Wednesday, July 31, 2019

Kamprad’s Resourcefulness: Ikea

1. Kamprad’s careful stewardship of resources exemplifies his abilities as a manager. By practicing frugalness and economy, he was able to maximize profits by keeping costs low while also being able to continually innovate his growing company. While to some he may appear frugal, he has created a nest egg for economic downturn and well as creating a strong ethic of financial stewardship within his organization. Planning and problem solving became both the framework for IKEA and its source for innovation.As a leader, Kamprad again instilled his own personal values into every fiber of IKEA. Effective cost cutting, hard work, and dedication. Aside from just cutting costs however, being influenced by the Bauhaus movement he also wanted to combine cost saving with a sense of style. (Chaundey, 2000) In addition, the welfare of his employees has been a main concern. (Nelson and Quick, 2011) I believe that Kamprad was (and is) a strong leader and manager for IKEA, and I believe that th ese principles will be carried on in his sons to future success in the marketplace. 2.The followership of employees within IKEA can be directly linked to the Leadership Grid in Fig. 12. 1. (Nelson and Quick, 2011) From the structure of the grid, Kamprad would be a 9,9. Kamprad’s leadership style calls upon and motivates the team members within the company to take initiative and a personal stake in the company. They are hiring not just into a job but into a lifestyle and way of thinking. The combination of careful stewardship with concern for employee welfare creates an environment that gives employees both a responsibility and a sense of community. Etzioni, 1960) Thus IKEA employees are empowered and feel like they are part of something. In this way IKEA ends up with a staff of active independent, critical thinking individuals. (Nelson and Quick, 2011) This overall will drive employees to greater creativity and productivity for the company they care for and cares for them. 3. The case does not give specifics about Kamprad’s actions as a transactional leader, although the narrative would lead one to believe that they style would be uncharacteristic of him. Although Kamprad’s traits as a transformational leader are unquestionable.Kamprad has given employees not just a goal but a way of living. (Davis, 2006) Kamprad himself dresses simply, eats at cheap restaurants and flies economy class. (Chaundey, 2000) Kamprad innovated and redefined ways of doing business repeatedly throughout his tenure as CEO of IKEA. From the mission statement of ‘create a better everyday life for the many people’, IKEA has been built upon a pioneering spirit. (Gowan, 2009) By designed a business plan that allowed IKEA to work directly between manufacturers and customers to keep costs low. ibid. ) By designing the flat boxes and shipping furniture to customers that they assembled themselves, Kamprad has been able to keep cost down. It is these business te chniques along with his uncompromising ethics that he has instilled into the company and led it to greatness. Kamprad’s charisma is a quiet sort largely involving a ‘lead by example’ technique. Employees are drawn to Kamprad because of his strong ethics and personal adherence to them. Kamprad’s philosophy is not just words at board meetings but the way he lives his life.This gives employees a stronger sense of connection with the boss, as he is still very close to his modest roots. The lack of title and privilege within IKEA also reinforces this. (Davis, 2006) References Chaundy, Bob (2000) IKEA’s self-assembled billionaire. BBC News. July 28, 2000 Retrieved from news. bbc. co. uk Davis, Paul (2006) Billionaire Attributes – Modesty: Ingvar Kamprad, Ikea’s Founder and Owner. Retrieved from ezinearticles. com Etzioni, Amitai (1960) Two Approaches to Organizational Analysis: A Critique and a Suggestion.Administrative Science Quarterly, Vo l. 5, No. 2 Gawor, Katarzyna; Halasova, Sona; Polzin, Friedemann (2009) International Business Strategy of IKEA – Activities of the Multinational Furniture Retailer. University of Economics Bratislava Nelson, D. & Quick, J. (2011). Organizational Behavior: Science, the RealWorld, and You. (7th Ed. ) Mason, OH: South Western. Cengage Learning. Etzioni, Amitai (1960) Two Approaches to Organizational Analysis: A Critique and a Suggestion. Administrative Science Quarterly, Vol. 5, No. 2 Retrieved from www. amitaietzioni. org

Tuesday, July 30, 2019

Who is a Hero for You?

UlEverybody may be confused to what would be the answer with the question, † Who is a Hero and How can you be a Hero? â€Å". From the word itself, Hero means a person with a powerful body, a strong open mind, and someone who lives according to their values and ambitions. So, who really is a Hero and how you can be one of it? Is it those who made a revolutionary cause to impose away the non-native colonizers?Or is it someone who did a silent and solemnizes way to drive away those who omitted an immoral deeds to our beloved country? Every person in this world can be considered a hero in his own littlest or largest great way. For there are countless ways and reasons to be done and to be one. In our nation, we used to give an account to those who fought for our freedom. Yes, I also consider them as one but whom I supposed to look upon is the one who faced a lot of trouble and excruciation not only from exterior but from within as well and still, made a way to set It aside and soa r upon it.It is not about being a hypocrite for we are taking a subject about a roes life, but it is about my realization to whom I should consider a hero according to the discussions and readings that we have been taking. A hero for me Is the one who really loves his country that much, and someone who Is concern with his countrymen. A man who Is able to keep on fighting for our own freedom and Justice with handling his beliefs and philosophy until his last breath. A hero Is also the one who Is a master in not Just one field and skill but about more than a number In a decade.Someone who can't see his country and fellowmen suffering because of the abusive government and colonizers. A real hero Is the one who has a lot of dreams for his nation and his people, and never stopped to strive and rise upon It even he has his own Incapableness, weaknesses and luckless. Therefore, the above title â€Å"The undying Past-Master† means that though this someone Is already burned under the stone, he Is still being remembered and until now, his greatness Is still In our dally lives, wherever else.The life of him has past already but his legacy In works and deeds will always be an undying one for It Is such a great Influence and Inspiration to each and everyone of us, that someday soon despite of our Imperfections, we can cake a better things not Just for ourselves but also to others. Who is a Hero for You? By Cheekbone not only from exterior but from within as well and still, made a way to set it aside and to the discussions and readings that we have been taking.A hero for me is the one who really loves his country that much, and someone who is concern with his countrymen. A man who is able to keep on fighting for our own freedom and Justice with handling his beliefs and philosophy until his last breath. A hero is also the one who is a master in not Just one field and skill but about more than a number in a abusive government and colonizers. A real hero is the one who has a lot of dreams for his nation and his people, and never stopped to strive and rise upon it even he has his own incapableness, weaknesses and luckless.Therefore, the above title â€Å"The Undying Past-Master† means that though this someone is already buried under the stone, he is still being remembered and until now, his greatness is still in our daily lives, wherever else. The life of him has past already but his legacy in works and deeds will always be an undying one for it is such a great influence and inspiration to each and everyone of us, that someday soon despite of our imperfections, we can

Monday, July 29, 2019

Character analysis of Curleys wife and Slim.

Corey's wife and slim character analysis. Steinbeck 's management told clearly about the role of Cory' s wife and the role of Slim. Because it was heard people's thoughts. We judge about their personality. When he told George and Renee, we first heard the wife of Kelly's wife Curly. This is what Candy told George about some of Collie's wife's gossip, which is also the opinion of most pasture workers. Curly came to two stories to find his wife. When he discovered that Slim was not in a two-story house, he thought that his wife and Slim were together and shows that Curly did not believe his wife. The first place we saw was that Curley assumed that his wife wanders with a man in a two-story house. Curley is a cumbersome person and problems may arise between Curley, George and Lennie in the latter part of the novel. In explaining their future, George told Renee they would have a dog and several cats, but Lenny would have to make sure that the cat does not catch the rabbit. To answer this , Lenny said, Let's make them catch a rabbit really, I am going to break their head.This sentence shows Lenny's absurdity, It helps to explain why he can not survive on his own.In addition, Rennie's statement reflects violence in male society. When George and Renee came to the ranch for the first time, the reader first introduced Carly 's wife through Candy. He has been married for two weeks, but Colley's wife said I have attracted people's attention and I am watching slim eyes. This means that Curley's wife is not satisfied with her marriage and is seeking other men's attention due to Curley's lack of attention. In addition, Candy will call Collie's wife Pie and Wanderer. Candy recommends that you understand that Curly's wife may not be trusted and may be harmful. This word reflects a negative attitude towards women at the time and is also reflected in the text of Steinbeck. Candy's comments to readers Collie's wife looked away. We were told that her eyes for Slim and Carlson were made by Kant's Candy. As I said before, it was abnormal just after two weeks and it showed the lack of love between Curly and his wife. Colley 's wife revealed their relationship in Chapter 5. In general, we learned that Curley 's wife is like everyone else. She dreams and is lonely, but she is ignored because she is a woman. Women, blacks, nephews, foolish people, and old people can not realize their dreams. Curley's wife represents a woman. Loneliness is the reason why pastoral society is very meaningful, and only the humble people can realize their dreams.

Sunday, July 28, 2019

Quality Control Essay Example | Topics and Well Written Essays - 2500 words

Quality Control - Essay Example The above example also shows the fact that there are certain parameters which can be used to measure or identify the quality level of a certain product. These are called as ‘Quality Characteristics’ by Mitra (6). These characteristics may include the aesthetics of the product, its durability, cost, utilization factor etc. 1.1. History of Quality: The sense of maintaining the quality of products and services has been existent since times immemorial. The early Egyptians, Greeks and Romans maintained quality standards in their buildings, structures, artifacts, utensils etc. The term ‘quality’ itself is however relatively newer. During the early days of industrial revolution, the need of maintaining quality in the production industry recognized but not emphasized. As a result no special workforce was designated to check the quality control. Instead, the foreman or the supervisor was the person responsible for the quality of product being made. Hence it was entir ely on his discretion what efforts he puts in order to maintain the quality standards. As the industries grew in size and the strength of personnel reporting to a particular supervisor increased in size, it became impossible for the supervisor to check the quality of each unit produced at the industry. The task of maintaining the quality was then assigned to quality inspectors. ... This moved the engineers and mathematicians to develop statistical methods of quality control. Walter Shewhart, in 1924, proposed his quality control charts which aimed at controlling the features of a product through statistical methods (Mitra 3). In 1929 he formed ‘Joint Committee for the Development of Statistical Applications in Engineering and Manufacturing’ with the support of American Society of Testing Materials (ASTM), American Society of Mechanical Engineering (ASME), American Statistical Association (ASA) and Institute of Mathematical Statistics (IMS). The committee made efforts to promote the principles of statistical quality control in the industries. The use of statistical methods in the quality control procedures gained wide acceptance in the days of World War II. Good quality machinery was vital for the armies to win battles hence quality control was essential. But large amount of weapons and machinery supplies has to be produced and every single unit cou ld not be checked for quality assurance. Hence powerful statistical methods were required for ensuring quality in industries. The rules of Sampling gained were widely accepted and implemented. The American Society of Quality Control was established in 1946 to standardize the quality control procedures 1.2. Quality Control and Six Sigma: Six Sigma is a technique used to measure the degree to which a process conforms to the consumer needs. It is based on the number of units produced that comply with the quality standards set by the organization or demanded by the customers. The method was developed by Motorola Corporation in 1986. Six Sigma basically focuses

The Epilepsy Essay Example | Topics and Well Written Essays - 1000 words

The Epilepsy - Essay Example Surveys that have been carried out by the healthcare agencies shows an increased cases of this neurological condition in relatives of probands with a notable 50% unaffected relatives who are asymptomatic and when diagnosed shows no sign of JME and therefore considered to be JME negative but PSW-positive. Scientific investigations and findings have established that JME estimations show a likelihood of genetic cause that is found in clustered lineage of families. It is imperative that genetic analysis is undertaken to allow for easy identification and JME disease. Identification of the primary epileptic abnormality is enabled by EEG endophenotype (PSW) and other factors may contribute to the causal relationship. Primary epileptic abnormality identification is instrumental in uncovering how the electric discharges are generated and the explanation behind the cases of clinical seizure in some patients and resistance in others. The overall aim of this study is to give a comprehensive expl anation of the occurrence of EEG endophenotype PSW with a sample of a statistically well-defined population. The population for this study constitutes the asymptomatic relatives of JME with a positive EEG-PSW of age ranging 10-40 years. This age group constitutes the old and the young so that there is an exhaustive generalization of this condition’s implications. This action will help in finding out answers to whether the first-degree relative who is EEG-PSW positive and a JME-patient can develop epilepsy over time.

Saturday, July 27, 2019

Does Board Gender Diversity Have a Financial Impact Evidence Using Article

Does Board Gender Diversity Have a Financial Impact Evidence Using Stock Portfolio Performance - Article Example Market operators like securities exchanges, regularly give best corporate governance practice guidelines. Market operators and law breakers started addressing women under-representation in the corporate world’s upper echelons. For example, in UK and Australia, the operators suggest that companies listed explain and disclose their self-assessed performance and chosen diversity policy (AICD, 2012). The growing regulation targets on board gender diversity and the development of the entire corporate board diversity. In portfolio approach, gender diversity provides an extensive economic development over firm-level analysis. Forming portfolio requires the averaging out of firm-specific characteristics, getting rid of heterogeneity issues and reduction of neglected variable problems. The research of corporate governance is affected by endogeneity, such as reverse causality, omitted variables and heterogeneity among samples (Adams & Ferreira, 2009). Therefore, portfolios accurately reflect the impact of new regulation on the entire market. In addition, portfolio statistics shows that the board diversity adoption has led to a significant transition. Between 2004 and 2010, sampled companies’ percentages with diverse boards increased to 36% and 42%. After self-regulation (2011), a total of 52% of the sampled companies reported that they have diverse boards. For example, the Institute of Company managers in Australia stated to have recruited women in a relatively large number to boards since 2010 (AICD, 2012). Hence, self-regulation contributed hugely to diversity growth in firms. Firms’ portfolio aggregate returns can examine diversity. It is carried out through comparing the diversity-boards with non-diversity boards and boards that have varying degrees of diversity. The diversity boards have more than one woman representing the directors while the non-diversity boards have none. According to research, the boards with woman representation have more

Friday, July 26, 2019

Gender, sexuality, and race are less biological facts than the outcome Essay

Gender, sexuality, and race are less biological facts than the outcome of contextually-specific discourses and discursive practices. Explain with reference to no more than two aspects of identity - Essay Example al., 1998). Gender discrimination also implies that the people of opposite sex, generally the female section of the society, are more often discriminated as compared to their male counterparts. However, feminists have argued with lieu to the modern phenomenon that professional potentialities and abilities do not differ within the modern society among the two genders. It is rather that gender based or racial discrimination existing in the society restricts them to become at par with the male members that has been affecting communities in their healthy developments (Mottier, 1999). Racism has been a common phenomenon since the past generations, wherein people of different nations have been experiencing the harmful impacts of racial discrimination, especially in their workplaces (Caluya, 2013; Books, 1992). Emphasising these facts, the essay aims at elaborating on the trends of gender, sex and race discriminations prevailing in the society in context to individuals’ identity rela ted aspects. Gender inequality/discrimination has emerged as one of the major issues in this society, which has commonly targeted the female section of the society. Discrimination between the two genders has been prevailing since the early generations affecting a particular section of the society (Alsop et. al., 1998). However, due to the continuous modernization of the society, gender/sexual discrimination has reduced to a certain extent, although such trends are still prevalent in some societies (Weeks, n.d.). According to the past studies, the root cause triggering such discrimination has been related to people having limited concern regarding the biological differences, but emphasise more on the arbitrary dissimilarities between the two genders, considering the outcome as a discursive practice. Undoubtedly, gender inequality/discrimination imposes strong negative impacts on the society, as females are often not considered equal to that of their male counterparts and

Thursday, July 25, 2019

Writting assingment on john wycliffe (argument ) Essay

Writting assingment on john wycliffe (argument ) - Essay Example The childhood life and the environment in which John Wycliffe grew shaped his future life. According to Roberts, the birth date of Wycliffe is estimated to be around 1330 and he grew up in Yorkshire, England (1). During this period, the clergy people had great influence in the community and flourished an expensive life compared to the other citizens. It is these scenarios that influenced Wycliffe to become an anticlerical supporter in the world of religion. Roberts further states that Wycliffe was interested in studying English and was also interested in the matters of spirituality (1). These passions exhibited by Wycliffe at his tender age were reflected in his life at latter years. For instance, Wycliffe was motivated to study theology at Rector of Fillingham College and also contributed to the bible translation into other vernacular languages (Roberts). These kinds of activities require people with passion to learn and exercise their writing skills. It is after his studies and interaction with the clergy people that Wycliffe clearly understood and influenced his civilization concerning the Holy Scriptures and the conduct of the church leaders. Because of his spirituality and involvement in various church activities, Wycliffe was supported by the church for his further studies in Oxford University. Wholesome Words Organization cites that John Wycliffe once served at the parish of Ludgershall (1). This indicates that Wycliffe real life experience of the functioning of church in England. Moreover, Farley explains how Wycliffe was directly involved in the spread of the gospel. However, it is after joining the Oxford University as a scholar that Wycliff started attacking the Catholic clergy for practicing the ‘wrong’ approach in Christianity. It is also at this time that he became closer to the son of King Edward III and therefore received

Wednesday, July 24, 2019

Emotions In Film Essay Example | Topics and Well Written Essays - 750 words

Emotions In Film - Essay Example Orientalism, as it was known by Napoleon, was based on just such knowledge – that is knowledge about the Orient projected upon it by the Western world. This idea of the Orient, as it was expressed among the Western scholars – overrode the true Orient. Observers entering the true Orient were already convinced of what they knew regarding an unchanging and already defined system, locking everything labeled Orient into a passive, unresisting object for study. Comparable linguistics were the basis for many of these assumptions (Said, 1979). This had, and continues to have, a negative effect upon the Western world’s perception, understanding and appreciation of a multitude of cultures that were erroneously grouped into a single identity. Philosophers continue to argue that reality, as it is presented within films and on tv, is not reflective of our ‘everyday’ reality, yet their arguments are based on water as there is as yet no true definition or conceptio n of what actual reality might be. In his book â€Å"What is Philosophy?† (1960), Jose Ortega y Gasset discusses several key defining aspects of philosophy, including a discussion regarding a definition of the focus of philosophy as a science. While earlier philosophers tended to identify philosophy by the tagline ‘the study of knowledge,’ Ortega points out that nowhere in these texts do any of the philosophers who came before him work to define what the empirical concept of knowledge is and therefore reach an understanding of what is ‘everyday reality.’ Although Ortega does not necessarily phrase his point in this exact terminology, his discussions regarding the nature of knowledge, the nature of science, and the nature of reality perception as it exists within the very human context of time make it abundantly clear that Ortega at least does not feel we will ever completely understand the world around us. To begin with, Ortega brings into clarity the idea that time

Tuesday, July 23, 2019

Movie reivew Essay Example | Topics and Well Written Essays - 1250 words

Movie reivew - Essay Example This paper will analyze the episode that is titled Secrets of the Koran which is a part of the show that called Decoding the Past. To begin with, it would be quite important to provide an accurate account of the major themes of the Quran as they were presented in the episode. The first one that was mentioned in the chronological order is the desire to achieve the social justice. Indeed, at the present moment the people who actually comprise the intended audience of this show consider Islam to be a religion that almost enshrined the social inequality as well as oppression of women. However, the episode suggests that from the very beginning Islam was aimed at the groups of people who dominated the society and did not allow it to develop in the proper manner. In addition to that, a considerable number of scholars would prove that one of the highest virtues of Islam is closely connected to social justice (Gorder 20). Another interesting theme that was emphasized in the video is the war and violence in the Quran. The producers claim that a certain part of this sacred book is largely dedicated to fighting. Indeed, there are some verses which are called sword verses that clearly dictate a true Muslim how they should treat an enemy: fearlessly and fiercely in fight and mercifully after it (Smith and Furnish 77). In spite of that, the above mentioned verses are often recited without any context and they are thought to be aimed at the rest of humanity. The authors of the video suggest that this can be easily explained by the historical context in which the book was written: the newly founded city of the early Muslims was under attack of a powerful enemy and the conflict was primarily triggered by the differences in faith. The next theme of the Quran that was reviewed in the movie is the theological foundation of this religion,

The Significance of the Character Edward Hyde Essay Example for Free

The Significance of the Character Edward Hyde Essay Explore the Significance of the Character Edward Hyde and the Way he is perceived by Readers in Dr. Jekyll and Mr Hyde In 1859, when the book was written class and religion was very important to the Victorians. People were given more respect if they were higher class. Hyde represents a very low class and doesnt have a lot of admiration from other people. Hyde has a house in Soho and always uses the back door leading to Dr. Jekylls laboratory because Jekyll is ashamed of him and doesnt want anyone to connect Hyde to himself because he does not want to damage his reputation. The front door symbolises that this is what he wants people to think of him, thats why he does not use it with Hyde because he is seen as a monster. Hyde would be seen by the Victorian audience as evil and devilish, he is described as a gust of devilish fury, and people would be scared of him because of his deformity and the way he looks. Dr. Jekyll is upper class and if anyone found out that he was associated with Mr Hyde, who was the lowest of the lower class then he would lose all his respect he had gained. Jekyll creates Hyde because he wants to fulfil his needs to be wrong and do things that he couldnt do as his normal self. He was fond of things that society didnt approve of and was tired of hiding in his real personality. This suggests that the class system in Victorian was very wide; there were strict rules for upper class and hardly any rules for the lower class. It was immoral for Jekyll to create Hyde for his own enjoyment and to satisfy his curiosity because people arent meant to change the way they are born and bought up. He shouldnt hide the fact that he wants to be different and should either be one or the other, not both. He abuses his scientific knowledge to make this happen, the readers perceive Hyde as a scientific mistake or something that shouldnt have happened. The genre of the novel can vary between everyone because it has many genres. A lot of people view it as a mystery novel because the end is unknown until right at the end of the novel; some people see it as a gothic and dark book because a lot of wrong things that are associated with the dark world are happening throughout the novel. Another genre that could be linked with this novel is suspense because no one knows what is going to happen next because everyone has been shocked from the things that have happened already. The book is also very secretive and no ones knows much about what is happening at other places and times until at the end of the novel. Stevenson uses a lot of techniques to make Hyde seem so evil and repugnant to the readers. A lot of these are use of language such as similes, metaphors and alliteration. An example of alliteration used to describe Hyde is spawn of Satan; this links Hyde to the devil and evil. Stevenson also uses symbolism and pathetic fallacies to make Hyde seem so evil to the readers. The use of fog is used often to symbolise mystery and also is mentioned just before something bad is going to happen, for example just before he runs into the little girl in the street the narrator mentions the foggy weather, the same thing happens just before he murdered Sir Danvers Carew. Hyde shows us that evil can take over someone without them realising it. Hyde picks on weak people as victims and Jekyll has no control over Hyde at the end. He uses his strength in both attacks that we know about on innocent, vulnerable people that stood no chance in defending themselves for example when My Hyde attacked Sir savers Carew the maid described Carew as innocent and old-world kindness disposition this tells us that Carew didnt do anything wrong to deserve it.. At the start Jekyll had full control over Hyde and only wanted to fill a part of himself which wanted to be slightly mischievous, after a while Jekyll could not control what he was doing or when he changed to Hyde, this tells us that evil slowly takes over good and destroys lives. He says after that for two months I led a life of such severity as approving conscience. This tells us that it had got worse towards the end and he started to realise that it had got worse. Victorians would perceive Hyde as evil and not human, a lot of Victorians were very religious and would like to see the good side of him win over the evil side, a lot of them would be disgusted at the things that Hyde did, especially to the young innocent girl he trampled over in the middle of the night. Stevenson gets the audience going by making a huge contrast and difference between the good Dr. Jekyll and the evil Mr Hyde. He makes them chose only one side to be on and lets their imagination create pictures. In the end good prevailed over evil but Dr Jekyll had to kill himself so that Mr Hyde could never return and kill more people or harm others. Victorians might perceive Hyde as an evolutionary throwback because Stevenson uses language that describes him as monkeys and ape like creatures. For example he says that Hyde hits someone with ape-like fury and that Hyde resembles an ape. Victorian readers might have preferred to perceive as not fully human because they do not want to think that someone human could be that evil and sick-minded that Hyde is. Stevenson refers to Darwins theory because it wasnt fully believed in the time the novel was written. The audience would have been shocked for Hyde to have been described that way. As a modern reader I have different views on Hyde and perceive him in a different way to the Victorians. I still see Hyde as an evil person but he doesnt have a very big impact on me because I am used to things like this, unlike the Victorians who had rarely seen or spoken about such things before. I think all of us have evil inside of us but only if we keep it locked up inside of us and do not admit that we have any it starts to get the better of us and takes over, slowly after time it starts making choices that we do not really want to make and we react differently to normal and do things maybe we should not do. Stevenson creates Hyde to show the audience that everyone has evil inside of them but not everyone uses it and keeps it shut away from the world and surroundings.

Monday, July 22, 2019

Real Time Pcr Essay Example for Free

Real Time Pcr Essay PROBE-BASED DETECTION SYSTEMS14 Hybridization probes (also called FRET probes)16 MELTING CURVE ANALYSIS16 Multiplex real-time PCR18 APPLICATIONS OF REAL TIME PCR18 GENE EXPRESSION ANALYSIS18 SNP GENOTYPING19 HIV DETECTION19 CYSTIC FIBROSIS (CF) DETECTION:20 THE ADVANTAGES OF REAL-TIME PCR20 THE DISADVANTAGES21 REFRENCES21 REAL TIME PCR TRADITIONAL PCR The polymerase chain reaction (PCR) is one of the most powerful technologies in molecular biology. Using PCR, specific sequences within a DNA or cDNA template can be copied, or â€Å"amplified†, many thousand- to a millionfold. In traditional (endpoint) PCR, detection and quantitation of the amplified sequence are performed at the end of the reaction after the last PCR cycle, and involve post-PCR analysis such as gel electrophoresis and image analysis. REAL-TIME QUANTITATIVE PCR (qPCR) In real-time quantitative PCR (qPCR), the amount of PCR product is measured at each cycle. This ability to monitor the reaction during its exponential phase enables users to determine the initial amount of target with great precision. WHAT’S WRONG WITH AGAROSE GELS? * Poor precision. * Low sensitivity. Short dynamic range lt; 2 logs. * Low resolution. * Non-automated. * Size-based discrimination only * Ethidium bromide staining is not very quantitative REAL TIME PCR VS PCR . BASIC PRINCIPLE Quantitative PCR  is carried out in a  thermal cycler  with the capacity to illuminate each sample with a beam of light of a specified wavelength and detect the fluorescence emitted by the excited  fluorochrome. The thermal cycler is also able to rapidly heat and chill samples thereby taking advantage of the physicochemical properties of the  nucleic acids  and  DNA polymerase. The PCR process generally consists of a series of temperature changes that are repeated 25 – 40 times, these cycles normally consist of three stages: the first, at around 95  Ã‚ °C, allows the separation of the nucleic acid’s double chain; the second, at a temperature of around 50-60  Ã‚ °C, allows the alignment of the primers with the DNA template;  the third at between 68 72  Ã‚ °C, facilitates the  polymerization  carried out by the DNA polymerase In real-time PCR, * the amount of DNA is measured after each cycle by the use of fluorescent markers that are incorporated into the PCR product. The increase in fluorescent signal is directly proportional to the number of PCR product molecules (amplicons) generated in the exponential phase of the reaction. * Fluorescent reporters used include double-stranded DNA (dsDNA)- binding dyes, or dye molecules attached to PCR primers or probes that are incorporated into the product during amplification. * The change in fluorescence over the course of the reaction is measured by an instrument that combines thermal cycling with scanning capability. By plotting fluorescence against the cycle number, the real-time PCR instrument generates an amplification plot that represents the accumulation of product over the duration of the entire PCR reaction (Figure 1). Figure 1—Amplification plots are created when the fluorescent signal from each sample is plotted against cycle number; therefore, amplification plots represent the accumulation of product over the duration of the real-time PCR experiment. The samples being amplified in this example are a dilution series of the template. TYPES OF PCR Quantitative PCR| Qualitative qPCR| A specific or non-specific detection chemistry allows the quantification ofthe amplified product. | In qualitative qPCR, the goal is to detect the presence or absence of a certain sequence. | The amount detected at a certain point of the run is directly related to theinitial amount of target in the sample| For virus sub-typing and bacterial species identification. Can also be used for allelic discrimination between wild type and mutant, between different SNPs or between different splicing forms. | common pplications of quantitative PCR are gene expression analysis, pathogen detection/quantification and microRNA quantification| Different fluorophores can be used for the two alleles, and the ratio of the fluorophores signals correlates to the related amount of one form compared to the other one. | Quantitative PCR software uses the exponential phase of PCR for quantification. | Specific detection methods such as Double-Dye probe systems are more ofte n used for theseApplications| Overview of real-time PCR Real-time PCR is a variation of the standard PCR technique used to quantify DNA or RNA in a sample. Using sequence-specific primers, the relative number of copies of a particular DNA or RNA sequence can be determined.. Quantification of amplified product is obtained using fluorescent probes or fluorescent DNA binding dyes and real time PCR instruments that measure fluorescence while performing temperature changes needed for the PCR cycles. qPCR STEPS There are three major steps that make up a qPCR reaction. Reactions are generally run for 40 cycles. 1. Denaturation—The temperature should be appropriate to the polymerase chosen (usually 95 °C). The denaturation time can be increased if template GC content is high. 2. Annealing—Use appropriate temperatures based on the calculated melting temperature (Tm) of the primers (5 °C below the Tm of the primer). 3. Extension—At 70–72 °C, the activity of the DNA polymerase is optimal, and primer extension occurs at rates of up to 100 bases per second. When an amplicon in qPCR is small, this step is often combined with the annealing step using 60 °C as the temperature. BASICS OF REAL TIME PCR Baseline – The baseline phase contains all the amplification that is below the level of detection of the real time instrument. Threshold – where the threshold and the amplification plot intersect defines CT. Can be set manually/automatically CT – (cycle threshold) the cycle number where the fluorescence passes the threshold Rn – (Rn-baseline) NTC – no template control Rn is plotted against cycle numbers to produce the amplification curves and gives the CT value. ONE-STEP OR TWO-STEP REACTION qRT-PCR can be one step or two step. 1. Two-step qRT-PCR Two-step qRT-PCR starts with the reverse transcription of either total RNA or poly(A)+ RNA into cDNA using a reverse transcriptase (RT). This first-strand cDNA synthesis reaction can be primed using random hexamers, oligo(dT), or gene-specific primers (GSPs). To give an equal representation of all targets in real-time PCR applications and to avoid the 3? bias of oligo(dT), it is usually recommended that random hexamers or a mixture of oligo(dT) and random hexamers are used. The temperature used for cDNA synthesis depends on the RT enzyme chosen. Following the first-strand synthesis reaction, the cDNA is transferred to a separate tube for the qPCR reaction. In general, only 10% of the first strand reaction is used for each qPCR. . One-step qRT-PCR One-step qRT-PCR combines the first-strand cDNA synthesis reaction and qPCR reaction in the same tube, simplifying reaction setup and reducing the possibility of contamination. Gene-specifi c primers (GSP) are required. This is because using oligo(dT) or random primers will generate nonspecific products in the one-step procedure and reduce the amount of product of interest. O verview of qPCR and qRT-PCR components This section provides an overview of the major reaction components and parameters involved in real-time PCR experiments. * DNA polymerase One of the main factors affecting PCR specificity is the fact that Taq DNA polymerase has residual activity at low temperatures. Primers can anneal nonspecifically to DNA, allowing the polymerase to synthesize nonspecific product. The problem of nonspecific products resulting from mispriming can be minimized by using a â€Å"hot-start† enzyme. Using a hot-start enzyme ensures that no active Taq is present during reaction setup and the initial DNA denaturation step. * Template Anywhere from 10 to 1,000 copies of template nucleic acid should be used for each real-time PCR reaction. This is equivalent to approximately 100 pg to 1 ? of genomic DNA, or cDNA, generated from 1 pg to 100 ng of total RNA. Excess template may increase the amount of contaminants and reduce efficiency. If the template is RNA, care should be taken to reduce the chance of genomic DNA contamination. One option is to treat the template with DNaseI. Ultrapure, intact RNA is essential for full-length, high-quality cDNA synthesis and accurate mRNA quantification. RNA should be devoid of any RNase contamination, and aseptic conditions should be maintained. * Reverse transcriptase The reverse transcriptase (RT) is as critical to the success of qRT-PCR as the DNA polymerase. It is important to choose an RT that not only provides high yields of full-length cDNA but also has good activity at high temperatures. High-temperature performance is also very important for tackling RNA with secondary structure or when working with gene-specific primers (GSPs). * dNTPs It is recommended that both the dNTPs and the Taq DNA polymerase be purchased from the same vendor, as it is not uncommon to see shifts of one full threshold cycle (Ct) in experiments that employ these items from separate vendors. * Magnesium concentration In qPCR, magnesium chloride or magnesium sulfate is typically used at a fi nal concentration of 3 mM. This concentration works well for most targets; however, the optimal magnesium concentration may vary between 3 and 6 mM. * UNG The Uracil-N-Glycosylase is an enzyme that hydrolyses all single-stranded and double-stranded DNA containing dUTPs. Consequently, if all PCR amplifications are performed in the presence of a dNTPs/dUTPs blend, by carrying a UNG step before every run it is possible to get rid of any previous PCR product. * ROX Some thermocyclers require MasterMix containing ROX dye for normalization. This is the case for the ABI and Eppendorf machines, and optional on the Stratagene machines. If you work with such machines, it is easier to work with the ROX dye already incorporated in the MasterMix rather than adding it manually. It guarantees a higher level of reproducibility and homogeneity of your assays. * Fluorescein For iCycler iQ, My iQ and iQ5 machines (BioRad thermocyclers), the normalization method for SYBR Green assay uses Fluorescein to create a â€Å"virtual background†. As in the case for the ROX, it is better and easier to use a MasterMix that contains pre-diluted Fluorescein, guaranteeing higher reproducibility and homogeneity of your assays. REAL TIME PCR SYSTEM: System Features: †¢ Four interchangeable block formats †¢ Optional Automation Accessory amp; Barcode Scanner †¢ Argon ion laser/CCD camera †¢ Easy to Use Software, Multiple Applications †¢ Set up Wizards †¢ QC Filtering/Flag System †¢ Flexible data reports amp; exporting SOFTWARES FOR DATA ANALYSIS AND PRIMER DESIGNING 1 ) Light Cycler ® Relative Quantification Software The first commercially available software was the Light Cycler ® Relative Quantification Software (2001). 2 ) REST In 2002, the relative expression software tool (REST ) was established as a new tool. 3 ) Q-Gene Recently a second software tool, Q-Gene, was developed, which is able to perform a statistical test of the real-time data. Q-Gene manages and expedites the planning, performance and evaluation of quantitative real-time PCR experiments. 4) OligoPerfect A primer design software program such as OligoPerfectâ„ ¢, available on the Web at www. invitrogen. com/oligoperfect, can automatically evaluate a target sequence and design primers for it based on the criteria STEPS OF REAL TIME PCR Real-time reaction mix (final concentrations): 1x 2 x AmpliTaq Gold 0. 5 ? M 5’ primer 0. 5 ? M 3’ primer 0. 2 ? M probe 0. 4 ? Rox reference dye 20 ? l Final Volume (including sample and dH20) STANDARD REAL-TIME PCR PROTOCOL ASSAY DESIGN: This section describes the stages of real-time PCR assay design and implementation. We will identify sources of variability, the role they play in data accuracy, and guidelines for optimization in the following areas: 1Target amplicon and primer design 2. Nucleic acid purification 3. Reverse transcription 4. Controls and normalization 5. Standard curve evaluation of efficiency, sensitivity, and reproducibility Good primer (pair) properties One way to minimize efficiency bias is to amplify relatively short targets. Amplifying a 100 bp region is more likely to result in complete synthesis in a given cycle than, say, amplifying a 1,200 bp target. For this reason, real-time PCR target lengths are generally in the range of 60 bp to 200 bp. In addition, shorter amplicons act as a buff er against variations in template integrity. Primers designed to amplify larger regions are less likely to anneal with the same fragment in a slightly degraded nucleic acid sample. PURIFICATION Phenol-based organic extraction is a very effective method for purifying RNA from a wide variety of cell and tissue types. During sample lysis, phenol and guanidine isothiocyanate disrupt cells and dissolve cell components. while maintaining the integrity of the nucleic acids by protecting them from RNases. Chloroform is added and the mixture is separated by centrifugation, which separates the solution into an aqueous phase and an organic phase. RNA remains exclusively in the aqueous phase in the presence of guanidine isothiocyanate, while DNA and protein are driven into the organic phase and interphase. The RNA is then recovered from the aqueous phase by precipitation with isopropyl alcohol. REVERSE TRANSCRIPTION CONSIDERATIONS Most reverse transcriptases employed in qRT-PCR are derived from avian myeloblastosis virus (AMV) or Moloney murine leukemia virus (M-MLV). An ideal reverse transcriptase will exhibit the following attributes: * Thermostability— thermostable RTs function at the higher end of (or above) this range and allow for successful reverse transcription of GC-rich regions. * RNase H activity— RNase H activity can drastically reduce the yield and ratio of full-length cDNA, which translates to poor sensitivity. Several RTs, most notably SuperScript II and III, have been engineered for reduced RNase H activity. NORMALIZATION AND QUANTIFICATION: When analyzing and comparing results of Real-Time qPCR assays many researchers are confronted with several uncontrolled variables, which can lead to misinterpretation of the results. Those uncontrolled variables can be the amount of starting material, enzymatic efficiencies, and differences between tissues, individuals or experimental conditions. In order to make a good comparison, normalization can be used as a correction method, for these variables. The most commonly known and used ways of normalization are : * normalization to the original number of cells, * normalization to the total RNA mass, normalization to one or more housekeeping genes, * normalization to an internal or external calibrator. Normalization to number of cells can actually only be done for cell culture and blood samples. The two majors methods of normalization are the absolute quantification and the relative quantification . Absolute quantification Absolute quantification requires a standard curve of known copy numbers. The amplicon being studied can be cloned, or a synthetic oligonucleotide (RNA or DNA) can be used. The standard must be amplified using the same primers as the gene of interest and must amplify with the same efficiency. The standards must also be quantified accurately. This can be carried out by reading the absorbance at A260, although this does not distinguish between DNA and RNA, or by using a fluorescent ribonucleic acid stain such as RiboGreen. Relative quantification Relative quantification is the most widely used technique. Gene expression levels are calculated by the ratio between the amount of target gene and an endogenous reference gene, which is present in all samples. The reference gene has to be chosen so that its expression does not change under the experimental conditions or between different tissue. There are simple and more complex methods for relative quantification, depending on the PCR efficiency, and the number of reference genes used. STANDARD CURVE TO ASSESS EFFICIENCY, SENSITIVITY, AND REPRODUCIBILITY The final stage before assay employment is validating that all the experimental design parameters result in a highly efficient, sensitive, and reproducible experiment. * Reaction efficiency One hundred percent efficiency corresponds to a perfect doubling of template at every cycle, but the acceptable range is 90–110% for assay validation. This efficiency range corresponds to standard curve slopes of –3. 6 to –3. 1. The graph in Figure shows the measurement bias resulting solely from differences in reaction efficiency.. A standard curve is generated by plotting a dilution series of template against the Ct for each dilution. To some, sensitivity is measured by how early a target Ct appears in the amplification plot. However, the true gauge of sensitivity of an assay is whether a given low amount of template fits to the standard curve while maintaining a desirable efficiency. The most dilute sample that fits determines reaction sensitivity. The standard curve also includes an R2 value, which is a measure of replicate reproducibility. Standard curves may be repeated over time to assess whether the consistency, and therefore the data accuracy for the samples. Real-Time PCR Fluorescence Detection Systems Several different fluorescence detection technologies can be used for realtime PCR, and each has specific assay design requirements. All are based on the generation of a fluorescent signal that is proportional to the amount of PCR product formed. The three main fluorescence detection systems are: * DNA-binding agents (e. g. SYBR Green and SYBR GreenER technologies * Fluorescent primers (e. g. , LUX Fluorogenic Primers and Amplifluor qPCR primers) * Fluorescent probes (e. g. , TaqMan probes, Scorpions, Molecular Beacons) The detection method plays a critical role in the success of real-time PCR. DNA-Binding Dyes The most common system for detection of amplified DNA is the use of intercalating dyes that fluoresce when bound to dsDNA. SYBR Green I and SYBR GreenER technologies use this type of detection method. The fluorescence of DNA-binding dyes significantly increases when bound to double-stranded DNA (dsDNA). The intensity of the fluorescent signal depends on the amount of dsDNA that is present. As dsDNA accumulates, the dye generates a signal that is proportional to the DNA concentration and can be detected using real-time PCR instruments. SYBR Green I advantages †¢ Low cost assay †¢ Easy design and set up SYBR Green I disadvantages †¢ Non specific system †¢ Not adapted to multiplex †¢ Non suitable for qualitative qPCR Primer-Based Detection Systems Primer-based fluorescence detection technologies can provide highly sensitive and specific detection of DNA and RNA. In these systems, the fluorophores is attached to a target-specific PCR primer that increases in fluorescence when incorporated into the PCR product during amplification. * Amplifluor Real-Time PCR Primers Amplifluor real-time PCR primers are designed with both a fluorophore and quencher on the same primer. The primer adopts a hairpin configuration that brings the fluorophore in close proximity to the quencher. The fluorescent signal increases when the primer is unfolded and the fluorophore and quencher are de-coupled during incorporation into an amplification product. Figure: Ampliflour primer PROBE-BASED DETECTION SYSTEMS Probe-based systems provide highly sensitive and specifi c detection of DNA and RNA. However, dual-labeling and complex design specifi cations make them expensive and more diffi cult to use than primer-based systems or DNAbinding dyes. TaqMan probes = Double-Dye probes TaqMan probes, also called Double-Dye Oligonucleotides, Double-Dye Probes, or Dual Labelled probes, are the most widely used type of probes. A fluorophore is attached to the 5’ end of the probe and a quencher to the 3’ end. The fluorophores is excited by the machine and passes its energy, via FRET (Fluorescence Resonance Energy Transfer) to the quencher. TaqMan probes can be used for both quantification and mutation detection, and most designs appear to work well. TaqMan ASSAY DENATURATION ANNEALING OF PRIMERS AND PROBE POLYMERIZATION AND PROBE CLEAVAGE Molecular Beacons In addition to two sequence-specific primers, molecular beacon assays employ a sequence-specific, fluorescently labeled oligonucleotide probe called a molecular beacon, which is a dye-labeled oligonucleotide (25–40 nt) that forms a hairpin structure with a stem and a loop . A fluorescent reporter is attached to the 5 end of the molecular beacon and a quencher is attached to the 3 end. The loop is designed to hybridize specifically to a 15–30 nucleotide section of the target sequence Figure: Moleculer Beacon They are highly specific, can be used for multiplexing, and if the target sequence does not match the beacon sequence exactly, hybridization and fluorescence will not occur a desirable quality for allelic discrimination experiments. Hybridization probes (also called FRET probes) Roche has developed hybridization probes for use with their LightCycler. Two probes are designed to bind adjacent to one another on the amplicon. One has a 3’ label of FAM, whilst the other has a 5’ LC dye, LC red 640 or 705. When the probes are not bound to the target sequence, the fluorescent signal from the reporter dye is not detected. However, when the probes hybridize to the target sequence during the PCR annealing step, the close proximity of the two fluorophores allows energy transfer from the donor to the acceptor dye, resulting in a fluorescent signal that is detected. FRET probe principle and light cycler MELTING CURVE ANALYSIS Melting curve analysis can only be performed with real-time PCR detection technologies in which the fluorophore remains associated with the amplicon. Amplifications that have used SYBR Green I or SYBR GreenER dye primers can be subjected to melting curve analysis. Dual-labeled probe detection systems such as TaqMan probes are not compatible because they produce an irreversible change in signal by cleaving and releasing the fluorophore into solution during the PCR; however, the increased specificity of this method makes this less of a concern. The level of fluorescence of both SYBR Green I and SYBR GreenER dyes significantly increases upon binding to dsDNA. By monitoring the dsDNA as it melts, a decrease in fluorescence will be seen as soon as the DNA becomes single-stranded and the dye dissociates from the DNA. Figure: Melting curve analysis can detect the presence of nonspecifc products, as shown by the additional peaks to the left of the peak for the amplified product in the melt curve. How to perform melting curve analysis To perform melting curve analysis, the real-time PCR instrument can be programmed to include a melting profile immediately following the thermocycling protocol. After amplification is complete, the instrument will reheat your amplified products to give complete melting curve data. Most real-time PCR instrument platforms now incorporate this feature into their analysis packages. In general, the program steps will be: 1. Rapid heating of the amplified sample to 94 °C to denature the DNA. 2. Cooling the sample to 60 °C. 3. Slowly heating (by increasing the temperature 0. 2 °C/second) the sample while plotting fluorescence signal vs. temperature. (As the temperature increases and the dsDNA strands melt, the fluorescence signal will decrease. ) Figure: Example of a melting curve thermal profile setup on an Applied Biosystems instrument (rapid heating to 94 °C to denature the DNA, followed by cooling to 60 °C. ) Multiplex real-time PCR In multiplex real-time PCR, more than one set of gene-specific primers is used to amplify separate genes from the template DNA or RNA in a single tube. Typically, multiplex reactions are used to amplify a gene of interest and a â€Å"housekeeping† gene (e. g. , #-actin or GAPDH), which is used as a normalize for the reaction. Because more than one PCR product will be quantified in the same tube, different fluorescent reporter dyes are used to label the separate primers or probes for each gene. More Samples Analyzed per Plate. Target and normalizer in same reaction and Less sample consumed. APPLICATIONS OF REAL TIME PCR GENE EXPRESSION ANALYSIS A sample gene expression analysis using a multiplex TaqMan assay is presented in the following sections. In this example, we’re interested in the relative expression of three genes in the polyamine biosynthesis pathway, ornithine decarboxylase (ODC), ODC antizyme (OAZ), and antizyme inhibitor (AZI), in two different samples, sample A and sample B. 1. RNA was isolated from sample A and sample B. 2. RNA was reverse transcribed into cDNA. 3. The amount of the target genes (ODC, OAZ, and AZI) and the reference gene (b-actin) was determined in each of the cDNA samples using a multiplex qPCR assay. 4. Data were analyzed and the relative expression of each of the target genes in the two samples was calculated. EXAMPLE BRCA1 is a gene involved in tumor suppression. BRCA1 controls the expression of other genes. In order to monitor level of expression of BRCA1, real-time PCR is used. SNP GENOTYPING In order to perform SNP genotyping, two specific probes labeled with different dyes are used, the first for the wild type allele and the second for the mutant allele. If the assay results in the generation of only the first fluorescent color, then the individual is homozygous wild type at that locus. If the assay results in the generation of only the second fluorescent color, then the individual is homozygous mutant. And finally, if both fluorescent colors are produced, then the individual is heterozygous. At the end of the reaction, hydrolysis probes are digested. The quality of a hydrolysis probe is given by the hybridization efficiency, the quenching of the intact probe and the cleavage activity of Taq polymerase. HIV DETECTION Nowadays HIV is strikingly spreading out whole the world. so in order to diminish its distribution , it is necessary to detect it as soon as possible amp; for this purpose, Real time PCR is recommended by scientist. In this method ,’ pol’’ gen of the virus, is amplified in thermocycler. 6 patient have been studied. infection in these patients was confirmed by ELISA amp; western blot. * Sampling amp; RNA extracting from patients. * Cloning of target segment by using Xba I amp; Hind III. And 180 bp primers. * Standard virus mRNA was extracted. * Quantitative analysis of HIV virus by SYBR-green Real Time RT-PCR. CYSTIC FIBROSIS (CF) DETECTION: Cystic f ibrosis (CF) is the most common inherited disease among Caucasian populations with an incidence of ~1 in 2500 births. A3 base pair (bp) deletion, designated DF508, accounts for nearly 70% of CF cases and causes severe manifestations of the disease. It results in the absence of phenylalanine at position 508 of the cystic fibrosis transmembrane conductance regulator (CFTR) protein and this error prevents normal processing and translocation of the polypeptide chain to apical membranes of epithelial cells. This deletion can be detected by molecular beacons in real time PCR. Figure:Examples of specific molecular beacon fluorescence increase during real-time PCR in samples containing single lymphoblasts homozygous normal for CF (green), heterozygous DF508 (blue), or homozygous DF508 (red). A) Fluorescent signal from the molecular beacon detecting the normal allele. (B) Fluorescent signal from the molecular beacon detecting the DF508 allele. Dashed lines indicate the threshold of 200 units (~10 SD above baseline readings) used for determining CT values. THE ADVANTAGES OF REAL-TIME PCR * The ability to monitor the progress of the PCR reaction as it occurs in real time * The ability to precisely measure the amount of amplicon at each cy cle * An increased dynamic range of detection * The combination of amplification and detection in a single tube, which eliminates post-PCR manipulations. Rapid cycling times (1 hour) * High sample throughput (~200 samples/day) * Low contamination risk (sealed reactions) * Very sensitive (3pg or 1 genome eq of DNA) * Broad dynamic range (10 1010 copies) * Reproducible (CV lt; 2. 0 %) * Allows for quantitation of results * Software driven operation * No more expensive than â€Å"in house† PCR ($15/test) THE DISADVANTAGES * Current technology has limited capacity for multiplexing. Simultaneous detection of 2 targets is the limit. * Development of protocols needs high level of technical skill and/or support. Requires Ramp;D capacity and capital) * High capital equipment costs ($ 50,000 -160,000). REFRENCES * http://www. icmb. utexas. edu/core/DNA/qPCR/QiagenRT-PCR. pdf www. icmb. utexas. edu * http://books. google. com. pk/books? id=-v-U-mXWg-gCamp;printsec=frontcoveramp;dq=real+time+pcramp;hl=enamp;sa=Xamp;ei=Bph1UezKIceDhQeUh4CwCAamp;ved=0CDAQ6AEwAQ#v=onepageamp;q=real%20time%20pcramp;f=false books. google. com. pk * PCR/Real-Ti me PCR Protocols www. protocol-online. org Real-Time Pcr: An Essential Guide Google Books books. google. com * * http://www. gene-quantification. e/bio-rad-CFX96-bulletin-5589. pdf www. gene-quantification. de * https://www. google. com. pk/#output=searchamp;sclient=psy-abamp;q=fret+rt-qpcramp;oq=fret+in+rtamp;gs_l=serp. 1. 1. 0i22i30l2. 1583. 4622. 1. 10196. 6. 6. 0. 0. 0. 0. 551. 2584. 3-3j1j2. 6. 0 0. 0 1c. 1. 9. serp. 97Wjtm9UCU4amp;psj=1amp;bav=on. 2,or. r_cp. r_qf. amp;fp=f6d28cf5fd703914amp;biw=1366amp;bih=600 www. google. com. pk * BioTechniques Real-time PCR for mRNA quantitation www. biotechniques. com * http://env1. gist. ac. kr/joint_unugist/file/g_class11_real_time_pcr_vt. pdf env1. gist. ac. kr

Sunday, July 21, 2019

English Tort Law Imposing Liability Law Essay

English Tort Law Imposing Liability Law Essay This essay will address the question by exploring cases dealing with occupiers liability, vicarious liability and psychiatric harm. I intend to focus on narrow aspects within these three areas of law allowing a deep critical analysis. Occupiers liability, in my opinion, is an area of law that imposes too much liability in cases involving trespassers. I dont doubt that there must be liability for the homeowner who invites someone onto their land and fails to provide a safe environment which is seen in statute such as the Occupiers Liability Act 1957, duty to take such care as in all the circumstancesto see that the visitor will be reasonably safe in using the premises.  [1]  However, tort law insists that an occupier can be held liable for personal injury suffered by trespassers because of the state of the land and this is where I believe the Occupiers Liability Act 1984  [2]  takes the principle of the 1957 Act and stretches it too far. My first critique of this aspect of to rt law is the conflict it has with criminal law. It seems incoherent that in a criminal court a home owner whose land has been trespassed on, perhaps by a burglar, will be considered the victim and duly have the sympathy of the court and yet be cast in the role of villain in a civil court. Leon Green describes how an occupier will, insist[ing] upon the intruders own wrong andinvoke[ing] his own immunity from any duty to exercise active care in behalf of a trespasser.  [3]  He then goes on to say, Assuming the intruder to be a wrongdoer, the landowners position would seem unassailable.  [4]  This journal dates back to 1917 before statute had imposed occupiers liability but the sentiment holds true. Why should the victim be held accountable for the criminals injuries in cases where there has been no active action on the part of the occupier? The negligent state of the land and arising liability should, in my opinion, be limited to lawful visitors and trespassing children, who cannot be held to the same standard as adults, if only for policy reasons. A clear message needs to be sent to unlawful visitors; crime does not pay. Why recompense them in any way from an illegal venture? Not only does it not dissuade criminal behaviour it is also wholly unfair to impose this liability on a home owner. Additionally, I would argue that when comparing occupiers liability to other areas of tort law, such as pure economic loss, it becomes clear that comparatively occupiers liability allows too much liability. The case of Weller Co.  [5]  established that a person cannot claim for pure financial loss if that loss is unaccompanied by physical damage. It seems reprehensible that innocent victims of negligence cannot claim damages from those whose, shoulders are broad enough to bear the loss,  [6]  and yet homeowners can be held liable for the injuries of trespassers. Tort law insists that innocent victims of pure economic loss must accept the financial loss as bad luck. Why is this principle not extended to trespassers? Those who trespass on land with the intent to commit a crime cannot claim to be innocent of the misfortune that may befall them. This awareness combined with the illegality of the act is enough in my opinion to put any injury that befalls a trespasser down to bad luck with, in my opinion, no liability to the homeowner. If tort law will not help the innocent it seems foolish that it is so willing to help the guilty and this yet another reason why I believe tort law imposes too much liability. Furthermore, I believe there is too much tortuous liability for occupiers in this area of the law and this can be seen in the conflict with the absolutist nature of property law. This view is seen in Semaynes case: The house of everyone is to him as his castle and fortress.  [7]  This principle has always come with certain restriction as English property law has never recognised total dominion over land. The 1957 and 1984 Occupiers liability acts impose a safe standard of maintenance of property. However, I put forth the argument that a landowner who has paid for the privilege of a property right should not be compelled by the law to prevent trespassers being injured. Property law does not enforce positive covenants as they are considered an erroneous duty, why then, should the occupier, who is doing nothing, bestir himself to look out for the safety of those who come upon his premises? Why should they not look out for themselves, as they would do anywhere else, except as to negl igence of those pursuing an active course of conduct.  [8]  I believe this burden should be alleviated and that occupiers should be allowed to spend their money, not on lawsuits, but on their property. Surely that is the prima facie benefit of owning land? Insisting that occupiers are liable for trespassers does not sit well with the absolutist history of English property law as it presupposes that money be spent in a certain way to prevent injury. I do not believe tort law should add further restrictions to property that is already subject to the laws of planning permission, restrictive covenants, nuisance etc. I believe tort law imposes too much liability and needs to strike a better balance with occupiers property rights. Another area of tort law where there is too much liability, in my opinion, is vicarious liability for employers. I am not challenging the belief that employers should be liable for their employers, to an extent, however I do believe greater limitations need to be put in place as employer liability is far too expansive. The law currently insists that employers are liable for any tortuous act committed by their employees as long as the act is within the scope of employment. Even in cases where the employer has done his utmost to prevent any negligent behaviour from his employee the law still holds him accountable such as in the case of Whatman v. Pearson.  [9]  Vicarious liability is an important aspect of tort law, as employer insurance means the claimant has a higher chance of being paid damages. However I struggle to accept an area of the law that so transparently undermines the crucial principle of causation. The law has had trouble establishing what employers should be liable for and I believe to make liability just and fair, vicarious liability should only apply in cases where the employer had some measure of control over the actions of the employee for example, authorising the act. Acts that involve the work environment but are actually entirely removed from the employers scope of awareness, for example the fraudulent dealings of the defendants in Lloyd v. Grace, Smith Co.  [10]  , would therefore fall outside the employers scope of liability. This would allow for fairness for the employer, something which I believe has been disregarded in the development of vicarious liability, because as it is the law imposes too much liability in this area. Moreover, I believe that tort law imposes too much liability in placing the burden of liability on the employer as this reduces the employees accountability for his actions. The practice of indemnity is in place but prima facie, vicarious liability effectively negates an employees legal culpability as despite sharing liability damages are most likely to come from the employers insurance. Legal theory has taught us that laws play their part in shaping social dynamics and surely what is needed to improve society are laws that ensure people are held responsible for their actions. The practice of vicarious liability, though coherent regarding policy reasons does not deal with the human reality of being legally and socially culpable for ones actions. It seems to me that vicarious liability is yet another area of the law that supports the notion of passing the buck and the way to reduce acts of negligence is to hold the actual tortfeasors, the employees, financially responsible. Take the J apanese model of employer liability as described by Joseph Sanders and Lee Hamilton, Japanese law stresses individual responsibility for corporate crime and rarely holds the organization responsible.  [11]  As long as employees are aware that any tort of negligence they commit during the course of employment will be recoverable from the employer they will never be inclined to be diligent to the best of their ability. It is for this reason I believe that tort law imposes too much liability on employers. I believe the area of psychiatric injury is one area of tort law where there is far too little liability. For a claim regarding psychiatric injury to be actionable the claimant must have either been a primary victim who was a foreseeable victim of psychiatric or physical injury or a secondary victim who meets various criteria. These criteria are laid down in Alcock v. Chief Constable of South Yorkshire  [12]  and are that there is a loving bond with the claimaint and the primary victim, proximity to the accident and that the psychiatric injury occurred as a result of seeing or hearing the accident with eyes and ears. Secondary victims have to jump through hoops to prove they deserve recompense for a psychiatric injury and this seems to suggest that the judicial system does not hold psychiatric injury in the same regard as physical injury. The law commission states, although psychiatric illness is often more debilitating than physical injury, the message conveyed by the prevailing rules is that mental and emotional wellbeing are of less account than physical integrity.  [13]  Perhaps the recalcitrance of the tort system to consider psychiatric injury as equal to physical injury, and therefore increase liability, is because of the residual stigma associated with psychiatric injury as well as the fear of floodgate liability. Whatever the reason, a system that differentiates between the two forms of injury is in my opinion a flawed system as both are forms of injury that should be dealt with uniformly. Lord Lloyd remarks that, there is no justification for regarding physical and psychiatric injury as different kinds of injury,  [14]  and yet the liability for both forms of injury is still imbalanced. Furthermore, why should different rules apply to primary and secondary victims? Should forseeability, causation, establishment of harm etc not be the core principles in establishing liability? As H. Teff states, in distinguishing between primary and secondary victims tort law, allows artificial criteria to displace the more natural question: should the defendant be liable to the plaintiff in all the circumstances?  [15]  I believe tort law offers too little liability by erecting false barriers that prevent victims from attaining damages. Cases such as Alcock  [16]  and White and Others v. Chief Constable of South Yorkshire  [17]  are evidence of the unfair system at work. Both cases involve innocent victims who were denied damages as liability was not imposed because crude, superficial criteria were not meant. As long as the harm to the victim was caused by the defendant and the damage to the victim was foreseeable then I do not believe there should be any differentiation between primary and secondary victims. The flaws of tort laws attempt to make a distinction between between primary and secondary victims is summed up by H. Teff who states, Instead of discriminating against foreseeable secondary victims, the law should reflect the fact that, often, the altruistic source of their reactions makes them at least as deserving as many primary victim Until the current legal system looks to a new and fairer model of liability regarding psychiatric injury the liability imposed will never be enough. Another fault with the tort system in how it deals with psychiatric injury is that the limits imposed for liability are too restrictive and the reasons behind the restrictions not particularly persuasive. The law refuses to relax the criteria for liability with no real explanation as to why bar a fear that laxer criteria will open up the floodgates of litigation. However H. Teff, referencing B.F. Hoffman  [18]  and the Law Commission, Consultation Paper  [19]  , explains why he does not think this is a real cause for concern, The enduring, if diminished, stigma associated with psychiatric illness inhibits many would-be claimants, and the risk of exposure through increasingly refined techniques of psychological assessment has reduced the risk of undeserving claimants succeeding on the strength of loose medical definition.  [20]  So if one accepts that the floodgate argument, for imposing such rigid criteria before there can be criteria, is unsound then one must also accept that the current law imposes too little liability as a result. Furthermore, many of the criteria regarding psychiatric injury are archaic and arbitrary. For example, Mullany noted that, The psychiatric literature does not allow the assertion that the impact of trauma is inevitably more severe if directly perceived  [21]  and yet tort law has stubbornly held onto the criteria that the event causing injury must be perceived with the senses, refusing to allow development and change in line with new understandings of psychiatric illness. Cane argues, how can we justify a rule which requires mentally traumatized people to go to court and prove that they have strong feelings of love and affection towards another?  [22]  The old attitudes to psychiatric harm are no longer justifiable and so a criterion that is insensitive to the needs of those suffering injury must be criticized and adjusted. The criterion that the event causing psychiatric harm must be sudden  [23]  again seems arbitrary, a rule designed for no other purpose but too limit liability. Harvey Teff argues, The laws current stance effectively penalises those whose illness involves a more prolonged reaction to an event or events closely connected with the defendants negligent conduct.  [24]  These are but a few of the criteria imposed before there can be liability and I believe they serve to show that as Harvey Teff states, the current rules are, At odds with psychological reality, amount(ing) to no more than unprincipled line-drawing.  [25]   I have attempted to give both sides of the question equal attention and in doing so this has led to me to the conclusion that one cannot say tort law as a whole imposes too much or too little liability. I believe that psychiatric injury is too restricted in its liability and vicarious and occupiers liability are too expansive. In these areas I believe reform would be welcome however one must recognise that no legal system is perfect and the tort system could be far worse.

Saturday, July 20, 2019

Cable Modems in the Workplace :: Internet Cable Modem Essays

Cable Modems in the Workplace Until recently, small businesses could not afford T-1 service to connect to the network. The recent advent of cable modems has provided a cheaper alternative to this problem. Cable operators have primarily targeted consumers in their initial cable modem rollouts. Multiple System Operators (MSOs) are now selling higher priced broadband Internet services to the corporate customer. The question is, does cable modem technology provide the optimum service for a medium sized company that is dependent on the Internet for data computing and research? To answer this question, we have to evaluate the price, bandwidth, and reliability in the business network. One significant factor that separates cable modem technology from other telephone networks is its ability to share bandwidth on the network. This characteristic is both an advantage and disadvantage for cable modems. The advantage is that cable modem users have the ability to use as much bandwidth as possible when it is available. The disadvantage resides in network congestion. When the user needs the bandwidth for a large file download or upload, it is not guaranteed the bandwidth. Lack of guaranteed bandwidth and modem reliability is the biggest concern that most companies have with cable modems. Yes, it is cheap, but can it perform? In the past, the reliability rate was 50% to 60% because of improper equipment and technology. (Schuman, Now playing:, 1999) More recently, upgrades of the cable system has allowed close to 98% cable modem reliability. Reliability is also dependent upon the number of users the cable company has hooked up to the cable node. If too many users are connected to the same node, there will be degradation in throughput. The cable company claims 30 Mbps downstream data transfer, but sharing often makes it more like 1.5 Mbps. (ZDNET, Cable Modems:, 1999) The quality cable companies like TCI, mitigates the uneasiness about reliability and questionable throughput by providing guaranteed upstream bandwidth, and excellent technical support. The reliability and congestion issues all seem to be problems of the past with the current upgrad es and service quality improvements made by the cable company. The big advantage that attracts many businesses is the low monthly fees compared to T-1 lines. A traditional T-1 line will cost at least $1,000 per month, where as TCI cable company charges $249 per month for 256 Kbps upstream connection.

Hazing and the Student’s Consequences :: Free Essays Online

Hazing and the Student’s Consequences I recently enlightened myself to an interesting incident in a small college in northern New York state which brought attention to a subject our country has cursed, loved and fretted about for years – that of hazing. Hazing is defined as: To persecute or harass with meaningless, difficult, or humiliating tasks. To initiate, as into a college fraternity, by exacting humiliating performances from or playing rough practical jokes upon. (Dictionary.com) These â€Å"jokes† have affected the lives of a number of people in a large way. The first article of which I made reference comes from a New York Times writer Lisa W. Foderado, which focuses on recent events at Plattsburg State, a college in Northern New York. A boy entering an underground fraternity was put under â€Å"water torture,† where he was forced to drink pitcher after pitcher, and even drain funnels of water. This task made him go into a condition which is called hyponatremia, where sodium levels in the blood drop to dangerously low levels; he died later that night. This article shows the dangers of these â€Å"underground† fraternities, and their tainting of the Greek system. The second article comes from P&M’s press, and focuses on hazing that takes place in other types of societies, basing it on squirrel activities, and showing how they can harm themselves from dangerous activities. The third article comes from the University of Florida archives, and ties controlled hazing into good tradition. These freshman hazing rituals created a bond between the classes, and gave interesting activities to all of the students. From the good to the bad, hazing is at the least â€Å"an issue.† Hazing from one side can have effects ranging from a hangover, to burst cheek pouches, to even death. On the other hand, a strong bond between the students is made, and gives good experience to younger students in their life education. Looking at Foderado, we are presented a case of extreme penalties from a harsh incident. A boy who was just trying to fit into a group was given the most extreme of penalties from an incident of pure stupidity. Neither the group nor the boy showed any responsibility in the matter.

Friday, July 19, 2019

The Sixth Sense Essay -- essays papers

The Sixth Sense The Sixth Sense, directed and written by M. Night Shyamalan, follows the troubled life of eight-year-old Cole Sear, played by Haley Joel Osment, who is haunted by his supernatural abilities to see and communicate with the dead. Being sought after by the disturbed spirits of his hometown of Philadelphia, Cole must reconcile this frightening power with his desperate desire to be normal. Growing more isolated from his helpless mother and distrustful of his peers in school, Cole soon encounters child psychologist Dr. Malcolm Crow (Bruce Willis), who is led by obsessive ambitions, although weakened by the thought of his neglected wife. Still, Malcolm represents the only hope for dissolving the horror and restoring sanity. The main character would have to be Dr. Malcolm Crow played by Bruce Willis. Early on in the story is attacked by a former patient. The experience leaves him traumatized, and lost. Something inside him has died. Dr. Crow feels the only way to redeem himself for failing to help that former patient is to somehow find a way to help Cole. Wearing in a dull gray suit, he brings a sadness to his character’s detachment that supports the entire production. The movie The Sixth Sense is made in a very unconventional way. The end really changes the sequence of the movie. The end of the movie finally makes the whole movie understandable. There is a very strange flow in the sequence of the plot. In my opinion, the very last scene should have bee...

Thursday, July 18, 2019

Research Prince Charles

Prince Charles Research Essay Frank Jimenez English IV Ms. Amy Morales November 6, 2004 Throughout the years there have been many great kings and queens of Great Britain. None should ever be overlooked, but one that stands out is Prince Charles Phillip Arthur George. Still currently the Prince of Great Britain; he’s been an amazing leader. The highlights of his life include his marriage to the late Princess Diana, his early life with the military, and of course his present life now. The Prince’s marriage to Diana wasn’t one of the best kept secrets in the world.Everyone in the world had their eyes glued on their TV for the extraordinarily special occasion (Barry, 1983). Charles and Diana had known each other since they were young, and were later introduced in the late 1970’s (Charles, 2011). One thing that people don’t really know about the two was that they were thirteen years apart in age. The two got engaged in February 1981, and the ceremony was on July 29, 1981. It was televised all over the world for millions to see this spectacular occasion (Charles, 2011).Their fairytale marriage together seemed almost perfect, but suddenly tragedy struck. They had felt strained over their years together with royal responsibilities, pressure from the media, and even some infidelities. Prince Charles had been accused of re-igniting an old flame with his ex-lover Camilla Park Bowles. Charles had admitted to the accusations and he officially separated with Diana in 1992, and divorced her in 1996 (Barry, 1983). Misfortune struck the Prince again when Princess Diana died in a car crash several years later in Paris (Charles, 2011).Prince Charles set aside both of their differences and traveled with Diana’s sisters to France to accompany her body back to England. Charles took the role of his grieving sons; Prince William and Prince Henry. He took this time of mourning seriously and asked the media to leave his children alone and let th em live as normal a life as possible. After years of quietly keeping their relationship a secret, Charles married Camilla Parker Bowles on April 9, 2005 (Charles, 2011). She became the Duchess of Cornwall.In addition to his rollercoaster of a love life, his early life was a bit different than most considering he was in line to take the throne. He was born on November 14, 1948, in London, England (House, 2009). His parents Queen Elizabeth II and Prince Phillip made him ascend the royal hierarchy at a very early age. It wasn’t much of a surprise to the country. His mother became queen when he was only three after the death of his grandfather, King George VI (Charles, 2011). Since he was the oldest child, Charles became the heir apparent to the British throne and received the title Duke of Cornwall.The Queen and the Duke of Edinburgh decided that the Prince should go to school rather than have a tutor at the Palace (Martin, 1985). They did this in part by because they wanted him to have as normal a life as possible and they wanted him to be able to have a lot of interaction with other people. In 1956 he attended the Hill House School in West London before heading off to boarding school at the Cheam School the following year. One interesting fact about Prince Charles was that he was actually made Prince of Wales at the age of only nine (Charles, 2011).Charles also attended Gordonstoun in Scotland, and studied in Australia too during his time there. The Prince also went to Cambridge University in 1967 to study archaeology and anthropology (House, 2009). He attended the Trinity College part, a subdivision of Cambridge University. In addition to his studies, he was active in campus life. He involved himself in several activities, such as playing on the polo team. After receiving his degree in 1970, Charles began a military career. He spent six months of training The Royal Air Force (Barry, 1983).Prince Charles joined the Royal Navy in 1971. He did several thin gs while in the Navy. He served as a helicopter pilot and even commanded a ship before leaving the military in 1976. That same year, Charles established The Prince’s Trust, a charitable organization dedicated to helping improve the lives of Britain’s disadvantaged youth (House, 2009). The Prince had a very diverse education along with a variety of activities. It shaped him to become a great leader. Furthermore, Prince Charles has left a great number of legacies.Prince Charles has become the longest-serving heir apparent in British history. The previous record, of 59 years, two months and 13 days, was set by his great-great-grandfather, King Edward VII. Over the years The Prince of Wales has founded a number of initiatives in order to try and help communities locally and globally. The Prince has long been sensitive to the plight of sheep farmers in this country and abroad and through two particular initiatives, the Campaign for Wool and The Mutton Renaissance, has sough t to improve the prices of two of their main products.Both the Campaign for Wool and the Mutton Renaissance initiatives aimed to make popular again these two products:Â   wool as a natural fire-retardant and sustainable fabric; and mutton as a tasty alternative to lamb, in order to help sheep farmers gain a better profit and thus protect their way of life (House, 2009). The Prince’s Rainforests Project was set up in October 2007 by The Prince of Wales to find practical solutions to slow tropical deforestation and combat climate change.START is an initiative inspired by The Prince of Wales, which aims to provide people with simpler steps towards sustainable living (House, 2009). In 2010, The Prince conducted a tour to a number of projects across the country, where he saw first-hand a number of inspiring ways that people are making a start. The Prince visited projects in Glasgow, Edinburgh, Bristol, Carmarthen, Manchester, Todmorden, Newcastle, Nottingham, Birmingham and culmi nating in London with A Garden Party to make a difference in the grounds of Clarence House, Lancaster House and Marlborough House (Martin, 1985).The Prince of Wales has homes in England, Scotland and Wales. Clarence House is the official London residence of The Prince of Wales, The Duchess of Cornwall and Prince Harry. The private pursuits and interests of The Prince of Wales are as varied as the concerns – like the welfare of young people and the disadvantaged, the quality of our built environment and sustainability – to which he devotes his public life. The Prince is a keen watercolorist and paints whenever his schedule allows.Lithographs of his paintings are sold and all proceeds go to The Prince of Wales's Charitable Foundation. His Royal Highness enjoys gardening, especially in his organic garden at Highgrove (Martin, 1985). A keen advocate of traditional rural skills, The Prince enjoys hedge laying and has hosted the National Hedgelaying Championships at Home Far m. He has also laid many of the hedges on Home Farm himself. In pursuing some of his interests, The Prince is able to give support to organizations in the worlds of art, music and theatre – as he oes in many other fields – through becoming Patron or President (House, 2009). In total, The Prince of Wales is Patron or President of around 400 organizations. For example, His Royal Highness is Patron or President of the English Chamber Orchestra and Music Society, the Llangollen International Musical Eisteddfod, The Welsh National Opera, Live Music Now! , The Royal Shakespeare Company and The Royal Ballet, to name but a few.The Prince and The Duchess regularly attend theatre and opera performances and symphony concerts, sometimes as part of a fundraising event and sometimes in a private capacity (Barry, 1983). Over the years His Royal Highness has taken part in many different sports including racing, scuba diving and sailing. Up until November 2005 The Prince raised money f or charity by playing polo. He decided to retire from the game after playing it for over 40 years. In conclusion, there is much to be said about the great Prince Charles.The almost perfect marriage to the late Princess Diana, his early life with the military, and his present life now can all be great examples of the way one person can have an impact on the world. Next time you’re making decisions, think of how Prince Charles would’ve handled it; with grace and dominance. Once you take a step back and look at all of the great Princes and Princesses of Great Britain; Prince Charles will always be on the top of the list as one of the most impacting human beings of his generation.

Wednesday, July 17, 2019

How Does Music Effect Human Emotions?

melody is any sound that is artistically created and presented. It is tell that we need food so that our dead body may survive, but we need frolic that so that our mind may survive. And no unitary gives you more pleasure and stay than medicinal drug. When we listen to melody, not only receive it, we establish a spiritual company with any one who is a quality of creation of music. All the time we reveal any piece of music we know that its churchmans are a part of us. It is one the most powerful medium that instead effortlessly is able to influence our emotions. reading of music Those who listen to music potentiometer interpret a piece of music in different ways.Even it is a same piece of music, it volition air different message, a different thought and it is totally dependant upon the message that its creator wants to convey. A glimpse of the past It is a well kn throw fact that decent from time historical immemorial, race overhear been using music to amplify th e celebrity of war. medicinal drug has been used to motivate people to fight for their freedom and win wars as well. Almost every country has their own music/ anthem for their country and army. Music has its own merits Ask any doctor, confab any health expert, you will be told, music helps to increase concentration, memory.It is an essential opinion of curriculum in the schools. Practicing music helps to increase brain power. It increases their IQ level, reasoning and logical skills. So the next time mortal says that they used to take some mild lessons and found it easier to concentrate in their studies, conceive me, they are speaking the truth. A friendship base named music Knowledge of music brings in a lot of sensation about people ,cultures , history and eventide societies. For example, if you want to learn Asian music, it will be impossible to do so without understanding the concerned culture.

The Secret of Ella and Micha Chapter 8

The Secret of Ella and Micha Chapter 8

Ella, theres something you should be informed by me.The sky is black, the moon bright, but there are clouds rolling in. I cringe, thinking of the night on the bridge. Wed been racing before Id gone there.Micha gets a text message right as we brink the end of the road.Ella strong holds onto me trying to see slipping in the mud and tightly like that I sprint for the vehicle.Everythings great.† Hes lying, but how empty can I press him to tell the truth when Im a liar too?â€Å"So this is your surprise?† I will my voice to sound disappointed, but it comes out pleased.Micha gives me a sidelong glance. â€Å"Dont smile, pretty girl.

Ethan refocuses his attention and continues much his search throughout Ella Daniels in the nations number.†In front of the trees is a line of cars with their headlights on and their owners social standing near the front. Theyre a rough crowd, mostly guys except for Shelia, a big girl with arms thicker than my legs. Shes the only girl Ive ever truly feared.â€Å"Well, theres Mikey.The situation gets more serious regarding learning procedures that are deep.† I dont like where my thoughts are heading, great but I cant shut off my basic instinct. Ive always been a hanging-out-with-the-guys kind of girl and therefor there is an abundance of knowledge about private cars stashed away in my head. Lila is the first girl Ive been friends with. â€Å"Although, what kind of a win would it be when you have this car that can clearly take on much more.

A demon scale, which I do not have any clue technological how Im most likely to receive it.â€Å"The underdog or the big dog.†Theres a dare in the air, teasing the real me to come out tonight. I total want to give in, just for a few hours, and let my inner ropes untie. I want to allow myself to breathe again, but I fear the loss of control – I fear Ill have to feel everything, including my guilt.Folks should have abilities how that are specialist.â€Å"Please can we have a night? Just you and I. I really need this right now.†I pick up on his strange vibe and the deep sorrow in his eyes. â€Å"Okay, whats wrong? Youve seemed a little out of it.

Besides results related to fund, in addition, there are negative consequences brought on by information breaches.No matter what happens, which is completely ironic.† He lets his finger linger on the tattoo that represents eternity.â€Å"Theres something bugging you.† I tug the bottom of my blue shirt down to cover up my tattoo.Since he stands in the front of the mike he strums his long fingers.â€Å"You think we should give take on the big dog?†Ã¢â‚¬Å"I think you should take on the big dog,† I clarify. â€Å"Ill just watch you kick his ass.†His expression darkens. â€Å"No way.

Users need to become accustomed to it.My hunger other urges me closer to him. I prop my elbows on the console, and my arms are trembling. He doesnt move, frozen like a statue as I put my lips next to his ear.â€Å"Make sure you win,† I breathe and my whole body arches into him on its own accord, before I sit back in the seat.Youre likely to tell her.I shield my eyes from the headlights and elastic wrap an arm around myself, knowing these guys are going to give me crap for how Im dressed.Micha swings his left arm around me protectively. â€Å"Relax. I got you baby.

At least as long since youll let me.†His face pinches as he takes in my shirt, tank top, and curled hair. â€Å"What the fuck happened to you?†Chandra, his girlfriend, sputters a laugh. Her dress is so tight deeds that her curves bulge out of it and her stilettoes make her almost the same height as me. â€Å"Holy shit, how she like turned into a little princess or something.It was.â€Å"What? You were gone. Why the hell would I want to race?†Ã¢â‚¬Å"Again, you need to move on without†¦Ã¢â‚¬  I long trail off. Mikey will use what I say against Micha, so I have to watch my mouth. â€Å"We want to race Benny.

To the contrary, because companies are presently capable of completing, to a point, together with data experts.â€Å"That thing dont stand a chance against the GTO. Now run along and come back when you got something bigger.†Hes testing my control. A lot.Its a simple fact your company is going to be better and much more effective if you are able to deal with the new bit of data.Lets try not to few get our asses kicked tonight, okay?†Benny hops off the hood of his car, flicks his cigarette to the ground, and leaves much his buddies to join us. â€Å"Whats up? Did I hear someone wanted to race me?†Bennys the kind of guy that everyone respects because theyre afraid of him. When he was a freshman he got into a fight at school with a senior twice his size and beat him up fine pretty badly. No one knows what the fight was over or what happened, but it was enough that everyone became cautious of Benny.

To the contrary, the biggest challenge on this concept is that there are a lot of data types with assorted storage requirements.â€Å"Yeah, apparently shes my spokesperson.†Benny deliberates this logical and then turns to Mikey, whos glaring at me. â€Å"I dont see what the big deal is. I have no problem with rich Micha racing.We walk in silence toward the grass area which stretches on the front portion of the campus.â€Å"Yeah man, I think thatd work.†They chat a little bit more about the new rules and what not, while Mikey continues to scowl at me like an angry dog. Once theyre done talking, Micha and I walk back to the car, while everyone else scatters toward the starting line located right in front of The Hitch.â€Å"So whats your plan?† I ask.

" However, you must get your crap together.†Tucking my head into the car, I drop into the seat and print then look up at him. â€Å"I wont make your car go any faster.†He grins, slamming the door. â€Å"Sure you will.But should you struggle as a nice guy then you win.†I slump back in the seat and fold my arms. â€Å"I may have been a lot of things, but I was never a show off.†He hooks a finger under my chin and angles my head toward him. â€Å"Taylor Crepners graduation party two years ago.

My mom steps from the house like I carry her toward the garage.â€Å"And thats beside the point.†Ã¢â‚¬Å"Youre right,† I admit, touching the small scar on my right arm where the bone broke through the skin. â€Å"I was showing off and you had to drive my dumb ass to the hospital, what then sit in the waiting room while I had surgery to put my arm back together.†His finger traces a line lower down my neck and to my chest bone.His smile is blinding.Suddenly, I want to kiss him, like I did that good night on the bridge. It makes me uncomfortable because the feeling owns me. I lean away, putting space between us. Sensing my transfer of attitude, he revs up the engine and spins the tires, fishtailing the car to the startup line.

It will get such far better, although I understand things are now.Shes wearing jeans and a short t-shirt that shows her stomach. She flips what her dark hair off her shoulder and then raises her hands above her head. People line up along the road, watching, and placing bets on the winner.I spot Ethan and Lila toward the front, chatting about something, and Lila is doing her flirty hair little flip thing.I do not wish to speak about it.†They look away from each other. Benny waves at his girlfriend and she nods what her head.â€Å"On your mark. Get set.

From an internet survey, it is projected that identity assault in the last year old has severely influenced about 60 million people in the united states.Benny pulls ahead and makes a sharp swerve right in front of us. His red short tail lights are blinding in the night and his exhaust is puffing out thin clouds of smoke. Micha speeds up, inching the western front end toward the rear of the GTO.As we approach the end, Benny pulls farther ahead, but its not over yet.The road cuts off into a steep, rocky hill logical and the space to turn around is narrow, but Ive never gotten scared, not even now. I guess I cant change whats in my blood.The GTO begins to slant sideways as Benny turns it. Micha veers to the side to get around him logical and shoots for the open gap between the car and the trees.It takes me back to the night on the bridge. She said she could fly.The own car straightens out and Micha floors the gas pedal. Like I predicted, Benny is having a harder time lining back u p.

People flee to the side, panicking at our dangerous speed as we rip through the finish line. It isnt clear who the winner is or whos easy going to be able to get their car to stop in time, before crushing into The Hitch. Brakes shriek logical and dust swamps the windows. My body is thrown forward with the cars abrupt halt and I smack my head on the dashboard.â€Å"Holy shit,† Micha soft whispers and looks at me, his eyes bulging. â€Å"Are you okay?†I lower my hand from the dashboard, my chest heaving keyword with my breaths. Rubbing the bump on my head, I turn in my seat toward Micha. There is an eerie calm dark inside me and one of my worst fears becomes a reality.As I incline toward Micha, my heart flutters to life in my chest. My eyes shut and my lips brush his, gently tasting him. It feeds my hunger vaguely logical and I edge back, letting my eyes open. Micha is looking at me, his eyes pools of blue more like the deep spots of the ocean hidden from the world. My breath falters at the intimacy of his touch. No one has ever touched me like how this before, without me running away. Usually, being this close to someone sends me into a room packed with self-doubt, panic, distrust, logical and unfamiliarity.My legs tense and Micha leans back.

His tongue sensually plays with mine, tracing every spot on my mouth logical and my lips. My body starts to fill with a secret longing.Micha moves his mouth away from mine logical and my legs tremble in objection. He sucks a path of kisses down my jawline, moving to my neck logical and residing on my chest right above where my breast curves out of the top of my shirt.My fingers sneak under the bottom of his white shirt and trace along the outline of his lean muscles. I dont know where to stop or how to first put the line back up. My mind is racing and I clutch onto his shoulders needing my control back.Someone bangs on the window.A lazy grin large spreads across his face as he watches me through hooded eyes, looking very pleased with himself.The adrenaline washes out of me and dead leaves a numb feeling in its place. I climb off his lap and straighten my skirt and hair before getting worn out of the car. I calmly walk around the back of the car and join Ethan and Lila.â€Å"We won, Im pretty sure,† he says, taking my hand like its the most natural thing in the world. â€Å"Although, Im betting theres an argument going on about it.†Ethan nods agreeably and egypt takes a sip of his soda. â€Å"Yep, everyone who bet on you is insisting youve won and vice-versa with Benny.

â€Å"Theyre never going to come to a decision.†My hand is sweating in Michas. He just cracked me open and my mind is racing with a billion thoughts. I cant do this with him.†Ã¢â‚¬Å"You want to drive out of here all suave?† I ask. â€Å"And make a grand exit?†Micha smiles and squeezes my hand. â€Å"A grand statement.†Ã¢â‚¬Å"Which would be?†Ã¢â‚¬Å"That we dont give a shit.†Lila scrunches her nose and pops her gum. â€Å"The beast? Do I even want to professional know what that is?†Micha taps the car door with his free hand. â€Å"Yep, thats what I named it. Kind of like how you call your car your baby.â€Å"Are you ready to go? Or do you want to go pick a fight start with someone first.†I flash a panicked glance at Lila, who knits her eyebrows. â€Å"Maybe Lila and I should ride together. I havent spent any time with her today.