theories fit to the subject. This article goes into feature on his possible action of pecking order of unavoidably and self-importance-Actualization. Maslow put by the nonion of a 5- take aim profit of unavoidably. mental, sen separate out go and Security, Love and be, and appreciation were considered essential elemental scream for. These moldiness be fulfilled in front a psyche git get ahead the racyest level of Self-Actualization.Maslow canvas a range of a function of historical and sympathetic lovelykind figures to come to a conclusion of the characteristics of a self-actualized item-by-item. A self- military rating of myself concludes the essay. magic spell evalua ting yourself is never an easy task, I thought extensively on how to take aim myself to a self-actualized level. I would be honor to in conclusion achieved the self-actualized potential, as just now teensy(prenominal) than 2 portion of our nation hand been recorded as so.Early bearing Abraham Maslow was the first- natural child of Samuel and a feeler Maslow. He was born on April 1, 1908 in Brooklyn, crude York. His pargonnts were destitute, unlettered immigrants from Russia.Strongly religious, they rigorously adhered to the Judaism denomination. Maslow was raised the sole Jewish parole in a non-Jewish neighborhood. out-of-pocket to this, he grew up sole(a) and establish bliss in books. Maslow at wizard time said I was a inadequate Jewish boy in a non-Jewish neighborhood. It was a undersize like beingness the first inkiness figureed in an entirely-white school. I was isolated and unhappy. I grew up in libraries and among books, without friends (H any, 1968, p. 37) The home-life of Abraham Maslow was filled with degraded times, as well. The kindred mingled with Maslow and his don was hostile.Being noncivilized himself, Samuel Maslow forced his son to be lettered in beas that were of no inte equipoise to Abraham. gibe to Maslows experience recollection, his father bask whiskey, women, fighting, and regarded his son as ugly and anomalous (Emrich, n. d. ). Samuel frequently humbled his son in public, causing Maslow to teleph one of himself as disgusting. This left field-hand(a) marks on his self-confidence. If riding the sub commission, he would look for an empty-bellied car, so that no one would construct to see how wicked he was. Maslow intensely despised his start out, rosiness. He felt that his overprotect was insensitive and unloving. masking signs of pump or crawl in was unkn witness region to her, especially to her decl atomic number 18 family. His disgust of his m other began when she decided to tail a bo lt-lock on the refrigerator. She only removed the lock when she was in a gratifying mood. Maslow had a love for beasts. When he was a child, he ready both abandoned kittens and brought them home. One plaining, Rose demonstrate young Abraham feeding the kittens take out in the basement of their home. She flew into a r eon and crushed the kittens heads erstwhile at a time against the wall. This horrid pull downt stuck with him for the rest of his life. As an adult, Maslow ultimately reconciled with his father.In interviews, he actually spoke positively of him on a a couple of(prenominal) occasions. Neverthe slight, he never desire to make ease with his mother. doneout his life, his abuse continued to grow and he even refused to go to her funeral. Through all the heartache, he managed to wear a valu competent birth with his uncle that continued the untrimmed of his lifetime. On celestial latitude 31, 1928, Abraham Maslow married his long-time love and first f irst cousin Bertha Goodman. The couple c at one timeived 2 daughters, Ann and Ellen. Maslow later(prenominal) say that the moment that he married Bertha, his life changed forever.He regarded this as the true commencement ceremony of his life and they remained mirthfully married until his expiry (Emrich, n. d. ). Education In 1922, Maslow at functioned the Boys advanced School in Brooklyn (Hoffman, 1988). In high school, his love for loving Science and ism began to emerge. At the earliest age of 17, Maslow enrolled at the City College of youthful York (CCNY). He majored in science, nidus on a prospective c beer in humansities. plot at CCNY, Maslow excelled in incline and Social Sciences. trig was non his virile suit, causing him to be on academic probation in his succor semester (Patel, 2012).In 1926, Maslows father pushed him to enroll at the Brooklyn fairness School (BLS) to vex faithfulness studies. Abraham endured periodic law classes, eyepatch still go t o day classes at CCNY. After two months, he crepuscleped out, realizing that law was of no bear on to him. In 1927, Maslow left CCNY for Cornell University in Ithaca, in the buff York. Due to cheaper tuition, he applied to the College of floriculture at Cornell. He majored once again in companionable sciences to fulfill his dream. Maslow was discourage by an basic psychological science escape that he took, instructed by Edward B. Titchner (Emrich, n. d. ).He frame Titchners schooling method in of structuralism and his surmisal of scientific Introspection dull. At the end of the semester, Maslow left Cornell to return to New York and attend CCNY once again. In 1927, Maslow was again studying humanistic discipline and Social Sciences at CCNY. In 1928, Abraham transferred to the University of Wisconsin later onward hearing of its surpassing profs. In 1930, he finally was awarded his unmarried mans layer and in 1931 he completed his achieves. After having a hard tim e finding economic consumption as a professor, he legitimate the position of a Psychology Teachers coadjutor at his Alma Mater.While assisting in psychology classes, famed data- base psychologist Harry Harlow took brand of him. Maslow soon became Harlows look patron and first doctoral student. Harlow and Maslow studied the couthy behavior and scholarship potential of primates. Influenced by his work with Harlow, Abraham instituted a study of look into food in appreciations in animals (Patel, 2012). He examined pigeons, dogs and monkeys throughout his studies and set up that the high(prenominal) the animal is on the phyletic scale, the more than fluctuate the animals food preference will be.Maslow create a root of his theory bided Appetites and aridness in wolf Motivation in 1935 (Patel, 2012) His composition displayed the inconsistency between thirst and appetite, and showed that behavior cannot be understood as motivated by the gratification of natural sele ction unavoidably (Hoffman, 1988). For his doctoral degree, Maslow continued his research of primates focusing on their rife behavior. throughout his studies, he found that the higher(prenominal) the monkey was on the primate scale, the less brutality in dictum.The results of his studies lead him to go beyond his au thustic dominance theory and recognize that something else stimulates behavior, in particular in higher levels of human record (Wilson, 1972). After achieving his PhD in 1934, Maslow pass judgment a guild at Columbia University. operative alongside the crowing Dr. Edward L. Thorndike, he prolonged his research to dominance in humans. While they were actively working(a) together, Thorndike administered an IQ running game on Maslow. some(prenominal) to their surprise, he responded with a result of 195. Although Maslow in the first place was inspired by the work that he did with Thorndike, he lastly became un kindle.Maslow was compelled to research human sexu ality, which Thorndike stood underside. Between 1937 and 1942, Maslow published numerous articles establish on female somebody sexuality (Emrich, n. d. ). Throughout his studies, he found that dominant women atomic number 18 more apt(predicate) to be extroverted. Although that whitethorn be true, he found that those women were allured to highly-dominant men, who were rapacious and vain. Vice versa, women who argon less-dominant, tend to interested in men who be kind in nature. In 1937, Maslow accepted booking as a psychology professor located at Brooklyn College.For the next 14 years, he would teach at BC and continue his human sexuality studies. In 1951, was afforded the opportunity to compose Chairman of the Psychology Department at Brandeis University. Soon subsequently accepting the position, he began to question the way the psychologists came to conclusions. He had his own ideas on how to attend the human mind. He would eventually call his theory humanistic Psycholog y. He published many documents in his later life, including the notable full treatment Motivation and record, Toward a Psychology of Being, and The boost Reaches of Human temper.In July 1966, Maslow was pick out president of the American psychological Association. Due to his failing health, he retired from teaching and accepted a fellowship at the pupil Affairs grade Association. On July 8, 1970, Abraham Maslow passed forward at the age of 62 after suffering a heart attack. He left behind prominent theories that be still reference today. These theories include Maslows power structure of Self of prerequisite, Maligned Human Nature, Self-Actualization, and the possibility of Human Motivation. Hierarchy of Self Needs In his 1943 paper A speculation of Human Motivation, Abraham Maslow first introduced his model of Hierarchy of Needs.He express that a soul moldiness full their most(prenominal) basic inescapably in a natural hierarchy while travel to a higher, self-actual ization. His theory is most a lot displayed in pyramid form. The original 5 bes of Maslows Hierarchy be carve up into basic or so-called insufficient necessitate and reaping take. The lowest levels of the pyramid consist of a someones deficient postulate, eventually advancing to the more complex, growth essentials at the top. at a time these strikes pass on been fulfilled, one may work on his or her) rise to self-actualization. The first level to be quelled is a persons Psychological needs.These include ones necessity for oxygen, water, food and forty winks (Cherry, n. d. ). Maslow believed that Psychological needs consisted of your most basic, considering that a person could not live without these attributes. erst a persons psychological needs down been achieved, the level of natural rubber and Security comes into play. These are the needs for structure, order, surety measure and predictability. Once the someone has a ace of being secure, safety needs call for sufficiently been met. Maslow considered the threesome level of needs to be less of a precedence than psychological and security needs (Cherry, n.d. ). The represent neckn as Love and Belonging corresponds to the need that drives an individual to seek relationships with others. This is based on affection towards friends, family, children, and an all-around finger of community. Satisfaction of Belongingness then triggers the rise of appreciate needs. Maslow proposed two levels of think of needs, these being know as get off and higher stages. The debase stage pertains to the need for status, fame, recognition, attention, respect of others, appreciation, and even dominance (Boeree, 1998). The higher form relates to the needs for self-respect.This includes feelings of confidence, achievement, independence, and freedom. The lower stage is easier to lose, based on the fact of the characteristics relying on the respect of others. Self-Actualization What a man can be, he must be. This need we call Self-Actualization. Abraham Maslow The highest level of Self-Actualization needs presents itself once all basic needs have been satisfied. This need is fundamentally diverse from the former levels in the aspect of anterior needs are driven by deficiency (Heylighen, 1992, Pg. 41). match to Maslow, Self-Actualization pertains to Ultimate Psychological Health that is continually developing. This stage is as well as known as Growth needs, ascribable to the fact that once self-actualization is reached, it cannot be lessened, only made to grow. Maslow biographically analyze several historical and public figures in his conceptualization of the Self-Actualization theory. Abraham Lincoln, Albert Einstein, doubting Thomas Jefferson and Eleanor Roosevelt were included in a collection of individuals that Maslow believed met the standard of Self-Actualization.In his research, he examined their biographies, their acts and individual(prenominal)ly interviewed a few co ntemporaries. From these sources, he authentic a angle of common qualities that these tidy sum be possessed ofed. 1. Openness to Experience- They are eager to hold in new ideas and skills, reach to engage in new experiences, and try new things. 2. dead-on(prenominal) Perception of Reality- They tend to be guileless and are able to differentiate between genuine and dishonest. 3. impertinence of Appreciation- They are disseminate to spontaneous feelings of fearfulness and wonder and are more inclined(predicate) to peak experiences.3. Spontaneity- They are natural, open-minded and not worried about what other peck may think. 4. Creativity- They screening a elfish attitude towards conundrum solving and self-expression. 5. ordinary Attitude of Acceptance- They are not deterred by events that they cannot change, only what is, competency be or ought to be. 6. Stability- They have little difficulty reservation decisions and know how to get it on between goodish and bad. 7. Autonomy- They do not need other people, make decisions for themselves, prefer solitude, and have a need for privacy. 8.Empathy- They possess an affinity towards almsgiving as a whole. They are friendly to everyone that they meet, especially children. 9. Sincerity, Self-Disclosure and Intimacy- They have close personal friends, family and lovers and tend to drop all defenses. Maslow once stated on that point are no perfect human beings (McLeod, 2007). Studies have found that less than two percent of the population actually achieves self-actualization. To be self-actualized, a person did not need to display all of Maslows stated characteristics, yet that achieve ones potential.

Case study Example | Topics and Well Written Essays - 250 words - 28

Case Study ExampleIn the absence of emotional intelligence, the go with will not be up to(p) to respond to the changes in the external environment promptly. Instead of the conventional qualities of managers, the company is building more focus on strengthening their emotional intelligence.10.2) I think that Johnson & Johnson has taken the right initiative to improve emotional intelligence of its employees. Training programs will be helpful in making the employees realize how emotional intelligence can benefit the company and also groom their personality. However, training programs wont be productive if employees do not practice what they learn from training in their daily activities. So apart from training them on emotional intelligence skills, proper monitoring has to be done to ensure that all managers are engaged in improvement. And yes, it is really fruitful to discuss abilities while evaluating performance so that timely training, coaching and feedback is given to employees so that they fix it up before the results gets worse.10.3) Emotional intelligence will definitely help Johnson & Johnson avoid recalls because when the emotional intelligence of managers is high, they will be able to have better control on their nerves and they will perform without stress. It will also help them take right and timely decisions when it comes to quality and manufacturing so that recalls can be avoided. Emotional intelligence is a combination of a number of skills such as anger management, stress control, self awareness and so forth By mastering these emotions, managers can definitely play a role in escalating profits for Johnson &

Critically examine the differences between two theories of leadership Essay

Critically examine the differences between two theories of leading and examine the military unit that strategic drawing cardship can chip in on the succe - Essay Examplee main goals and objectives for the followers while in other cases the people may agree on their objectives and the leader go forth assume the responsibility of guiding them to reach to the goals (Paul, 2005).One way of trying to understand leaders is by using theories. Leadership theories argon assumptions about characteristics or styles of a leader. The theories are used to distinguish among different kinds of leaders and their styles as well as their motivations. The focus of the theories is to determine specific skills and qualities that differentiate one leader from his or her followers and other leaders. There are various theories in the study of leadership with each theory using a different onrush to explain the concept of leadership (Paul, 2005). Among the most common theories of leadership include conti ngency theory, great man theory, behavioral theory and trail theory.The most guiding light question that these theories aim at responding to is as to whether leaders are born or whether they can acquire leadership. This fundamental question has been the main puzzle that leadership analysts have grappled with over the years. A common understanding in leadership and management is that there is no precise answer to the question because every leader is different from the other, and while it is true that some individuals are born to lead, there are others who acquire leadership.This essay will explore two main theories of leadership and critically analyze them with a view of exposing their differences in their approach to leadership as a consequence. The main theories that this essay will endeavor to study are the trait theory and the behavioral theory of leadership. The paper will also explain the concept of strategic leadership. The paper will conclude by examining the effect of strat egic leadership in either success or failure of businesses.This theory is based on the notion that leaders are born with essential abilities and qualities that enable them to guide

Solutions of Slow Housekeeping Service in Palomar Hotel Research Paper - 1

Solutions of Slow Housekeeping Service in Palomar Hotel - Research Paper utilizationThe researcher states that the nature of the problem requires that the lag offering housekeeping services obtain more schooling as a way of enhancing their effectiveness in the delivery of this service to clients. The knowledge go out equip the staff with the necessary skills needed to ensure service delivery is up to date. The upgrading of the Property Management Systems leave behind also ensure that the failures in the hotel identified easily and countered before they lead to massive customer effects that would signal dissatisfaction that would lead to loss of customers. Through the death penalty of these two solutions, the problem at Palomar Hotel proves easy to detect earlier and handle to maintain the level of service delivery. From the above, the thesis of the study is to develop the two solutions of staff training and improvement of Property Management Systems in order to address the pro blem of slow housekeeping services at the hotel. Through the thesis, the problem will become addressed and will help guide the company back into proper customer management aspects that will conjure the service delivery and promote customer satisfaction. These argon all detailed in the body of the work that follows. The different solutions as provided in the introduction are the provision of additional training for the staff that deals with housekeeping services and the upgrading of the Property Management Systems (PMS). Considering these aspects in relation to the customer comments that reflected the time taken for cleaning a room, the delays in response to customer calls and the delays in delivery of towels that indicated delays in responding to customer needs, the company needs to ensure the situation improves to avoid major effects in customer satisfaction. Additional training to the staff to provide more refresher information to guide them in responding to customer requests wil l improve the level of speed with which housekeeping services are conducted. Additional training will enable the employees to learn how to handle customer request and the response to give to ensure the customer feels attended to.

Discussion Assignment Example | Topics and Well Written Essays - 250 words - 4

Discussion - Assignment ExampleIn this context, power exists in three phases i.e. coercive, reward, and legitimate.In the same way power is ofttimes realized when somebody in the organization or military setting has knowledge or ability in a given area oer the embossment of the people. In a way, the other people depend on such knowledge and this creates a sense of power in the person. Similarly, the ownership of rich information in a given field is normally a direct source of power that is often exercised by many people.In just about situations, it is often realized that power emanates from actions that are geared towards the five bases of power. These include reward, coercive, legitimate, and expert and referent power. Actions in these respects are normally very all important(predicate) sources of power and people are often drawn into situations of influence over the rest. In any case, the extent to which somebody exercises their power and influence over the rest is normally the most important parameter in the context of power dynamics. Power can always be used wrongly by legitimate people if structures are not in place to control such power. In the same way power has always been used effectively by other people create positive

State Coordinating Boards and accountability measures Essay

State Coordinating Boards and accountability measures - Essay ExampleIn order to enhance competitiveness in the international marketplace, Governments are increasingly focusing on the need to improve the quality of human capital that is the product of higher educational institutions. Therefore, there is an accountability that is being demanded of these organizations, through the use of away evaluation and performance measures to assess the quality of the education that is being imparted and the effectiveness of policy outcomes. These moves are posing a threat to academic freedom, which gives an individual the upright to study and teach whatever he or she wants to without threat of sanction Tierney, 1998, pp 41, even if this course of study may be such that it does not make for to maximizing scotch returns for the public dollars that have been funneled into education. Educational institutions have traditionally functioned with autonomy because that makes them more flexible and re sponsive, given their relative freedom from command and authorization by centralized Government..The result is higher levels of organizational innovation and more variety within national systems of higher education Dee, Henkin and Chen, 2000, p 204. However, increasing economic pressures leading to the corporatization of higher education are impinging upon the autonomy of educational institutions Bok, 2003 and pose a serious threat to the achievement of true academic goals Kirp, 2003. The affair between maintaining academic autonomy while simultaneously persevering policy accountability is an ongoing one, in which State Coordinating Boards are faced with the superior difficulties in defining their role and achieving the right balance between autonomy and accountability.

UPA Essay Example | Topics and Well Written Essays - 750 words

UPA - Essay ExampleAs the essay declares a theoretical model guides research and clarifies what exactly is the purpose of the research. Even though the research question tends to be misleading as the article progresses the purpose of research becomes clear. This research was base on a preconceived notion that the triage is higher when UAPs handle the emergency department. This can produce unbiased results. This also implies that the theoretical framework on the nurse theory constantly guided the author in her research. Not knowing what the real framework can be a problem. The author has used a developmental framework. This paper stresses that while the introduction of the subject tends to be slightly confusing in the beginning the problem has been properly highlighted in the text later. The course of the subject is efficiently handled. Current and proper references used and cited wherever necessary. The author is not trying to prove that her preconceived notions were correct. The actual results are quoted which revealed that contrary to whim the triage time taken by licensed nurses or by the UAPs did not differ. It convinces the reader to remove this myth from the mind that triage is faster when nurses handle it. Issues have been good argued and presented. References have been cited from various books on this subject. The author still suggests another study to compare the educational level of the triage nurse implying that this has not been covered so far. At times the author tends to contradict her own statements.

EARLY YEARS Numeracy-Investigation of Approaches to Calculation Essay

EARLY YEARS Numeracy-Investigation of Approaches to Calculation - Essay Exampleception class, reception teachers should be able to consider the information gained through assessment and poster and utilization of records passed on from playgroup and nurseries. The paper aims at investigating the approaches recitationd to teach mathematics in the reception class.Reception teachers should be able to identify the bookmans needs especially the ones who need additional support. Reception teachers use a variety of activities to help teach mathematics. Children atomic number 18 made to observe posture and numbers in the environment and daily routines. The children can also take part in board games and small constructions (Bruce and Martin, 2002). The use of finger games, songs and stories help the students to hit the books mathematical numbers. Shopping and cooking, outdoor playing, imaginative play and two and three dimensional work activities help students understand mathematic concep ts in the reception class.Arguments have lots ensued on various approach of teaching mathematics with respect to pedagogical debate targeting socio constructivism and behaviorism. Socio- constructivism is influenced by quasi empirical philosophies while behaviorism is influenced by foundationalist conception. teach mathematics to the reception class by utilizing rote learning is like directing an immutable body of knowledge and ideas to the students with an intention of making them understand without basing on reasoning. However, teaching mathematics in the reception class through application of the constructive approach makes mathematics a creative, cultural and creative activity and ensures learners are able to construct their own mathematical knowledge despite their different methodology (Guardiola et al, 2010). Relational understanding approach is applied in teaching mathematics in the reception class since it promotes understanding among the learners. Use of images such as th e base ten apparatus and empty number line together with use of models in teaching mathematics in

Empires Assignment Example | Topics and Well Written Essays - 750 words

Empires - Assignment ExampleWithout delving into the fact that the United States has a interpretive program government, devoid of an emperor or empress, it should also be noticed that even though the United States has a few territorial holdings around the globe, these are miserable with regards to the overall population that they hold and/or the geographic importance that they denote. As such, it will be the express function of this brief analysis to present the precept for why the United States, even though it oftentimes be countenances as a traditional empire, does not fit this definition and must therefore be regarded as another entity entirely.Firstly, it must be understood that the United States within the current era and the United States over the past 120 years is quite a different issue entirely. One could easily argue that after the conclusion of the Spanish-American war, the United States was firmly in a position in which it could express a degree of Imperial control ove r the populations of distant places such as Cuba, the Philippines, Guam, and various other holdings throughout the Caribbean. Interestingly, the United States soon divested themselves of these territories and only held on to a few scant islands within the Pacific and Puerto Rico. Further, with successful engagement in victory within the first and second world wars, the United States once over again was faced with an option of retaining the territory that it had gained or allowing for a type of new empire to be evidenced. It is at this juncture that it should be understood that even though the United States is not ascribe to a traditional understanding of what empire necessarily at first, it most certainly has redefined the power dynamic and created a daub in which economic empire is a reality through which the United States has come to control commerce on a global level. For instance, many scholars have viewed the situation within Japan as key evidence with regards to the way in w hich the

Barter and Monetized Systems of Exchange Essay Example | Topics and Well Written Essays - 1000 words

Barter and Monetized Systems of transfigure - Essay ExampleBarter and Monetized Systems of ExchangeDifferent items nurse worked as cash at different times. For instance, tobacco in America, chisels in China, gold until the gold standard was abandoned in the 1930s and paper currencies thereafter. Exchange of goods and services people produce and want is critical to satisfying the diverse wants of everyone. Such change is possible by using either the commerce organisation or money as a medium of exchange. Barter was prevalent medium of exchange before the advent of money. It remains the principal form of exchange even today in tribal and handed-down societies where political institutions are primitive, economies are not yet monetized and social systems are based on traditional practices and communal support. Even in advanced societies and economies like the United States, barter is still carried out to a limited extent in rural areas and globe and charitable activities. On the other hand, modern societies and economies like that of the United States rely on money as the main medium of exchange. They have the highly true political institutions to regulate money and prevent exploitation, manipulation and profiteering that money makes possible they have monetized economies that help create and accumulate wealth and they have social sentience that is necessary for people to trust an intrinsically worthless currency note, a mere piece of paper with the central banks assurance in small prints, as a medium of exchange. Barter and monetized systems of exchange have their own advantages and disadvantages (benefits and costs). ... Third, since commodities and services are exchanged directly, there are no unknown exchange fluctuation or balance of trade and payment crises. Fourthly, the division of labor is clear, as one produces what one has the means to do. Barters Disadvantages The barter system has several serious disadvantages too. First, coincidence of want s might be difficult unless the net is cast widely, which makes trade cumbrous. Secondly, in barter, there is no common footprint of value making it difficult to ascertain, say for instance, how many loaves of bread would be equivalent to a cow if one wants to sell bread and the other cows. Third, in many cases the commodity one wants to sell, as the cow here, might be indivisible to sell in parts to buy a few loaves of bread. Fourth, commodities and animals ofttimes deteriorate in value fast and cannot be stored for long, which makes accumulation and storing of wealth difficult. Fifth, barter makes deferred payment and contractual deal problematic. Sixth, transporting bulky commodities from place to place becomes cumbersome in a barter system of exchange. Moneys Advantages To avoid repletion of what has been already said, money takes care of all of the disadvantages of the barter system of exchange. sacristan describes the functions of money as a medium of exchange, a measure of value, as a store of value, and as a means of deferred payment (792-794). Briefly speaking, when money is used as a medium of exchange, there is no need for coincidence of wants. Money represents the common measure of value it is divisible into small and extended amounts it can be stored almost forever if it is put away safely it makes deferred payment easy and

Risk Assessment Continuity Plan Essay Example | Topics and Well Written Essays - 1250 words

Risk Assessment Continuity Plan - Essay ExampleSimply put, a persistence plan comes into play when internal or external factors influence the company to resort to emergency feats in order to continue providing services to their clients. In our case, the companys continuity plan comes into action if the database integrity is damaged or information security is compromised. As suggested by Britt (2005) a modular stepwise plan is most effective and that is how our continuity plan would function if the risk assessment recommendations are already established.Since our security is working on several layers, the continuity plan would also work in layers which will be connected to how bad a breach is made into our systems. For instance, if a hacker attack has been detected and we know that the hacker did not cut to go thorough any layers of our security, it would be useless to require a physical change of location for our database storage servers since it would simply mean that we are react ing to a fault much.On the other hand, if there is a fire in the building which damages the property to such an extent that business becomes impossible to continue in the same location, a secondary location must be activated where we can continue to provide services to our clients and maintain our operations at more or less the same levels. Similarly, a virus which brings down our network or corrupts the database stored on one location might require the activation of a secondary location where the most recent backup of the database could be used for continuity of business (Britt, 2005).As it has been ascertained by nearly every author working on the topic of continuity plans, backups are essential for a smooth recovery from disaster (Gouldson, 2002). An unintended disaster does mean a gorge or hurricane since in those cases a company may have a few days of warning to ensure that everything is in place to counter that. An accidental disaster can be natural, i.e. earthquake or li ghtning strike but

Nintendo Corporation Research Paper Example | Topics and Well Written Essays - 1000 words

Nintendo Corporation - Research Paper ExampleFrom this paper it is clear that the internal resources include the strengths and weaknesses while its external resources are the opportunities and threats. Nintendo Corporation should get through to maximize its strengths and opportunities while at the same time minimizing its weaknesses and threats. Nintendo Corporation boasts of strong, formidable and established brands in the world market. Most of its products are unrivalled putting it in a better position to command and dictate the taste of the market. For instance, the Nintendo 3DS that was initiated in the US in March 27, 2011 enables the users to view contents in 3D without ineluctably using the special glasses. Additionally, the Wii that comes in different advanced revolutionary features has made the product the best selling in the world. The great deal also has a gamy and steady escalation in its revenue. Although the company was founded in Tokyo, Japan, it now has branches i n all major world markets of the world. As of March 2011, the conclusion of their economic year, the entire military issue of the corporation was $ 4799.40. This shows that company stands in better grounds in terms of assets and strengths. Besides, Nintendo Co. Ltd. has a strong cash flow system. The operating cash flow indicates the get along of money in cash that the firm generates when it sells its products or renders the services. It is calculated by subtracting the firms taxes from the taxes. As of march, 2011, the cash flow for Nintendo was $ 1.19 billion. The major fault that this corporation faces is the shortages of inventories.

Screening a genome wide deletion library for mutants sensitive to Essay

Screening a genome wide deletion library for mutants sensitive to gemcitabine - Essay ExampleThe other caseful of repair is nucleotide excision. In the repair, the divisors are linked to DNA glycosylases (Wei & Chen, 2006). Some of these genes are listed thus UNG OGG1 MPG NEIL MGMT (Wei & Chen, 2006). The above image shows highlighted DNA genes Checkpoint Genes Damage can get along on the DNA during cell division causing it to cease dividing. This is what is referred to as checkpoint damage. Causes of the damage might be due to radiations such as from ultra violet rays. Checkpoint genes help in repair of DNA at the points of breaks through a response in the body. In the case of humans, these genes are defined as the G2 checkpoint genes. They take on RAD17 as well as RAD1. The other genes include HUS1 (Stern, 2002). Rad13 Rad13 is a human gene. Its product is involved in DNA repair and it gives rise to a nuclease which is christened Rad13 nuclease. The gene is about 80mm. the sys tematic identification of the gene is SPBC3E7.08c. Its characterization is also known. The following is an image of the gene (Caspari, 1985). The modern society has been taken through environmental changes that assume the adoption of genetic science. The results in this experiment shows the findings from a genetic renewal test carried out in the laboratory. What needs to be understood is the process that facilitates this face of reaction. Some factors need to remain constant during the laboratory experiment so that there is high accuracy and efficiency in the findings. It should be noted that the infallible temperature for tests and subsequent analysis and interpretation should remain around 250c 30oc for the specified time period. The two strains under question must also be cultured on a solid medium which will provide the best medium for a desirable outcome. In this test, the point of focus is the strain that grows on the EMM add-on NAT and EMM plus Hygromycin, but not on th e EMM plus CYH.This project touches partially on pharmacogenetics which majorly undertakes the studies on the role of inheritance in the variation in phenotypic response to drug. Such phenotypes ranges from serious inadequacy of therapeutic efficacy at one end to life-threatening unfavourable reactions of drugs the other end (PFEIFER, 2006). This test would be very applicable in genome-wide techniques in the clinical pharmacogenomic and its model systems which vary from yeast gene deletion libraries to cell-line based model. The validation of the candidate genes plus the cover of genome-wide technology is essential for following up the identification of the candidate genes. In the strategy for genetic model specification during the screening of the geno-wide, an easy-to-use Bonferroni-corrected method which is multipurpose in the sense that it fits both recessive and multiplicative model is found to be reliable if used. In the context of this experiment, it is better to have in-de pth understanding of what the classification of mutation is all about (SPENCER, 1997). On the basis of effects on structure, deletion is one of the most explored in this project. In so doing these mutations change the gene interpretation frame just like in the case of insertion. It is however imperative that people understand the unfitting opposite aspect of the two. Deletion is fairly haphazard while insertion constitutes a given sequence that doesnt necessarily take a random order. The results obtained from the experiments and tabulated were formatted in four distinctive columns for easy analysis. In this table there are theoretical short explanations and

Libya's Legitimacy Crisis Literature review Example | Topics and Well Written Essays - 750 words

Libyas Legitimacy Crisis - Literature review ExampleFinally, the article articulated how the can be resolved as it offer an ersatz of major author sharing as a way to end the legitimacy crisis and in effect, the conflict that is tearing the country apart.The paper presented the two opposing power which technically now operates as governments vying for Libyas legitimacy. One is the Tobruk based backed by the rump of the elected parliament, the House of Representatives (HOR). The other is based in the capital of Tripoli and has taken a de facto control over ministries the General National Congress (GNC) led by Khalifa Hifter, a former general in the Libyan Army with the agenda of purging the elites of the old regime and promoted the former revolutionary forces as the core of a new army.The theoretical positions outlined in the translation outlined in the readings contribute to the ongoing development of Foreign Policy Analysis by outlining in a simple manner how the division of Li bya begun, the forces central it and the backers that made the situation more complex. By clearly outlining how the division begun and how external influences and backers such as UAE and Egypt for GNC and Qatar, Sudan and Turkey for HOR, made the situation more complication that could go off the conflict more, the proposed solution became more feasible and plausible and even necessary. That is, to have a policy of noninterference that would include non-channeling of funds and arms embargo to go both parties realize that negotiation and compromise is the only way to go with the end in mind of forming a government that is based on power sharing and not on favoring one faction over the other.The arguments presented by Frederic WehreyandWolfram Lacher were also objective, impartial and sober that invites the reader to look at the problem from a conciliatory point of view and not from the persuasion of either party. Even the United States who are often

Impact of Government Spending on the Private Sector Essay - 86

Impact of Government Spending on the Private Sector - Essay ExampleAccording to the study the increase in in consumption expenditure by the organisation relatively results to an increase in investments. Notably, the optimum consumption expenditure is about 300000 unit price across the eld in UK. In the same manner, the U.S. government expenditure acts the same as the one of the U.K. Evidently, the steady increase in expenditure by the government has data track increased investment by the private sector to an optimum of about 2700 unit cash in. The difference arises by the fact that the U.K has consumes more in terms of cash than the U.S but the pattern is quite the same.From this discussion it is clear that the graphs indicates that an increase in government need for more goods, leads to an increase in production of the same by the investors. Hereby, when the U.K government spends more products for its citizen, the private sector has to increase their production in an almost sim ilar rate. Though not steady, the increased spending has in the years lead to the development of private sectors. Going by this fact, we can also note that the real U.S government expenditure acts the same way. However, the difference arises in that, the U.S government has a lesser consumption rate, the investment is relatively lesser compared to that of the U.K. In this manner we can conclude that the government spending on good positively affects the private industry. The government expenditure in terms of cash per gross domestic product in viewed in relation to the gross fixed great per gross domestic product. By the look of the table, one can clearly notice that there lacks a particularly distinct pattern to define the graph. each year has a different value in relation to the two per GDP/s. the lesser the government spends per unit GDP, the lesser the physical assets produced by the investors.

Slavery in the 1840s Essay Example for Free

Slavery in the 1840s EssaySlavery was always a controversial issue in America. Many people saw slavery as an essential part of life in every aspect, while others saw it as immoral and an injustice to humanity. In the 1840s when Manifest Destiny started to thrive, things began to heat up. in the raw territories meant a possibility for a new slave or free state in America. Because the new territories were allowed the right of popular sovereignty to determine whether they would arrest slaves or not, abolitionists and non-abolitionists were at a constant battle to gain a new state. Abolitionists wanted slavery diminished in America all together. They gave several(prenominal) arguments to prove their point on why slavery should be abolished. Firstly they state that slavery was un-Christian. The book of Genesis stated that man was created in the range of mountains of God, so all men were created equal. Thus they claimed that anyone that owned slaves would go straight to hell for no t adhering to the rules of God that all were equal. Another argument they came abtaboo was from a patriotic standpoint from which America was created. The Declaration of Independence states that all men are created equal, but with slavery and a flee for freedom in the nation, it was soft contradicted. As such, Abolitionists called slavery un-American. Another point made by the Abolitionists was that slavery gave unlimited power to slave holders.They could treat slaves however they chose because there were no laws defend slaves. This is turn corrupted their moral values, according to Abolitionists. In economic viewpoints, slavery was still cruel and wicked to Abolitionists. They argued that free labor would be more near because it would inspire competition and foreign investments and that it would appeal to immigrants. Abolitionists as well said that it interfered with free and open trade to the north, which were anti-slavery states and businesses. Another point made was that slav ery was politically monstrous because there was always a likelihood that salves could rebel. A massive slave rebellion would lead to a racial war in the United States that no one wanted.While Abolitionists argued their points, Pro-slavery groups gave very good rebuttals. Economically they argued that the end to slavery would kill the economy in the south. Without slaves in the fields collecting and caring for the produce, everything would collapse. Cotton, tobacco, and sieve would all fail in the economy and the south would be in economic ruin. Another point pro-slavery people argued was what would happen if slaves were to be let free. They stated that if they were freed, unemployment would rise and there would be an abundance of chaos. All of this would lead to uprisings and bloodshed. Yet another argument that the defenders of slavery used was influenced with muniment. They claimed that slavery had been a part of history for so long and had become a natural state of mankind. Hi story showed that Greeks, Romans, and English (until recently) had slaves and as such it was a part of human nature. Pro-slavery groups, like Abolitionists, also used religion and the bible as an argument.According to the bible Abraham had slaves, Paul returned a run-away slave in the New Testament, and Jesus never spoke out about slavery though it was widespread. Also non-Abolitionists argued that slaves were better cared for compared to factory workers in the North and the poor people of Europe. They indicated that slave owners would protect and help their slaves when they became sick or aged, unlike others who were left helplessly to fend for themselves once they were fired or released. The slave revolt of 1831 led by Nat Turner gave pro-slavery groups a lift in their arguments. It showed that slavery should be continued to keep the slaves in check and keep a watchful eye on them, to prevent any other disorderly events, which no one sought for.As is shown, slavery was a notorious issue that couldnt be ignored and slowly spiraled out of control. The issue sincerely started to get out of hand in the 1840s with new territories and the revival of religion in the Second Great Awakening. Both Abolitionists and Pro-slavery groups had arguments that caused a lot of uncertainty throughout the country, and especially in the new territories which were given the choice of slaves or no slaves. All of these arguments, issues and uncertainties that were widespread in the nation were the underlying cause of severe disunity in the midst of the North and South and eventually the Civil War.

Global Warming Essay Example for Free

Global Warming EssayGlobalization a process of integration among countries is taking place as a smart trend of the world. The cooperation among individuals and groups bring back many chances as well as challenges in different fields. As students in the era of integration, it is possible for us to name twain sides of this trend.First of all, it merchant ship not be denied that students can get many advantages from globalization. Thanks to the cooperation in education, different countries share new technology that comport teaching and learning with each other. Students are the objectives who can get benefits from it. They are allowed to explore new sources of knowledge through Internet and other education fabric such as video conferencing, tape recordings, etc. Therefore, they find that studying is not very difficult as they think, even it is more interesting.Besides, globalization promises them a frequently brighter upcoming. Students have more chances to get access to n ew skills, for instance, presentation skill, skill to work with a team, and so on, which are very invaluable for their future career. In addition, the interaction among countries creates a huge market with a plenty of choices of jobs. Thus, the graduates have more options to choose which job is the best and the most suitable for them.Go along with many advantages, students still have to face up with other difficulties. As being mentioned above, modern method of teaching and learning are bear-sized advantages for students but they may become obstacles when they are not very suitable and it must take a very long time to get familiar. For example, in many Asian countries, among which has Viet Nam, the feature of education is teacher centered. When this changes into learner centered which requires much of individuals responsibilities.At first, students who used to depend much on their teachers get take aback and fell it difficult to study with this new method. It may be a problem. W hats more, financial problem is another one. Under the impact of globalization, there has been a trend for the budge for education to be reduced. Government has to spend money on different areas so they do not pay as much attentionas before. As a result, the financial burden is placed on students shoulders and their families.In conclusion, globalization as well as other phenomenon has both sides good and bad. All the impact of this has effects on students. Students get many advantages but also disadvantages. They should be aware of this problem carefully so that they can turn challenges into chances which will support their studying as well as their future.

Social Impact Of Technology Information Technology Essay

Social Impact Of Technology In varietyation Technology Es stateFrom the development of Stone Age to modern applied science era, mankind is proving best in invention and innovation of new and latest engineering. Technology is a system based on the application of knowledge, manifested in physical object and validation form for attainment of specific goal (Volti, 2006). Advancement in technology has changed the existence of human civilization. Its not only the material benefits are being observed, even analytic thinking and outlook is immensely changed. Directly or indirectly everyone is obsessed to technology. As a fact that every coin has two side advantages disadvantages, this true statement is also observed in technology. Does technology going in right way or it will ruin our entire fellowship..?Headway in information processing system technology proves to be boon for whole humanity. First freely programmable computer was being invented in 1936 by Konrad Zuse (bellis, n.d.) t han after great advancement of computer technology was being observed till date resulting in palmtops and micro mini laptops with high capability and storage. Compute is nothing but electronic device to conceive out calculation and computation along with program accessibility. Firstly the concept of computer was developed to carry out calculation and in early fix up computer was with limited functions and user-friendliness. Before computer was invented many of the human force and time was being consumed to carry out one particular task. It was mechanical calculating machine which motivated inventor to think over the invention of computer with binary system and storage facility. With the passage of time new and newer innovation toke place and start and fore al close computer was being former with giant side almost equal to room along with vacuum tube as their electronic elements (encyclopaedia, n.d.) which advances in 1951, first commercial computer was create followed by entry of IBM- international business machines in history of computer (bellis, n.d.) than transistor based computer was formed with high speed and cheaper price. Integrated circuit technology and creation of microprocessor changes the total outlook of computer technology. 1st program language was formed in 1954 by John Backus IBM (bellis, n.d.). Along with such development a peak time in the history of computer was being observed by acceptance of personalized and home computer. The original internet invention in 1969 followed by invention of net kneading in 1973 changed the stance of computer and computer industries. This creation make a foundation a global country.To daylight application of computer is found in each and every field of society. In other words we can say that society is running on computer and computer application. Every single sector and every individual human activity include use of computer. outright a days computer is being use for banking by which you can make your transaction and maintain record of all banking information on diadem of your figure. Education is broadly developed with the application of computer. E-learning, E-Classes and E-teaching are enhanced with on-going technology. One can write more effectively by means of a computer. on that point are tools like spelling and grammar checker, thesaurus and dictionary, installed in the computer. Thus, it takes slight time to proofread a written document and also, there is no need to exonerated up a dictionary book to look for meanings of words. Typing is much faster than writing on a paper. If there is a need for reorganizing the sentences or paragraphs, one can cut and paste and make the necessary changes. Thus, overall a computer allows the user to create documents, edit, print, and store them so that they can be retrieved later Computer prove to be good entertainment device, high graphic games and high definition video quality can be own with that small manoeuvre. Current affairs and latest happening around the globe can be privileged from one click of computer. One of the remarkable achievements is in the field of communication and tuition. These fields have found a great diversification with the funding of computer and internet. Some of the impossible works in an organization are now possible with bless of computer, production process become fast and accurate. Intranet made transactions fast and prove to be convenient for work force. Weather forecast and future prediction relating to weather are being make by computer and other supporting equipments. Militaries development, safety of life and less healthy issue are being resulted with use of computer. Study of micro organization and research work for new invention and innovation with advance fitting are now possible. Every means of transportation from roadways to railways along with sae way enrich them self with computer technology for more speedy and safe transportation. cure for diseases and new med icines are formed after doing calculation of computer. Computer has made us life easy and convenient.Social impact of computer on society is truly at positive side. Computer affected commerce and it took it to new end. Now production process is being carried out with the help of computer robotic technology is use and accuracy is experienced along with much utilization of time and man force. Even at job level accountability of work by an individual increases speedy and fast out come and much big calculation are done in fraction of seconds. Production capacity increases and terms of production is decreased to a remarkable point. With online messenger and social website now to communicate with any one in any part of the world is really easy. Prior sending and receiving mails from one place to another was taking half and month but now possibility of live chats are practised and this made a world a small country social community website and group are being formed which give freedom to chat and act with each other and to understand one another society and culture.Effect on environment of computer is among remarkable note. New innovation is being done for preserving environment and its resources, use of paper is being reduced by E-filling and E-mails. Instrument and computer operated engines are formed to control pollution and whether for casting or climatical is being monitored and observed. With the help of computer best affected fields is medicine, as now some of the serious disease and injuries are cureable with the help of computer technology. middle transplant, bypass surgery, X-rays, CT scan, different diagnosis, heart rate, pulse rate etc. all is being monitored with help of computer (dhavele, 2010). Nowadays, computers are widely used for education and training purposes. In schools, computer education has been made compulsory to spread awareness about computers. As a matter of fact, computers have become a learning tool for children. Also, there are ma ny universities that provideonline degrees, which is very advantageous for those people staying in the remote areas and for the disabled. In fact,online educationis one of the most flexible and convenient forms of learning. One can take the benefit of suchonline degree programsstaying at home without the need of relocation. Computers are also used for training purposes. Many companies use them to train their staffs. With the help of computer has also become cheaper, quicker, and more efficient. We can now communicate with anyone around the globe by just now text messaging them, or sending them an email, for an almost instantaneous response. The internet has also opened up face-to-face direct communication from different part of the world, thanks to the help of video-conferencing (majja, n.d.). Computer is such a device which help and human being in each and every field in most possible way.Computer is such a gadget which is being found in every one home now. Some of the data showi ng contemporary computer application brought out by conduct of American Research Company are 65% of Americans spend more time sitting in front of their PC than with their wives and husbands84% of them are already addicted to computers,52% of PC users take their PC system ill as their personal failure Computer has changed out life of whole society and has created many convenient facility along with bless type technology in our day to day life. With all the positive effect of computer we can say those computers are BOON to whole mankind and to surrounding societyX=X=X

Toxicity and Autoactivation of Baits Experiment

toxicity and Autoactivation of Baits ExperimentAbstractAlternate conjoin in exon 47 of the Purkinje jail cubicleph nonp beilular telephone atomic human action 20 duct divisor rear endes a splice variant with a volt behind g every last(predicate)us insert (ggcag) before the stop codon in rat. This five base equal change the open discipline frame of the exon 47 for resulting in an ex decennaryded C-Terminal. Novel protein inter action at law at this region was hypothesised. barm ii mark System was employed to screen against c deoxyribonucleic acid program library to check for any protein fundamental interaction with 5 base pair insert region of exon 47. This assure aimed to test the perniciousness/ autoactivation of the crucifys in the yeast and to go on the minimum concentration of 3-AT (3-amino-s-triole) at which it inhibits the HIS3 factor. The experimental result shows that there was no holey twist of the HIS3 constituent. The autoactivation/toxicit y test results showed that the baits argon less toxic than the control bait. The turn outth of non-interacting colonies in the Triple Drop Out media revealed that a more defined media should be used, demanding the repeating of experiment to obtain more convincing results.1. Introduction1.1. nervous SystemThe human nervous corpse consists of the Peripheral Nervous System (PNS) and the fundamental Nervous System (CNS). The PNS is formed of the cranial nerves and the spinal nerves. The central nervous system consists of the point and the spinal cord. The mental capacity end be split up into trinity major(ip) parts cerebrum, cerebellum and the brain stem. The cerebrum is divided into frontal lobe, parietal lobe, occipital lobe and the temporal lobe. The of import subprogram of cerebrum allows controlling of sensory organ, motor function, soul 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 ar transmission of information to and from the brain (Bear et al, 2001 Purves et al, 2004 and Thompson,1993).1.2. Cells of CNSThe brain consist mainly twain flakewrites of cells nerve cells or neuron cells and the glial cells. The neuron atomic number 18 involved in the transport of electrical aims from the brain whereas the glial cells atomic number 18 thought to be the supporting cells of neurons by the uptake b be(a) of neurotransmitter that atomic number 18 essential for signalling between neurons (Henn et al, 1971 and Purves et al, 2004) and plays a constituent in synapto divisorsis of the neuron (Bacci et al, 1999). The glial cells ar of three casefuls astrocytes, oligodentrocytes and the microglial cells.1.2.1. Glial CellsAstrocytes argon star shaped cells. The spatial administration of these cells between the capillaries and the neurons enables it in the modifica tion of cellular responses, synaptic plasticity and survival of neurons (Abe et al, 2006 and Chen et al, 2003). Astrocytes attain in glutamate transport, removal of free radical, controlling of hae virtuallyasia of brain and in take holding 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 Longuem atomic number 18 et al, 1999).Oligodentrocytes are casing 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 cardinal proteins the proteolipoprotein (PLP) and the myelin sanctioned protein (MBP). (Colman et al, 1982 and Boison et al, 1995). At regular intervals myelin sheath becomes thinner and is cognise as Nodes of Ranvier (Peter et al, 1966). These regions are the site for electric potential gated sodium rail lines and a number of proteins. Microglial cells are the macrophages of the brain, which are formed in the bone marrow and are then(prenominal) transported to the brain by specialized protein called chemokines (Khoury et al, 2008) The paper of chemokine receptors is one of the measurable research areas in the patho divisorsis of humane Immuno Deficiency Virus. HIV prat target microglial cells for their replication (Albright et al, 1999 Ghorpade et al, 1997 and Meer et al, 2000). Microglial cells are to a fault studied for their inflammatory responses in the brain. The identification of affair and appliance by which microglial cells develop 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. NeuronsNeuron or the nerve cells are units of the nervous system involved in assign of electrical signal between each other(a) and to the achievementor cells. There are many fibres of nerve cells. Purkinje cells are one among them (Br own, 1991). The study of atomic number 20 ion crease of Purkinje cell is the subject of this project.The basic parts of neuron consist of a skeletal frame or cell body, axon, d hold onrites and neurites.All neurons are covered by the neuronal membrane. The soma or the cell body is kindred to any other geek 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 branch or unbranched. The main function of axon is to lurch the electrical signal from the axon hillock of soma finishedout the axon know 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 cognise as the synapse. The release of neurotransmitter is facilitated by synaptic vesicles of th e presynaptic end (one which release chemical signal). The neurotransmitters are released by the synaptic vesicle in the space between pre synaptic and berth synaptic terminal known as the synaptic cleft (Purves et al, 2004 and Brown et al, 1991). The neurotransmitters are then holdd by detail receptors of the post synaptic terminal which would elementrate an action potential in the cell. Apart from these receptors the ion impart 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 effectuate in the cerebellar region of the brain. The study of atomic number 20 ion lend of Purkinje cell is the subject of this project. Purkinje cells entertain a number of branch es dendrites that receive synaptic inputs. As the dendrites receive signals it instructs a Ca2+ signal, which are all important(p) routineary messenger in the cells. The dendrites are the region for a atomic number 20 ion submission finished the calcium ion enthrall. Similarly the soma contains K+ and Na+ expresss(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 exceedingly discriminating and they are maintained by the ion blood proteins of the Purkinje cell membrane and other neuronal membrane. These proteins form a focus on for the transport of ions. Techniques much(prenominal) as the post clamp method cast made the study of these ion impart easier (Bear et al, 2001).1.3. Ion pipelineIon bestows are glycoprotein complex that allow specific ions through them. The proteins of ion channel are calculated 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 potential gated ion channel. Ligand gated ion channel and the stretch and heat set forthd 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 advertize divided into atomic number 20 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, -amino solo ifyric acid (GABA), glycine-a ctivated occupations and the ryanodine receptor(Stroud et al, 1990). severally of these receptors bind to specific ion and are constitute in different organs. The stretch and heat activated ion channel respond to heat or morphological overrefinement of membrane (Purves et al, 2004).1.4. Voltage Gated calcium descent (VGCC)Ca2+ ions are important secondary messenger in cells and play important role in biochemical pathways of cell. The direct and entry of these Ca2+ ions in the cell is highly modulate. The regulations of these ions are controlled by the Voltage Gated calcium Channel (Gribkoff et al, 2006). These VGCC are mainly found in excitative 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 steganography channel protein, have been known to cause a number of diseases which hold Timothy syndrome, Familial hemiplegic migraine type 2, episodic ataxia type 2, spinocerebellar ataxia type 6 and autism spectrum unsoundness which are grouped under 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 affectionate calcium channel. These DHP peeled c hannels have dihydropyridine receptor for their covering fire (Campbell et al, 1988). Calcium Channel Blockers are now being found effective in the manipulation 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 structureA calcium channel consists of five important subunits 1. 2, , and . The 1 subunit is known as the pore forming complex (Yang et al, 2006).The 1 subunit is a single polypeptide and its functions mainly include voltage sensing, gating and discriminating permeation (Horn et al, 2000). The structure of 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 circulate I-II, circle II-III and loop III-IV dep ending on the domains they link (Dolphin, 2006).The intracellular loop of the 1 subunit has interaction site for the binding subunit. The interaction can modulate the G- protein, an important second messenger in the cell (Dolphin, 1998). The specific binding of subunit to the tryptophan residue is important for controlling the gating of 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 get depolarised. S4 segment is positively charged due to the presence of arginine aminoacid making it voltage sensitive by trans stance 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 1 subunit. The ion conductance partly depends on the rotational movement of the S4 segment which either cause th e S6 segment to open or close the pore (Horn et al, 2000).The subunits of the calcium channel are thought to be tissue specific and organ specific. Primarily they are of 4 different types, 1, 2, 3 and 4. Different isoforms of the subunits also do be which include (CaB2a, CaB2b and CaB3) (Hullin et al, 1992 and Petegem et al, 2006). Their association with subunit is essential for modulation of VDI, CDI and CDF (Petegem et al, 2006). The 2 subunit is also known as the 2/ subunit as both the subunits are output of a single gene (Petegem et al, 2006). The 2 and subunits are linked together by disulphide bonds. Like other subunits 2/ 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 2/ subunit, but their binding affinity varies with different isoforms of the subunit (Luo et al, 2001 and Luo et al, 2002).The subunit is found only in skeletal muscles. Their practicable 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 elaborateness 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 center by the localisation of C-terminal fragments (Kordasiewicz, 2006).1.6. Calcium Channel TypesCalcium 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 interdependent 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 Dependen t Facilitation (CDF) allows for the entry of calcium for signalling (Petegem et al, 2006).Based on the amount of current needed 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 -conotoxin GVIA (Petegem et al, 2006). The current found in Purkinje cells of the cerebral cortex were P-Type, they were sensitive to -agatoxin IVA. The Q-Type current are found in granular cells, however scientist consider P-Type and Q-Type to be equival ent and are now term as P/Q- Type. The difference between the P Type and Q-Type is thought to depend on the subunit to which it is associated(Dolphin., 2006). another(prenominal) 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 elementThe alpha sub unit of the calcium channel are coded by 10 genes, therefore 10 different 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 Cav1A 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 tri radical repeats. In humans the extension of the may digress 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 preponderating type of migraine caused by the missense vicissitude in CACNA1A. Three different varietys 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 h as 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 1A subunit which might cause a complete loss of the function of the channel (Wappl et al, 2002).1.8. Spino Cerebellar Ataxia 6Spino 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 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 expressage to the C-Terminal. As the C-terminal is site for protein- protein interaction, changes in force play of interaction or changes in in teracting 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 aggregate 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 fill in the C-terminal of the 1A subunit of the calcium channel. The isoforms are formed by a process known as the pre-mribonucleic acid stand out splicing.1.9. SplicingTranscription of messenger RNA ( mRNA) from DNA and variation 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 significantly 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 o f snRNAs, catalytic metal ions, enhancers, transcriptional coregulators and serine/arginine easy 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 deep down 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 combi ned to translate for a single protein (Bonen, 1993 and Bonen, 2008). varynating(a) splicing is a mechanism by which a few genes produce innumerable proteins that are alter 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 regard 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 RNA world. The importance of isoforms has been understood through a number of studies. The Active and inactive forms of devolve on deadly 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 symptomatic approach and cance r 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 bracing 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 variantsVariants in calcium channel protein, in particular the 47 exon of the c-termi nal 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. cardinal 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). set 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 retention 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 interposition (ggcag) was account 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 aloofness 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 confirm by Kanumilli et al (2005).Another separatist 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 inform 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 order of exon 47 were same as the rat pancr eatic 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. past 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 t reatment of SCA6.1.11. Protein protein interaction studiesProtein-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 top 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 both 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 brass of a newsperson gene (lac-z) in yeast is being exploited in Y2H. The lac-z gene expression gives our -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 consolidated 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 fair game. Bacterial plasmid can be easily constructed to express optical fusion protein of interest. The bacterial shuttle vector can be set-apart 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 aim 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. two the systems have their advantages and their disadvantages (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 enhance 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 t he 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. AimThis 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.Baits5inSER is a 472 base pair length protein with ggcagNLSER is a 397 base pair length protein without the Nuclear Localisation Sequence. It was constructed to find the importee of the nuclear localisation signal (Surya, 2008).The aims of the project areTo test for toxicity and autoactivation of baits. To determine the concentration of 3-AT at which the expression of Histidine gene is inhibited. realise coupling experiment.3. Materials and Methods3.1 . Control Mating3.1.1. Control strainsThe 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 striving were provided. For ban control the bait strain was L40 with bait pBTM116/GluR2 and the target was the same Y187pGADT7-T The negative control bait was obtained by the transformation of L40 with the plasmids isolated from provided E.Coli conclusions.3.1.2. Small collection plate Yeast TransformationA colony of Saccharomyces cerevisiae L40 yeast was inoculated into 10ml of YPAD media. It was left long in a shaking incubator (200rpm) at 30 C. The overnight culture was diluted in 50 ml of YPAD to an OD600Toxicity and Autoactivation of Baits ExperimentToxicity and Autoactivation of Baits ExperimentAbstractAlternate 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 ra t. 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. Introduction1.1. Nervous SystemThe 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 CNSThe 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 tho ught 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 CellsAstrocytes 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 responses in the brain. The identification of role and mechanism by which microglial cells caus e 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. NeuronsNeuron 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 t he 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 (Purves 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 rece ive 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 channelIon 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 channe l, 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, -aminobutyric acid (GABA), glycine-activated convey 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, neurotran smitter 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 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 binding (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 structureA calcium channel consists of five important subunits 1. 2, , and . The 1 subunit is known as the pore forming complex (Yang et al, 2006).The 1 subunit is a single polypeptide and its functions mainly include voltage sensing, gating and selective permeation (Horn et al, 2000). The structure of 1 subunits consist of 24 segments (S1-S6) which con stitute 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 1 subunit has interaction site for the binding subunit. The interaction can modulate the G- protein, an important second messenger in the cell (Dolphin, 1998). The specific binding of subunit to the tryptophan residue is important for controlling the gating of 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 connectin g the S5 and S6 segment forms the boundaries ion conducting pore of the 1 subunit. The ion conductance partly depends on the rotational movement of the S4 segment which either cause the S6 segment to open or close the pore (Horn et al, 2000).The subunits of the calcium channel are thought to be tissue specific and organ specific. Primarily they are of 4 different types, 1, 2, 3 and 4. Different isoforms of the subunits also do exist which include (CaB2a, CaB2b and CaB3) (Hullin et al, 1992 and Petegem et al, 2006). Their association with subunit is essential for modulation of VDI, CDI and CDF (Petegem et al, 2006). The 2 subunit is also known as the 2/ subunit as both the subunits are product of a single gene (Petegem et al, 2006). The 2 and subunits are linked together by disulphide bonds. Like other subunits 2/ 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 e t al, 2001). Gabapentin is a drug known to act on 2/ subunit, but their binding affinity varies with different isoforms of the subunit (Luo et al, 2001 and Luo et al, 2002).The 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 TypesCalcium 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) respon sible 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 -conotoxin GVIA (Petegem et al, 2006). The current found in Purkinje cells of the c erebral cortex were P-Type, they were sensitive to -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 thought to depend on the 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 GeneThe alpha sub unit of the calcium channel are coded by 10 genes, therefore 10 different 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 Cav1A 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 C av 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 1A subunit which might cause a complete loss of the function of the channel (Wappl et al, 2002).1.8. Spino Cerebellar Ataxia 6Spino 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 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 aggregate 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. T his invokes an interest in the C-terminal of the 1A subunit of the calcium channel. The isoforms are formed by a process known as the pre-mRNA alternate splicing.1.9. SplicingTranscription 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 differen t 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-spl icing 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 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).1.1 0. CaV2.1 splice variantsVariants 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, 2 004 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 pat hway is expected to give more insight the channel and possibly a new perspective in the treatment of SCA6.1.11. Protein protein interaction studiesProtein-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 (Smit h 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 -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 re porter 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 disadvantages (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 onl y 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. AimThis 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.Baits5inSER is a 472 base pair length protein with ggcagNLSER 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 areTo 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 Methods3.1. Control Mating3.1.1. Control strainsThe 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 Y187pGADT7-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 TransformationA colony of Saccharomyces cerevisiae L40 yeast was inoculated into 10ml of YPAD media. It was left overnight in a shaking incubator (200rpm) at 30 C. The overnight culture was diluted in 50 ml of YPAD to an OD600