My Vocational Profile Essay Example | Topics and Well Written Essays - 500 words

My Vocational Profile - Essay Example No one else can set the principles of one's ethics other than oneself. I give respect to people irrespective of their social standings and demand the same from others; though not in a reciprocative manner. Another thing is that I value work no matter however small and whatever kind it might be. Presently I am working part time as a waiter in a restaurant because it helps in getting some income and gives me a new opportunity to meet new people all the time. The activities of my interests might sound to be somewhat common but this is what they are. I like to visit new places and meet new people; especially people who are unlike me in thinking, in behaviour and in lifestyle. I try understand why they are what they are. I like to travel to different places, possibly alone, which gives me enough time to think and digest what I see. I like to learn new things from different sources. Reading also interests me a lot. The kind of readings includes current affairs, business trends, readings about various personalities and science related readings regarding latest discoveries and inventions. My dreams are the biggest source of my motivations. I am ambitious by nature.

Strategic Management Process Paper Essay Example for Free

Strategic Management Process Paper Essay The strategic management process consists of four major components; they are environmental scanning, strategy formulation, strategy implementation, and evaluation and control. They each are broken down into their specific roles within the strategic management process. The Environmental Scanning is the monitoring, evaluating, and disseminating of information from the external and internal environments to key people within the corporation. An easy way to use this component would be SWOT, this acronym stands for Strengths, Weaknesses, Opportunities, and Threats and it shows the strategic factors for each individual company. The next component of the process is the Strategy Formula and that is the development of long-range plans for the effective management of the environment opportunities and treats, in light of corporate strengths and weaknesses, this process also relay’s on the SWOT process. This process helps define the corporate mission, helps specifies achievable objectives, helps develop strategies and helps setting policy guidelines. Strategies Implementation is the process of which strategies and polices are put into action through the development of programs, budgets, and procedures. This process can cause changes to happen within a company such as, overall culture, structure, and management system of the entire organization. When a corporate wide change is needed the implementation of strategy will be conducted by the middle and lower level management, and then the higher management team would review those changes. This can be called operational planning as well and that would implement day to day changes. The last process of the strategic management process is evaluation and control; this is the process which corporate activities and performance results are monitored so that the performance that is happening now can be compare to what is expected. This would apply to managers on all levels and  they use this information to take corrective action and resolve any problems. This part of the process can also pinpoint weaknesses in previous implemented strategic plans and can cause the process to start all over again (Wheelen Hunger, 2010). Strategic management is important for many reasons. First of all these processes help leaders to implement the vision and mission of the company, and what goals are needed to get the company where it wants to go. Typically created by the owner or top management in a company, the strategic management plan offers direction and guidance to the employees, sets up measurable goals and time lines and designates duties of all personnel. Marketing and sales projections are included in the strategic plan as well as plans to review the achievements of each department (Ray, 2012, p.1). BMW Strategic Process BMW is a German automobile and motorcycle manufacturer. BMW automobiles are considered luxury vehicles with endless amounts of options and expensive price tags. The company, which was at one time was severely struggling financially is extremely successful. The organization continues to sell more care almost every year, created strong brand recognition, and a loyal customer following. BMW created what they call corporate Strategy Number One; this includes plans for the future, survey results, and performance evaluations. The purpose of the strategy is to pursue the vision of being the leading provider of premium products and premium services for individual mobility (BMW Group†, n.d.). The strategy continues to describe the objective and how BMW plans to reach those objectives. To clarify the organization included the results from surveys distributed to stockholders and employees. The challenges the company faces were also clearly laid out, along with previous achievements. Conclusion After examining all the necessary elements to a strategic management process one can understand why each is necessary to achieve success. The elements work together and when done properly do so with little effort. However, to deflect any challenges from competition the elements need examining frequently to see if changes are necessary. BMW provides an excellent  example of an organization with solid strategic management. The organization has clear-cut processes and evaluates current process and is looking to the future. References: Wheelen, T. L., Hunger, J. D. (2010). Concepts in Strategic Management and Business Policy Achieving Sustainability (12th ed.). Retrieved on September 15, 2014 from The University of Phoenix eBook Collection database. Ray, L. (2012). Strategic Management Process, eHow Contributor. Retrieved September 15, 2014 from http://www.ehow.com/how-does_4964358_strategic-management-process.html BMW Group. (n.d.). Retrieved on September 15, 2014 from http://www.bmwgroup.com/e/0_0_www_bmwgroup_com/verantwortung/publikationen/sustainable_value_report_2008/_pdf/SVR_2008_engl_Kapitel_1.pdf

Toxicity and Autoactivation of Baits Experiment

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

The Design of Taipei 101 Essay -- Structural Design

Stiffness and comfort (steel outrigger trusses) The 101 story building, 500 meters high Taipei 101 is located in Taipei, Taiwan. Taipei 101 is the pride and identity of the people of Taiwan. The building represents â€Å"concept of striving for beyond perfection† (Taipei 101 anon 2005) as it outsmarts nature with its technological features. 101 building achieved a remarkable world record for the highest building in 2004 with 101 floors hence the name Taipei 101. Designing and building the 101 building was very challenging and rewarding, as it is located in one of the most hostile places in the world. The tall building is located in Taiwan, which is constantly under attack by Mother Nature, earthquake potential and typhoon winds are just common problem which the architect, C. Y. Lee & Partners, and the structural engineers Thornton Tomasetti Inc needed to solve. The engineers of the Taipei 101 made the impossible possible (Shieh et al n.d). The initial plan was to make three building with 60 storeys building in centre, but t he 60 building soon turned into 101 storey single tower building to meet investor’s demand. In order to be the most unique skyscraper in the world Taipei 101 building represents its culture (Taipei 101 Binder, 2008). In this part of the essay we are going to investigate about how is stiffness and comfort maintained in Taipei 101, we’ll be looking in depth some of the features of Taipei 101 that work together to provide stiffness and comfort. Firstly we will be looking at the stiffness and then move on to comfort features. How is Stiffness and comfort maintained by Taipei 101? To provide comfort the 101 building has to deal against the nature. Taipei 101 is located in a hazard prone environment. Typhoon and ... ...tlUvYTVMMmicsorE9tDlv1VgdZWCJDAKPfx3AOTuaIEUkm4piLesN_8NVrM131TbT8wyyRQ_0MIcIx7yVN5ho3D9NbHoZjiAx477&sig=AHIEtbQNaZZ1eKBA82LxMn_p6RZZMKxmvA&pli=1 (accessed 3.4.11) †¢ Anon. what are steel moment frames? (N.d). [online]. Available from: http://www.propertyrisk.com/refcentr/steel-side.htm (accessed 03.04.11) †¢ Brooks. C., Huff .I. The effect of high altitude or relative humidity. (2006). [online] available from: http://www.physics.umt.edu/borealis/RH%20Lab%20Report_06.pdf. (Accessed: 03.04.11). †¢ Anon, Taipei 101 expected to receive LEED platinum certification( 2010).[online]. Available from: http://www.taipei-101.com.tw/NEWSV/en/news_in.aspx?News_Sn=24 (accessed 03.04.11). †¢ Mizuguchi.h., nakagwa.t. and fujita.y. breaking the 1000mom barrier. (2005). [online]. Available from: http://www.elevator-world.com/magazine/PDF/sept05/sept05.pdf (accessed 03.04.11)

The Last of the Mohicans as a Mixture of Genres Essays -- English Lite

James Fenimore Cooper's The Last of the Mohicans as a Mixture of Genres James Fenimore Cooper's The last of the Mohicans is often seen as a simple adventure story within the historical frame of the French and Indian war. Only if we analyze the novel in a closer way, we will realize that it goes beyond this label and that its sources are many and varied, giving the work the richness of the genres on which Cooper's novel is based. These are romanticism, western, (being its author one of the forerunners of these genres in the U.S.A.), captivity narratives and epic. In works belonging to Romanticism, nature is given a great important role. In fact, the action takes place in the open air, except for the chapters of the siege of Fort William Henry, so it is the setting which predominates along the work. The close connection between the characters of romantic novels and nature is exemplified in the characters of Chingachgook, Uncas and Hawkeye, which apart from knowing the place where they live and being completely adapted to it, they consider nature as a divine entity. In his introduction of this novel in the Oxford Classics edition, John Mcwilliams agrees with this affirmation of the concluding that for Cooper it was more than the place where they move; 'it was the very condition of life, the shaper of moral values and of human behavior, for good and for ill'. In a similar way, the same happens in other important romantic American novels such as Nathaniel Hawthorne's The Scarlet Letter and Herman Melville's Moby Dick. In the former, Hester and hi s daughter Pearl live in close connection with nature as a source of moral freedom, and in the latter, the Nantucketeers consider themselves as part of the sea. The theme of nationalism, a... ... In conclusion, The Last of the Mohicans does not belong totally to a particular genre since it displays some key aspects of epic, it is one of the most important forerunners of western and inherits some aspects of characters of captivity narratives, facts which help to create an special example of romantic literature close to Herman Melville's Moby Dick, works which seem to be adventure stories but in fact they are richer than that due to both their quality and the variety of their sources. WORKS CITED Cohen, Hennig and Levernier, James eds. and comps. The Indians and their Captives. Wesport, Connetcticut: Greenwood Press Inc, 1977. 299 pp. Cooper, James Fenimore. The last of the Mohicans. 3rd ed. Oxford: Oxford's World Classics., 1998. 433 pp. LÃ ³pez Estrada, Francisco. Poema del Cid. 12th ed. Fuenlabrada, Madrid: Odres Nuevos, 1986. 164 pp. www.britannica.com

Occupiers Liability Essay examples -- Papers

Occupier's Liability Objective The objective of this document is to outline the obligations of the RSPB, as occupier of property. The obligations are set out in two Occupier's Liability Acts 1957 and 1984, and are owed to persons who enter RSPB property either as licensed visitors, or as unlicensed trespassers. The document does not purport to cover every particular situation and those in any doubt should consult Legal and Compliance as to secure their own position. Overview The RSPB owns large quantities of real estate, including many public reserves. It also owns many offices to which employees have access. As Occupier, the RSPB owes a duty of care to any persons who use RSPB land, irrespective of authorisation. Legal Analysis RSPB is the Occupier for the purposes of both the "Occupier's Liability Act 1957" and "Occupier's Liability Act 1984." This is because the RSPB as an organisation has immediate supervision and control and the power of permitting or prohibiting the entry of persons onto its land. Thus, the Law imposes equitable liability upon the RSPB for its defective premises and death or personal injury (and loss to property) that incurred by persons upon its land This responsibility upon the RSPB to secure the welfare of persons upon its land, is affected by the status of such persons. Indeed, the duty owed to lawful visitors is of a higher standard than the duty owed to unlawful trespassers. However, in accordance with the law, 'reasonable measures' need to be taken to secure the welfare of both groups. The Duty of Care owed to Lawful Visitors Section 2 (2) of the Occupier's Lia... ...tandard of care owed differentiates. The standard for an invited person is higher as the RSPB undertakes to secure his welfare, the standard for a trespasser is objective and thus lower, as the trespasser is deemed to be an average person, and no regard is given to the idiosyncrasies of the trespasser: age, mental health. Evaluation The RSPB must take all reasonable measures to ensure its grounds are reasonably safe and that any disclaimers of safety offer greater protection to the visitor rather than just highlighting the risk. However, for trespassers, there must still be no overtly dangerous objects/circumstances upon RSPB property, though regard need not be given to particular characteristics of trespassers. Moreover, disclaimers ought to warn of the dangers but not necessarily offer any alternatives.

Marketing Framework

What Is It? Marketing Framework Market Research and Buyer Behaviour Understanding the behaviour of buyers in your target group is crucial in marketing. Now more than ever, success is dependant upon market research and identifying and meeting customer needs at a profit. ‘Zara' have recently become the world's largest clothing retailer, ‘by focusing on understanding consumer needs and behaviour and by providing value to the customer'. Through intense market research, they developed an important knowledge of their target market's buying patterns and recognised the need to constantly update their styles.Furthermore they understood new concepts such as fast fashion' and ‘disposable clothing, the idea that teenagers and young adults craved new styles quickly and cheaply. It is now more important than ever for marketing managers to respond swiftly and decisively to changes in consumer patterns. Nowadays, an excellent method a company can monitor their target market and con duct extensive market research is through social media. Never before have enterprises had such powerful technologies to interact with customers.Almost every company has their own ‘Facebook or ‘Twitter' account (combined with ver one billion active Facebook users globally), where they can receive immediate customer feedback about the faults in their product online. Furthermore, customers can chat online about these products. 53 percent of people on Twitter in Ireland recommend companies and/or products in their tweets'(ROl Research for Performance, June 2010). ‘Calgon' dishwasher tablets recently produced an advertisement for television, where two women chat in an online forum about their dishwashers.The first woman complains about her dishwasher, while the other recommends she starts using ‘Calgon' tablets. Calgon certainly demonstrate their nderstanding of the importance of Word of mouth'; recent statistics show that 84 percent of customers trust peers over advertisments. Social media is both an opportunity and a challenge for businesses. Consumers used to have to go looking for the product, but now the product will find us, with excess information hurled at us from every direction! We don't have a choice on whether we do social media, the question is how well we do it. There are many other concepts which are particularly noteworthy when developing a successful marketing strategy. Marketing managers must take into account the ountless amount of factors that influence consumer patterns and buyer behaviour, trom gender and age, to social and economic backgroun s c ae O'Leary, CEO ot Ryanair, flourished in recognising an untouched market segment of consumers looking for low quality air travel at exceptionally low prices by comparison to the likes of Aer Lingus.He understood that people on lower incomes neither needed nor wanted to pay high prices for comfort, such as more leg room and nice meals on flights. This has become even more evide nt since the recession, with Ryanair climbing above high fair competitors. O'Learys market strategy succeeded because of is insight and understanding he had of the buyer behaviour of his target group. Segmentation, Targeting and Positioning Marketers developed the processes of segmentation, marketing and positioning so that they could understand their customers and their buying behavior. Segmentation is the first step in this process of product promotion.The goal of market segmentation is finding out where the firm should compete and in what segments should they focus that will result in a comparative advantage. Segmentation leads to comparative advantages in a number of ways. Firstly, it reduces rivalry due to reduced competition in any given segment. It allows for efficient resource allocation as resources can be effectively focused on concentrated targets. Finally it can allow a firm no matter what size to become the product of choice in the given segment. Target marketing is at its most basic level is when a firm directs its marketing message at a specific market or segment.It means that a group of customer has been chosen to whom the firm will direct most of their marketing resources, times and attention. Target marketing means that firms avoid wasting resources and time by giving the consumer what they need or want rather than making a product that the irm believes the consumer needs or wants. Finally positioning, which requires the firm to establish the brand in the minds of the consumers in order to maximize the potential benefit. Businesses use positioning analysis to understand how their target customers see their product and to see where they are in relation to their competitors.All three of these components must be completed correctly or else the whole marketing campaign will be destined for failure. Branding Branding plays a major part in the marketing world. Branding exists so as to differentiate a company's product or service from that of its om petitors. It performs many roles both for the consumer and for the business. In relation to the consumer ,brands allow the consumer to shop with a reduced level of risk due to familiarity with the quality of each brand and this also would lead to a reduction in time spent shopping.Concerning businesses, branding performs a lot of valuable functions. The main function, is brand loyalty as this leads to predictability and security of demand, increased security against competitors and can allow the businesses to charge higher prices. Other roles it plays are, that it offers legal rotection due to trademarks and it can give the business a comparative edge . Companies use brand to install an image in the consumer's head when they hear of ything associated witn that brand. To make sure this is a success they must match all the elements of marketing, such as pricing policy, distribution methods etc. to the image that this wish to create. 7 P's Of Marketing An essential element of the marke ting strategy is the marketing mix. Comprising of product, price, place, promotion, process, people and physical evidence, it allows enterprises to deliver value in conjunction with satisfying consumer needs. Firstly, the product is some good or service that a company offers in the market.. A product satisfies a customers' want or need, and to what degree depends on the quality of the product which is usually quite subjective.Price is the next P and it denotes the monetary value a good is exchanged for. Price plays a major part in the decision making process of consumers. High prices, usually convey a luxurious consumption experience contrasted to affordable low prices offering basic products. Enterprises communicate their products to their target market through adopting and implementing a promotional strategy. Promotional activities seek to inform, persuade and influence the target market. Strategies include; branding, personal selling and advertising.Following on, place is the loc ation of the transaction between the consumer and business, ultimately it is the accessibility of the product to the consumer. There are various channels of distribution both direct and indirect an enterprise may adopt, in order ensure its product reaches the consumer. During the provision of a service, it is the people or staff who exert a major influence as to the outcome of the consumer experience. Therefore, staff must be adequately trained nd equipped with the necessary skills, approach and attitudes in order to achieve the desired outcome an enterprise strives towards.The process an enterprise implements is the way in which business is conducted within an organisation and is seen through the flow of activities. For example, customer-cultivating companies are designed to serve customers and customer segments, this is one part of a potential business process, as seen in the Zara fashion retail example. Finally, physical evidence is how a business presents itself to its target ma rket. It is the outward appearance of the organization and creates an impression on the consumer.