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

Discussion - Article Example This is evident in the passage as it is confirmed by this statement â€Å"thus, intersubjectivity emerges as part of a larger problem (141).† It is a reality that subject reacts with other subjects without mediation of material elements such as body or sense media. This concept is implicit in the notion of ‘intersubjectivity’. Recognition that the person uses the materials of culture and is impacted by other people does nothing to impose on the reality that such a notion is essentially individualist (Williams, 1992). Hegel does not believe that every person has to involve in a life and death fight for recognition. This is evident as gave a suggested a different concept in substitute of a ‘formation of consciousness’, he proposed an individual level and a communal social-cultural world-chronological level.’ This can be analyzed as an attempt to prevent people from seeking recognition to much. The concept of consciousness is brought in the passage, however, not as self-consciousness but people living in self-sufficient communities. Undeniably, consciousness remains a prerequisite for recognitive self-consciousness. Hegel regards material culture as products of contemplation, and never spent bothered to understand the interrelationship between materials. While he discarded the labor procedure as a pattern of the dialectic he did not discard apprehension with the creation and use of material civilization, but changed it into a logical stature. Thus, when the promoters of ‘intersubjectivity’ expunge the usage of artifacts from unrestrained action, they ignore Spirit itself. Williams asserts that Hegel changed subjectivity to intersubjectivity. However, ‘intersubjectivity’ is incomprehensible and incoherent with core fundamentals of Hegel’s perception of subjectivity (Williams, 1992). It is accurate to conclude that recognition is an extremely un-Hegelian concept since it assumes the nonexistence of effective mediating media and artifacts. It

Racism and Kathryn Hess English Essay Example for Free

Racism and Kathryn Hess English Essay In the beginning of the movie Freedom Writers, the students initially only trust their peers from their racial groups. This is because they only trust the people in their gangs. Almost all the kids were in racially segregated gangs. At first, Ms. Gruwell has difficulty getting anything accomplished. Many of these students have never been shown any respect in the past. Eva and some of the other students tell Ms. Gruwell that they will not just hand her their respect, she must earn it. Ms. Gruwell begins to earn the respect of her students when she moves the students around, out of their racial divisions. She attempts to show the students that they are united by playing the â€Å"Line Game† with them. She puts a line of thick red tape across the classroom and tells the students to move forward when a statement she makes applies to them. Her most effective form of reaching out, however, comes in the form of a composition notebook. Trust is an important component of a teacher-student relationship because if a student doesnt trust the teacher, nothing the teachers teaches is affective. Ms. Gruwell eventually managed to get her students to show tolerance for one another. She teaches them about the Holocaust and that despite the students’ ethnic backgrounds, they aren’t all that different from each other. For example, she takes them to the Museum of Tolerance. This shows that the students aren’t realistically the different despite the color of their skin or their ethnic background. This is important because Ms. Gruwell could not effectively teach the class until they could get along. As a result, the students begin to build up a tolerance for one another. Part of Ms. Gruwell’s outlook on racism was affected by her father. For example, growing up, her father was a civil rights worker. This shows that his work most likely influenced her views on racism as she grew up. This is important because it taught her not to discriminate against others. As a result, she is able to change the views of her students for the better. At one point, Ms. Gruwell confiscates a racial caricature that was circulating the class. For example, the drawing was of a black student drawn with thick, exaggerated lips. Ms. Gruwell then compared the sketch of the caricatures that the Nazis used to draw of the Jews during the Holocaust. This showed that none of the students even knew what the Holocaust was. This is important because it allowed Ms. Gruwell to teach her students how serious racism really was. As a result, the students became more tolerant. Ms. Gruwell knew that all the students were suffering from physical violence, emotional abuse, substance abuse, poverty, homelessness, gang violence, and deaths of family and friends. She felt sorry about them and wanted to help them sincerely. She did it, and as a result, the class was getting better and better, and the classes grades turned up quickly. Others teacher didn’t believe Ms. Gruwell despite she did a great job. They thought she was a new teacher and had no idea about teaching. But the main point was, they had serious racial prejudice in their minds, they disliked the students in Room 203. For example, one of the teachers refused to lend books to Ms. Gruwell. This shows the racial discrimination was really serious. Hence, Ms. Gruwell had to do everything by herself. Even when Ms. Gruwells husband left her, the students made her life better. Problems still came up, she was denied to teach Room 203 in junior and senior year. But after the tough fight with other teachers, she was allowed to stay with them till the end of high school time.

Cesare Lombroso :: criminology

Cesare Lombroso was an Italian university professor and criminologist, born in Nov. 6, 1835, in Verona, who became worldwide renowned for his studies and theories in the field of characterology, or the relation between mental and physical characteristics. Lombroso tried to relate certain physical characteristics, such as jaw size, to criminal psychopathology, or the innate tendency of individuals toward sociopathy and criminal behavior. As such, Lombroso's approach is a direct descendant of phrenology, created by the German physician Franz Joseph Gall in the beginning of the nineteenth century, and closely related to other fields of characterology, such as craniology and physiognomy. His theory has been scientically discredited, but Lombroso had the merit of bringing up the importance of the scientific studies of the criminal mind, a field which became known as criminal anthropology. Lombroso studied at the universities of Padua, Vienna, and Paris, and was later (1862-1876) a professor of psychiatry at the University of Pavia and of forensic medicine and hygiene (1876), psychiatry (1896) and criminal anthropology (1906) at the University of Turin. He was also the director of a mental asylum in Pesaro, Italy. Lombroso's main idea was partly inspired by the evolutionary and genetical studies at the end of the nineteenth century, and proposed that certain criminals had physical evidence of an "atavistic" (reappearance of characteristics which were present only in distant ascendants) or hereditary sort, reminiscent of earlier, more primitive stages of human evolution. These anomalies, named as stigmata by Lombroso, could be expressed in terms of abnormal forms or dimensions of the skull and jaw, assymmetries in the face, etc, but also of other parts of the body. These associations were later shown to be highly inconsistent or plainly inexistent, and theories based on the environmental causation of criminality became dominant.

Politics in Joseph Conrads Heart of Darkness :: Heart Darkness essays

Politics in Heart Of Darkness Anyone can read Heart Of Darkness and easily sense the attitude of Conrad toward English politics. Many times throughout Heart Of Darkness Conrad points out the pointlessness and savagery of English colonization. Conrad also comments a bit on society as a whole. With these two ideas added to the book, there is no wonder of why Heart of Darkness is such a touching novel. Through several examples, Conrad often shows the pointlessness and savagery of the English colonization in Africa. Probably the first instance of this is when Marlow comes up to the French-man who is "shelling the bush". In this scene, the French see something move and so they start shelling it for that reason. The shelling really does no good; if fact, it probably does not even kill what is out there. This represents what the English are doing in a way -- they are trying to conquer a land by shelling it to death and by trying to kill all the people who live there. The next example that Conrad gives is when he sees the black guard, who is leading the black slaves in a chain gang, straighten up when he sees a white man. What this shows is how everyone tries to look better than they are when they are in front of a supposed superior person. Also it shows that if a person can suck up enough -- and sometimes betray their own people -- they can move up in the world. Probably the biggest example of the pointless of colonization is when Marlow is walking around and he sees big holes just around, a train and tools rusting to pieces, and when he hears blasts that seem to do nothing. What this shows is that the English presence in Africa does no good but create a empty hole in it (the big hole), that the English are just was wasting their time and money on a needless project (the rusting objects), and that the English seem to do nothing in Africa (the pointless blasting). One the final examples that he gives is the manager. This manager is all dressed up in "proper" clothes, must have everything in a perfect order, and complains about the sick man in the corner of the room. This symbolizes how uncaring the English are in their pursuit for ivory.

Homework Should Be Banned Essay

Homework has been given to students because teachers believe it helps them remember what they learn at school, and helps them learn the material better. â€Å"Homework is super helpful in learning and processing the material learned in class.† Said no kid ever. Too much homework is not helpful, and can be unhealthy. Excessive amounts of time spent on homework can take away from kids social lives, family time, and sports or other activities. Homework should be outlawed. Critically acclaimed author Tami Ansary says, that since 1981, the amount of homework given to sixth graders has increased by more than fifty percent. In 1957, the Russians launched Sputnik into outer space. The new competition made schools try to increase the difficulty of the curriculum. With harder classes came more homework. Homework takes away from time that kids could be spending with their families. Rather than spending time bonding and building strong family relationships, parents and children argue over homework. Time is taken away from important elements of daily life. This time is particularly precious in families with two working parents whose time with their children is limited. This time could be better spent going out to dinner, or doing other activities that most families are not able to do because the kids have too much homework. My opposition might say that lots of homework prepares them for the â€Å"real world† and lots of homework helps them learn the material better. When difficult work assignments are given frequently, it causes students to lose interest in the subject. Negative results can also occur when someone is not able to finish his homework. Usually they will resort to copying homework, having others do their assignments, or cheating on tests. Bad habits like these are likely to follow them through their lives. With no consequences to these actions students will almost always take the easy way out when it comes to homework. The use of homework is effective when used by the rule†¦

The Humanistic Perspective Essays

The Humanistic Perspective Essays The Humanistic Perspective Paper The Humanistic Perspective Paper The humanistic approach takes an optimistic view of human nature, seeing people as essentially good. Believing in the more noble aspects of human existence, it insists on the value of qualities such as choice, free will and self definition. Humanistic psychologists believe that we are all unique individuals and that each person is shaped by their own personal experiences. This approach is also notable for being open to more philosophical influences, stating that we must define our lives for ourselves. Humanistic psychologists also believe that experiments on humans are inappropriate because everyone is unique. They believe that if people are treated like objects they may as well become like objects. Instead clients should be treated as whole human beings. Humanistics often object to calling those who participate in studies subjects, since this implies that they are merely powerless objects in a psychologists game. They prefer words like clients and participants, and they prefer to emphasise mutual cooperation and respect. One key idea related to the idea of the whole person is self actualisation. The idea of self actualisation focuses on human motivation. Maslow (1970, cited in Haralambos Rice et al 2002) developed a theory of human motivation. He believed that humans are motivated to satisfy a series of needs. The needs are hierarchical in the sense that lower level needs must be satisfied before the needs above can be motivated. The diagram below shows Maslows hierarchy of needs from the most basic needs to the highest level. Unconditional regard is the other key idea in the humanistic approach. One factor that helps us to achieve our potential is unconditional positive regard. Rogers was responsible for developing client focused therapy this centres on people as individuals who can shape their own lives. Rogers (1951, cited in Haralambos Rice et al 2002) suggested that the therapist must not give advice or tell the clients what to do. The therapist instead, must create a supporting and accepting environment allowing the client to discover themselves and search for their own individual meanings. Unconditional positive regard must be shown by the therapist, being accepting and uncritical, sincere and genuine. This encourages the clients to become honest and accept themselves. By this route they will recognise and become their true selves. These ideas have encouraged many self help groups. Although the humanistic approach has many positive factors it also has weaknesses. The humanistic ideas are very vague and untestable. Some critics see the humanistic approach as a collection of recipes for living containing advice for getting the best out of life, rather than an approach which explains human behaviour. Also many self help therapy groups can be time consuming and are often only available to those who are willing to pay.

Its Is a short summary about Sherlock Holmes for Hounds Of The Baskervilles

Its Is a short summary about Sherlock Holmes for Hounds Of The Baskervilles Sherlock Holmes is a very complex character, he has many different qualities. You can almost think you know him, and then he might do something unexpected, like later in the book. I can not give his physical description, because the book does not go into great detail about his appearance. He approaches evidence in a very good manor. He always looks everything over; He always looks for every piece of evidence that he would be able to pick up and use. He also thinks outside the box when he is investigating, that is one reason why he is so good at his job as a detective. When he questions someone, he ask them every single question that can do with that case, because Sherlock Holmes know that he might not be able to question them again later. Holmes thinks a lot of himself and he makes it very evident. Some time he almost even shows off and brags to Watson about how he is better than him at solving crimes.Sherlock Holmes in "The Adventure of the Dying Det...Such as, in the first chapter, Holmes asks Watson what he thinks about the walking cane that was left behind. Watson examines the cane and give his best thought about what happened. Then, Holmes just said that that was a nice try, but this is what he belives what happened with his stick, and he was almost saying what he says, is right, always. Holmes does have a few quirks about him. He is almost unnaturally intelligent and can think of a solution to a case in a very short time. He cocaine addiction is also a very peculiar part about him. You wouldn't think that someone that smart and with that sharp of a mind would resort to cocaine when in need of assistance in thinking. I think the...

Learning Dates and Days in Mandarin Chinese

Learning Dates and Days in Mandarin Chinese The Mandarin Chinese calendar is relatively easy to learn. The days of the weeks are numbered 1 – 6, so once you’ve learned your Mandarin numbers, weekdays are a snap. The same thing with months – all the months are numbered from 1 – 12, so once you’ve learned these numbers, you simply add the word for â€Å"month† and you have the complete Mandarin calendar under your belt. Throughout this article, audio files are marked with ââ€" º. Number Review 1 ââ€" ºyÄ «2 ââ€" ºÃƒ ¨r3 ââ€" ºsÄ n4 ââ€" ºsi5 ââ€" ºwÃ… ­6 ââ€" ºlià ¹7 ââ€" ºqÄ «8 ââ€" ºbÄ 9 ââ€" ºjià º10 ââ€" ºshà ­11 ââ€" ºshà ­-yÄ «12 ââ€" ºshà ­-à ¨r Days dayââ€" ºtiÄ nÃ¥ ¤ ©todayââ€" ºjÄ «n tiÄ nä »Å Ã¥ ¤ ©yesterdayââ€" ºzuà ³ tiÄ næ˜ ¨Ã¥ ¤ ©tomorrowââ€" ºÃ¢â‚¬â€¹mà ­ng tiÄ n明å ¤ © Weeks weekââ€" ºlÇ  bi / ââ€" ºxÄ «ng qÄ «Ã§ ¦ ®Ã¦â€¹Å" / 星æÅ"Ÿthis weekââ€" ºzhà ¨i gà ¨ xÄ «ng qÄ «Ã©â‚¬â„¢Ã¥â‚¬â€¹Ã¦ËœÅ¸Ã¦Å"Ÿlast weekââ€" ºshng gà ¨ xÄ «ng qÄ «Ã¤ ¸Å Ã¥â‚¬â€¹Ã¦ËœÅ¸Ã¦Å"Ÿnext weekââ€" ºxi gà ¨ xÄ «ng qÄ «Ã¤ ¸â€¹Ã¥â‚¬â€¹Ã¦ËœÅ¸Ã¦Å"Ÿ Months monthââ€" ºyuà ¨Ã¦Å"ˆthis monthââ€" ºzhà ¨i gà ¨ yuà ¨Ã©â‚¬â„¢Ã¥â‚¬â€¹Ã¦Å"ˆlast monthââ€" ºshng gà ¨ yuà ¨Ã¤ ¸Å Ã¥â‚¬â€¹Ã¦Å"ˆnext monthââ€" ºxi gà ¨ yuà ¨Ã¤ ¸â€¹Ã¥â‚¬â€¹Ã¦Å"ˆ Years yearââ€" ºninÃ¥ ¹ ´this yearââ€" ºjÄ «n ninä »Å Ã¥ ¹ ´last yearââ€" ºqà ¹ ninåŽ »Ã¥ ¹ ´next yearââ€" ºmà ­ng nin明å ¹ ´ Weekdays Mondayââ€" ºxÄ «ng qÄ « yÄ «Ã¦ËœÅ¸Ã¦Å"Ÿä ¸â‚¬Tuesdayââ€" ºxÄ «ng qÄ « à ¨r星æÅ"Ÿä ºÅ'Wednesdayââ€" ºxÄ «ng qÄ « sÄ n星æÅ"Ÿä ¸â€°Thursdayââ€" ºxÄ «ng qÄ « sà ¬Ã¦ËœÅ¸Ã¦Å"Ÿå››Fridayââ€" ºxÄ «ng qÄ « wǔ星æÅ"Ÿä ºâ€Saturdayââ€" ºxÄ «ng qÄ « lià ¹Ã¦ËœÅ¸Ã¦Å"Ÿå… ­Sundayââ€" ºlÇ  bi rà ¬ / ââ€" ºlÇ  bi tiÄ n / ââ€" ºxÄ «ng qÄ « rà ¬ / ââ€" ºxÄ «ng qÄ « tiÄ nç ¦ ®Ã¦â€¹Å"æâ€" ¥ / ç ¦ ®Ã¦â€¹Å"Ã¥ ¤ © /星æÅ"Ÿæâ€" ¥ /星æÅ"Ÿå ¤ © Months of the Year Januaryââ€" ºyÄ « yuà ¨Ã¤ ¸â‚¬Ã¦Å"ˆFebruaryââ€" ºÃƒ ¨r yuà ¨Ã¤ ºÅ'æÅ"ˆMarchââ€" ºsÄ n yuà ¨Ã¤ ¸â€°Ã¦Å"ˆAprilââ€" ºsà ¬ yuà ¨Ã¥â€ºâ€ºÃ¦Å"ˆMayââ€" ºwÇ” yuà ¨Ã¤ ºâ€Ã¦Å"ˆJuneââ€" ºlià ¹ yuà ¨Ã¥â€¦ ­Ã¦Å"ˆJulyââ€" ºqÄ « yuà ¨Ã¤ ¸Æ'æÅ"ˆAugustââ€" ºbÄ  yuà ¨Ã¥â€¦ «Ã¦Å"ˆSeptemberââ€" ºjiÇ” yuà ¨Ã¤ ¹ Ã¦Å"ˆOctoberââ€" ºshà ­ yuà ¨Ã¥  Ã¦Å"ˆNovemberââ€" ºshà ­ yÄ « yuà ¨Ã¥  Ã¤ ¸â‚¬Ã¦Å"ˆDecemberââ€" ºshà ­ à ¨r yuà ¨Ã¥  Ã¤ ºÅ'æÅ"ˆ What’s the Date? What’s the date today?ââ€" ºJÄ «n tiÄ n shà ¬ jÄ « yuà ¨ jÄ « ho?ä »Å Ã¥ ¤ ©Ã¦Ëœ ¯Ã¥ ¹ ¾Ã¦Å"ˆå ¹ ¾Ã¨â„¢Å¸?Which day of the week?ââ€" ºLÇ  bi jÄ «?ç ¦ ®Ã¦â€¹Å"Ã¥ ¹ ¾?Which day of the month?ââ€" ºJÄ « ho?Ã¥ ¹ ¾Ã¨â„¢Å¸?What month is it?ââ€" ºJÄ « yuà ¨?Ã¥ ¹ ¾Ã¦Å"ˆ? Practice Dates What’s the date today?ââ€" ºJÄ «n tiÄ n shà ¬ jÄ « yuà ¨ jÄ « ho?ä »Å Ã¥ ¤ ©Ã¦Ëœ ¯Ã¥ ¹ ¾Ã¦Å"ˆå ¹ ¾Ã¨â„¢Å¸?Today is May 10.ââ€" ºJÄ «n tiÄ n shà ¬ wÇ” yuà ¨ shà ­ ho.ä »Å Ã¥ ¤ ©Ã¦Ëœ ¯Ã¤ ºâ€Ã¦Å"ˆå  Ã¨â„¢Å¸Today is June 22.ââ€" ºJÄ «n tiÄ n shà ¬ lià ¹ yuà ¨ à ¨r shà ­ à ¨r ho.ä »Å Ã¥ ¤ ©Ã¦Ëœ ¯Ã¥â€¦ ­Ã¦Å"ˆä ºÅ'Ã¥  Ã¤ ºÅ'號Today is December 24.ââ€" ºShà ­ à ¨r yuà ¨ à ¨r shà ­ sà ¬ ho.Ã¥  Ã¤ ºÅ'æÅ"ˆä ºÅ'Ã¥  Ã¥â€ºâ€ºÃ¨â„¢Å¸

Reactions and Reasons Essay Example | Topics and Well Written Essays - 1000 words

Reactions and Reasons - Essay Example This piece is quite impressive due to the number of feelings and associations it triggers by its sounding and instrumentation. It is visibly divided into 3 sections, which structure perceptions regarding music. The first and the third parts dwell on the same idea; however, they represent it differently. In this way these two sections are not perceived as identical. The second section of the piece is aimed at opposing the fist and the last sections. A number of binary oppositions characterize the differences between the second and two other sections of the piece. All of them create unforgettable atmosphere of the piece and help to reveal the meaning each part displays. From the very beginning the piece engages listeners by its full and brings sounding. This piece is composed for the orchestra; the music has rich texture and polyphony, which is visible from the first chords. The first impression of the music is that the part 0:00-0:51 has slight oriental tints in its sounding, as they have elements of pentatonic scale in string arpeggios. Such a confident beginning (forte, allegro), reminds of an overture – an introduction to opera or ballet. High contrasts in pitch, switches from major to minor element in music and rich instrumentation associates this piece with parade or celebration where many people are involved. It contains a controversy; reiteration of major arpeggios in minor in this part creates ambiguous and tensed feelings as music is unstable and highly changeable.

Main Character Analysis - The Yellow Wallpaper Essay

Main Character Analysis - The Yellow Wallpaper - Essay Example THE CENTRAL CHARACTER OF THE STORY REPRESENTS AN ‘OUTSIDER’ WHO DOES NOT WANT TO CONFORM TO THE TRADITIONAL NORMS AND HENCE FREES HER SOUL FROM THE PRETENSE OF THE CONVENTIONAL WIFE WHO ABIDES BY HER HUSBAND’S DECISIONS WITHOUT INDIVIDUAL JUDGMENT. Women have attempted to get rid of the conventional definitions of their roles and the ideology forced upon them, thus seeking for a redefinition of Womanhood. Charlotte Perkins Gilman illustrated the struggle of the female character of the story to overcome the patriarchal constraints in order to be able to cultivate her writing talents. Here she talks about the dominating care of her husband as she says, â€Å"He is very careful and loving, and hardly lets me stir without special direction†¦ There comes John, and I must put this away, --he hates to have me write a word. We have been here two weeks, and I haven't felt like writing before, since that first day. I am sitting by the window now, up in this atrocious nursery, and there is nothing to hinder my writing as much as I please, save lack of strength† (Gilman, 1891, 2) Gilman also points out the lack of cooperation from the female counterparts which have helped the male reinforce their dominance. She has mentioned the case of the sister of the woman’s husband in this regard. ... From the fact that John would not encourage her writing let alone appreciate them, she gradually suffered from low-self esteem. The following lines express her feelings, â€Å"But these nervous troubles are dreadfully depressing. John does not know how much I really suffer. He knows there is no REASON to suffer, and that satisfies him.† (Gilman, 1891, 3) The work represents the Cult of Womanhood, which ties up the women folk to the ambience of the home and family. Here the woman has been confined to the defined parameters that have been set by men. Constant domination negatively affects her creativity, as she has to put in the extra effort in order to overcome the mental set back and arouse the self-encouragement within her. The character Gilman creates in her story is an inspiration to all the women who have confined themselves within the shackles of dominance. She depicts the challenges thrown towards patriarchal ideologies and how women could move beyond the conventional ba rrier of ideology imposed upon them. She cites a reflection of this situation as she describes the changes in the wallpaper, saying, â€Å"There are things in that paper that nobody knows but me, or ever will. Behind that outside pattern the dim shapes get clearer every day. It is always the same shape, only very numerous.† (Gilman, 1891, 6) she wonders whether all women would be able to walk out of these artificial misleading designs created deftly by men – â€Å"I wonder if they all come out of that wall-paper as I did?† (Gilman, 1891, 12) The central character of the story ‘The Yellow Wallpaper’ therefore represents the ‘outsider’ to the set norms of the society

Company Law 2 Assignment Essay Example | Topics and Well Written Essays - 4000 words

Company Law 2 Assignment - Essay Example In other words, the general duties of the directors do not differentiae duties between them.5 These duties are imposed by law as the shareholders have limited control over them at the general meetings and they cannot participate in the day to day management of their companies.6 Foremost is the fiduciary duty under which duty of care and skill, duty to act bona-fide in the interest of the company and the rule of proper purpose have developed as a common law from judicial decisions. Duty to act bona fide (Section 171) When the directors act within the powers conferred by the constitution of their company, they are expected to act for the benefit of the company. They must act bona fide always what in their opinion is in the best interests of the company. 7 Directors are permitted to exercise their discretion in their decision making provided they act bona fide as held in Re Smith & Fawcett Ltd 8 This case involved directors’ absolute discretion in registration of share transfers. Court refused to interfere with the refusal to transfer of shares saying that it cannot draw an inference that directors refused to transfer shares with mala fide intention. By virtue of articles of association, the directors had enjoyed unlimited discretion to decide on the registration of transfer of shares. The directors acted within the powers conferred by the constitution and there was no way a court could interfere. This was despite the fact that directors insisted that the transferor who sought to transfer the shares in his name on the death of his father should sell 2001 shares out of 4001 shares to one of the directors. 9 In Rolled Steel Products (Holdings) Ltd v British Steel Corporation10, one of the two directors of Rolled Steel Products had borrowed from British Steel Corporation through his own company. But he did not disclose the fact when Rolled Steel Products made an agreement of guarantee with the British Steel Corporation for any debt owned by the directors of th e former. Since the director concerned did not disclose of the fact at the board meeting at which the guarantee was made, it was held that guarantee was invalid. The British Steel Corporation also did not disclose of the money owed. The director concerned had not acted bona fide. The British Steel Corporation as a third party might have had actual notice or constructive notice of the Rolled Steel’s director who was agent of the company. Therefore, the company was not bound when the third party knew that its representative either had no authority or exceeded it.11 Browne-Wilkinson LJ observed A third party who has notice- actual or constructive- that a transaction, although intra vires the company, cannot enforce such transaction against the company and will be accountable as constructive as constructive trustee for any money or property of the

Society gets what it pays for and the cost of reducing social risk is Essay

Society gets what it pays for and the cost of reducing social risk is regulation, leading to higher taxes and higher prices. Discuss - Essay Example Really, it is with the help of tax payments that social protection through the redistribution of social resources becomes possible. However, those who complain that social protection through regulation raises taxes and increases prices should remember that nobody is secured from various social risks, and it is through effective redistribution of social resources that individuals can insure themselves from the existing and future social risks. Social protection encompasses a series of public measures intended to reduce social risks and enhance income security of individuals (Holzmann & Jorgensen, 2000). Social protection measures rely on the payments provided by taxpayers to state budget; in other words, it is through the reallocation of limited social resources that social protection through regulation becomes possible. It is no wonder that not all taxpayers agree with the way social protection measures are provided to the vulnerable populations. However, all taxpayers should remember that they are not secured from the existing and emerging social risks (Holzmann & Jorgensen, 2000). Recent financial crises have shown that when social protection programs are not in place, individuals become extremely susceptible to the consequences of reduced GDP (Holzmann & Jorgensen, 2000). To a large extent, the presence of extensive social protection systems is both necessary and inevitable for any state or society that considers itse lf developed. Not all the people realize the principle of society-state relations. It is possible to say that society gets what it pays for, and the cost of reducing social risks is regulation, leading to higher taxes and increased prices. The latter are inevitable components of the social protection systems in the developed world. It is a reflection of the ongoing tension between the need for social protection and the overall non-affordability

Project Postmortem Essay Example | Topics and Well Written Essays - 250 words - 1

Project Postmortem - Essay Example The training session’s goals were effectively completed as per the two day schedule. At the end of the session all the participant had acquired the expected skills and proved to be competitive in their work execution. During the session however, there were some changes that occurred to the schedule in that we had to stop the training session for six ours due to the delay in arrival of some training equipment. We however solved this loss of time by extending the training sessions by some few hours into the night. The two day training session was of high quality as can be seen by the massive improvement in the performance of the trained employees. This has been seen by the large volume of the company’s products that are being produced every day. The number of errors and unintended mistakes by the employees have been greatly reduced. We there recommend for frequent similar training sessions to improve on the productivity of this company (Jackie L,

Society gets what it pays for and the cost of reducing social risk is Essay

Society gets what it pays for and the cost of reducing social risk is regulation, leading to higher taxes and higher prices. Discuss - Essay Example Really, it is with the help of tax payments that social protection through the redistribution of social resources becomes possible. However, those who complain that social protection through regulation raises taxes and increases prices should remember that nobody is secured from various social risks, and it is through effective redistribution of social resources that individuals can insure themselves from the existing and future social risks. Social protection encompasses a series of public measures intended to reduce social risks and enhance income security of individuals (Holzmann & Jorgensen, 2000). Social protection measures rely on the payments provided by taxpayers to state budget; in other words, it is through the reallocation of limited social resources that social protection through regulation becomes possible. It is no wonder that not all taxpayers agree with the way social protection measures are provided to the vulnerable populations. However, all taxpayers should remember that they are not secured from the existing and emerging social risks (Holzmann & Jorgensen, 2000). Recent financial crises have shown that when social protection programs are not in place, individuals become extremely susceptible to the consequences of reduced GDP (Holzmann & Jorgensen, 2000). To a large extent, the presence of extensive social protection systems is both necessary and inevitable for any state or society that considers itse lf developed. Not all the people realize the principle of society-state relations. It is possible to say that society gets what it pays for, and the cost of reducing social risks is regulation, leading to higher taxes and increased prices. The latter are inevitable components of the social protection systems in the developed world. It is a reflection of the ongoing tension between the need for social protection and the overall non-affordability

Public Health in Nursing Practice Essay Example | Topics and Well Written Essays - 2000 words

Public Health in Nursing Practice - Essay Example This essay approves that there are several characteristics of the population of the United Kingdom that can draw attention as they pose threat to the public health. Among these is the increasing concern that the population’s abusive patterns with regard to drug abuse is drawing. In a report of the UK Drug Policy Commission, the people of UK were identified to be the highest consumer of prohibited drugs in all Europe. This has also resulted to the increase of drug-related deaths which pushed it to the second spot among the European countries with high drug-related deaths statistics as well. Such deaths have been identified to be due to â€Å"poisoning, drug abuse, or drug dependence† This report makes a conclusion that drug abuse and dependence is indeed a public health concern that does not only affect the United Kingdom but the rest of the world as well. As a result, local, national and even international programs to respond to its negative threats are needed. The demographic trends and the behavior of the British people towards the use of recreational drugs which is identified to be â€Å"cultural† in origin is indeed alarming since it has been related to many severe health conditions and even risks to security that puts the health of the community as a whole in jeopardy. Although improvement is now apparent, the Government, health organizations and other related agencies must not only make policies to fight drug abuse as public a concern but also continue to monitor and evaluate these policies for the purpose of public health maintenance and improvement through the use of indicators such as demographic trends.

Describe marketing situations in which the use of qualitative research methods would be appropriate Essay Example for Free

Describe marketing situations in which the use of qualitative research methods would be appropriate Essay Describe marketing situations in which the use of qualitative research methods would be appropriate. According to Zikmund, Ward, Lowe, Winzar, Babin Qualitative research is a methodology that elaborate interpretations of phenomenal of interest without depending on numerical measurement ( Zikmund, Ward, Lowe, Winzar, Babin, 2011, pg 65 ) . It is characterised by its aims, which relate to understanding some aspect of social life, and it’s methods which generate words, rather than numbers, as data for analysis ( Patton, Cochrain, 2002 ) . In terms of marketing situations, qualitative research methods are appropriate to be used when the organization root for new product idea generation and development, strength and weaknesses of products/ brands and studying emotions and attitudes on societal and public affairs issues (qrca. org). To maximize understanding on the situation, there are two qualitative research methods that can be consider is Focus Groups and Questionnaires. With these methods, we identify the benefits as well as limitations of using these research methods. Focus groups can be define as an unstructured, free-flowing interview with a small group of people ( Zikmund, Ward, Lowe, Winzar, Babin, 2011, p71) . An approximately 60 to 90 minute discussion is led by a trained moderator with 8 to 12 relatively homogenous but unacquainted individuals who are brought together to discuss a specific topic ( Gunn, 2004) . Focus groups allow people to discuss their feelings, anxieties and frustrations, as well as the depth of their convictions, in their own words ( Zikmund, Ward, Lowe, Winzar, Babin, 2011, p72 ) . The benefits of focus groups can be presented for an in-depth exploration of new ideas, opinions, perceptions, and reactions to concepts and messaging. Focus groups often serve as exploratory research to assist survey design of subsequent quantitative research methods. Similarly, they can also be useful in validating and/ or clarifying results garnered from previous quantitative research and can be use to elicit â€Å" in their own words descriptions of products, services or issues being discussed. Conferences and other events present relatively low-cost opportunities to conduct focus groups with target audiences. There tend to be few interviewer effects on dialog because individuals tend to be influenced more by the group discussion than by the moderator. Participants are usually enthusiastic and spontaneous in their responses and groups tend to naturally cover more questions, opinions and comments than researchers could have anticipated. The disadvantage of the focus group however shows that the results from focus groups are qualitative and nonprojectable to larger populations. They require well-trained moderators to manage discussions, maintain focus, and minimize affects of the personalities and behaviours of individual participants on others and/or the entire group. Logistical and cost considerations include room and food set-up, and incentives are typically needed to encourage response. Questionnaires are not among the most prominent methods in qualitative research, because they commonly require subjects to respond to a stimulus, and thus they are not acting naturally. However, they have their uses, especially as a means of collecting information from a wider sample than can be reached by personal interview. Though the information is necessarily more limited, it can still be very useful. For example, where certain clearly defined facts or opinions have been identified by more qualitative methods, a questionnaire can explore how generally these apply, if that is a matter of interest. Ideally, there would then be a qualitative check on a sample of questionnaire replies to see if respondents were interpreting items in the way intended. Alternatively, a questionnaire might be used in the first instance, followed by qualitative techniques on a sample as a check and to fill out certain features of the questionnaire replies. Interaction among techniques in this way is typical of qualitative research.

How Musical Theatre Has Developed

How Musical Theatre Has Developed To discuss the historical development of musical theatre, this report will present an analysis of Leonard Bernsteins musical West Side Story. It will demonstrate how the 1961 screen version has been adapted for the stage in the 2009 Broadway revival to suit modern day audiences and show its reflection through time with the use of ideas and different styles as well as social and economic influences affecting the works. The conflict between the two rival gangs that is so central to the story could be further interpreted as a conflict between Catholic and Jewish communities, which was Bernsteins intention in the beginning. However, Bernstein seized on the idea that current racial tensions in New York caused by immigrants from Puerto Rico would provide a more powerful story and he undoubtedly saw that this would also offer him the opportunity to use a range of Latin-American dance rhythms, for he later said: it all sprang to life. I heard rhythms and pulses, and most of all I could sort of feel the form. (Bernstein, 2009, p.58) Much about West Side Story in that time was new and revolutionary. Instead of the old fashioned romance, akin to the earlier musicals, this is a story of bleak despair. Extended dance sequences convey the drama, and in a place of rousing finals, both acts end in murder. Although Shakespeares text is not used, his characters are clearly identifiable: They say Shakespeares plays are timeless because they still speak to the human condition today (Fischbach, 2010) Accordingly Arthur Laurent, author of the original book, got a second chance at his creation, and turned the 2009 Broadway West Side Story revival into the musical he always wanted to make. The Daily Telegraph found: There is nothing new about this production of West Side Story aside from the hot young cast and it is a prime example of why new is not always best. (Lalak, 2010) Social constraints of the time prevented certain usage of language, so some dialogue and lyrics were delivered in Spanish. This made a considerable difference without substantially changing the story. Laurent has given the show a more intimate feeling and made the Puerto Rican Sharks and their girls, more complete as characters. They are frustrated as strangers in their own land, and the use of Spanish immediately evokes their separation from the English-speaking Jets and their girls. The fundamental problems havent changed much since the start of West Side Story 50 years ago, as we continue to wrestle with new cultures arriving. More importantly, though, this production sets the story of the two gangs and a brief and star-crossed love affair between Maria and Tony as a young persons game. The casting here has made the lovers, both breathtaking in their roles, enormously youthful, which gives us more gleeful moments. It isnt to be, of course, but from the moment West Side Story begins with the legendary dance prologue through the neighbourhood, faithfully reproduced by Joey McKneely from the original choreography by Jerome Robbins, the show glows with redolent memories and enchants with brilliant new moments. It plays against a remarkably flexible set by James Youmans, used for maximum effectiveness by director David Saint. the fundamental problem with the show is its depiction of gang violence and the mean streets of New York.: as being said in the Mercury News. (Craig, 2010) That was always a signature of West Side Story, even in the 1961 screen version, but still well presented with fantastic dance sequences which were half-danced and half-mimed. This new form of dance became a visual symbol of a mode of thought. What the characters were feeling and thinking was expressed by their movement and their identities became inseparable from it. However, when the movie was released in 1961 it wasnt the dance which made a big impression, but the social tensions. It showed a fight for urban space, a space that has already been impregnated with cultural symbols and political significations for the relations, interactions, and social actions according to the American Way of Life. In this sense, the movie projected how the Puerto Rican migration to New York City in the forties and fifties not only took over the order of the Anglo-Americans, but how it also constitutes a threat for the assumed monolithic identity of the Anglo-American subject. New York City was shown as a divided territory, economically, racially, and ethnically. Each social-economic group inhabited a space and even neighbourhood border crossings were avoided. West Side Story is up to date not only because of its lengthy dance numbers, emotionally drenched songs and scores, and a contemporary plot that speaks so much of the place and time where the story took place, but also because we are now in a place and time where social-economic issues continue to prevail in every household and community and where racial discrimination is still rampant. The new Broadway revival speaks not only of this shows unwavering popularity but also of the timeliness of the message it delivers, a message of love, peace and harmonious co-existence regardless of race and social-economic backgrounds. This message clearly transcends five decades of an ever-changing political, economic, social and cultural landscape and is still relevant up to this day. West Side Story shows how dancing, acting, singing and design could blend together in unity. Whether its an old version of the musical or a new one, we might say that: it marked the most impressive body of choreography in a single show, and it was acclaimed as Leonard Bernsteins strongest work for the Broadway stage. (Garebein, 2000, p.9)

The Educational Philosophy I Will Incorporate in My Classroom Essay

The Educational Philosophy I Will Incorporate in My Classroom Education serves as the foundation to a lifetime of learning. Since every child is unique, I believe that it is important for them to learn in an environment that is both secure and stimulating. By creating this type of atmosphere, the students will be able to realize their intelligence and use it constructively. As a future educator, it will be my goal to establish a classroom that is, 1) non-authoritarian, 2) student-centered, and 3) focused around student experience. These three elements are part of Progressivism, the educational philosophy I plan to incorporate in my classroom. In a classroom that is non-authoritarian, the teacher serves as a facilitator, or supervisor of learning. By assuming this role, the teacher provides access to information rather than being the primary source of information. In this aspect, the students will be able to discover knowledge through problem-solving, cooperative learning activities, and decision making. I believe it is important for a teacher to provide students a sense of freedom so that they may choose what they want to learn according to their interests. By making a child feel as though they have a choice, rather than an obligation to learn information, it will help them constructively build their knowledge. I believe it is equally important to have a classroom that is student-centered. I do not imagine my classroom arranged with rows of seated desks all facing the front of the classroom. Instead, I think that students should have the opportunity to work amongst their peers in small groups, studying a wide array of topics. Students will not be required to focus their attention on on... ...my education at Concord University studying Elementary Education. After graduating with my Bachelors of Science in Education, I plan to get a job as a teacher in an elementary school. I believe that there is a need for compassionate and caring teachers in elementary schools. At such a young age, I believe that it is important for elementary students to have positive role models in their lives. I am very interested in pursuing my Masters Degree at some point, but my main goal is to get out into the school system as a teacher. The most important thing that I have learned in my life is the importance of education. Living in a world bursting with advantages and opportunities for those in education, I find it both a priority and privilege to share the gift of knowledge with the future of society. Even more, I find it an honor to be able to have the chance.

The Janjaweed of Darfur :: essays research papers

The millions of refugees due to violence in Sudan have been produced by the Janjaweed.The word Janjaweed is a term that is used to describe certain armed gunmen in Darfur. The word Janjaweed means 'a man with a gun on a horse'. The janjaweed are an army formed of men from tribes in Darfur and Chad that have become infamous due to their tendencies to be racist, rape, and massacre. The Janjaweed are a group who are against African farmers who have settled in Darfur.The janjaweed is made up of Sudanese and Chadian horse riding nomads. The janjaweed militias are known to attack in large numbers on camels and horseback. The janjaweed push people from their land, often towards town centers. The janjaweed have developed over the years, now having more sophisticated weapons but the same ideals. The janjaweed were first formed in 1988 after the Chadian president defeated the Libyan army. This ended territorial designs on Chad. By 2006 many Janjaweed have joined the Sudan Armed Forces. Many world leaders believe that the Janjaweed killings in Darfur are considered genocide. The janjaweed militia has killed about 450,000 civilians in the course of 3 years. The UN Security Council decided that they want the Janjaweed disarmed. Informally, the Janjaweed were created to run the non-Arabs out of the country. Janjaweed have also been known to steal cattle from non-Arab farms. The Janjaweed were a militia that was tolerated by the Sudanese government in the attempts to control land. It has been said that the Sudanese government formed the Janjaweed but they deny this, saying that they only formed self-defense armies after they were attacked. Although, the Sudanese government did in fact use the Janjaweed at one time or another to protect and serve Sudan. After the Sudan Liberation Movement (SLM) and the Justice and Equality Movement (JEM) attacked the Sudanese government, they armed the Janjaweed and sent them to counterattack. The Janjaweed executed a ?scorched earth? attack meaning they destroyed everything useful to their enemy and killed many people.

Elements of the Play Essay

Eugene O’Neill’s The Hairy Ape play revolves around Bob Smith, or Yank, showing that the Transatlantic Liner fireman has had an extremely painful past. Having been brought up in an unstable family, Yank nurses feelings of hatred towards society in general and utter desperation. To begin with, Yank’s father was abusive, a situation that forced the son to escape following the death of his mother. Yank also explains that his parents used to fight so hard that they usually damaged the family furniture. Eventually, Yank is isolated by all persons, a scenario that compels him to seek companionship with an ape that finally kills him. The theme of O’Neill’s play is how Yank – a contemporary man, struggles in an industrial society. To illustrate this theme, Yank sadly realizes that he is unable to transcend existing ideological or class barriers. Moreover, Yank is unable to develop new barriers. In effect, Yank is merely an outsider who finally closely resembles an entity that is caged in a zoo at which people point. To illustrate Yank’s inability to rise above class barriers, Mildred Douglas becomes so scared of Yank’s physical appearance that she flees in apprehension. Mildred has gone to check on Yank and his fellow Ocean Liner firemen as they feed coal into the sea vessel’s engine. The sight of Yank visible frightens Mildred such that she seeks protection from Yank’ colleagues. Mildred even calls Yank a huge ‘hairy ape’ (O’Neill 154). This incidence thus shows that Yank cannot be socially at par with Mildred despite the fact that he works towards facilitating the continuation of the business interests of people of Mildred’s class. This struggle continues throughout the play up to the time Yank desperately seeks companionship with an ape that ultimately kills him. O’Neill’s play is set in various locations which serve to demonstrate that low class people like Yank endure get suffering under their upper-class counterparts. Initially, Yank and his colleagues are aboard a sea vessel enjoying themselves by drinking. Yank is however contemplatively quiet, a fact that makes his colleagues to urge him to ‘Drink, don’t think! ’ (O’Neill 142). Yank also laments that ‘de woild owns me’ alluding to his save-like condition in the sea vessel (O’Neill 278). Mildred also comes in as Yank works on the Ocean Liner alongside his colleagues. Next, Yank accompanies Long as the 2 tour New York. As he tries to capture attention, Yank makes someone miss a bus. Yak is thus arrested and incarcerated on Blackwell’s Island. As he converses with fellow prisoners, Yank explains his desire to revenge in Mildred. He is thus advised to become a member of the Industrial Workers of the World (IWW). Yank thus realizes that Mildred’s father has metaphorically and physically caged him. Afterwards, Yank visits the IWW offices. He is however rebuffed on suspicion that he is spying for the government. He then visits a zoo whereby he tries to seek the companionship of an ape that kills him. Yank’s hopelessness sis reflected in his statement ‘I couldn’t belong in dat’ (O’Neill 298). All these incidences, from the Ocean Liner all the way to the zoo, illustrate Yank’s situation of being segregated and detained by society. Yank seems to be illiterate based on his mispronunciation of the word ‘think’ whereby he inserted says ‘tink’ (O’Neill 96). In addition hi desire to engage in thought shows that he is a contemplative person. Moreover, through his reaction to Mildred’s unkind gesture and statement, Yank is shown to be very sensitive. The philosophical side of Yank plays out when he tries to informs the ape that both Yank and the gorilla are alike since they are caged. Works Cited O’Neill, Eugene. The Hairy Ape: Anna Christie And The First Man. Whitefish, MT: Kessinger Publishing, 2004.

The Humane Conditions of the United States’ Japanese Internment Camps

In response to the attack on Pearl Harbor, Canadian and American governments took extreme actions to prevent possible Japanese attacks, first and foremost are the internment camps. Japanese internment camps housed Japanese US and non-US citizens from 1942-1945. The economic and social factors surrounding the camps were unprecedented. The United States managed the affair with somewhat of a dignified perspective while Canada on the other hand fully implemented dispossession, discrimination, but ignored a redress of any sort. In contrast to the United States, Canada completely exploited the Japaneses’ economic resources. Shortly before their evacuation to the camps the â€Å"to-be-interned† Japanese would quickly sell some or all of their personal possessions whether to the government or other white civilian buyers. Under the War Measures Act of 1943, the Japanese were required to pay taxes for every sold item which would later be auctioned; their land and other properties, if not sold, were immediately confiscated. Later, the property was resold to white Canadians and never returned. â€Å"Dispossession of Canadian citizens, was contrary to British principles of justice and to the Atlantic Charter,† announced Dr. Henry F. Angus, in opposition to Japanese internment. He demonstrates that even then were there individuals that recognized the unjustness of the camps. The taxes aforementioned were used for the payment of government employees and also to fund the internment camps and pay businessmen who took over maritime industries normally monopolized by Japanese. The United States was responsible for confiscating some private properties, but not nearly the amount of which Canada was responsible. The Canadians took economic advantage of the camps to their fullest extent. In Canada the social conditions of the Japanese internment camps were different from the Unites States camps which had the necessities such as food, shelter, and water. The ten Japanese internment camps in the United States were incomparable to the intolerable conditions in which Japanese Canadians were forced to live. Japanese Americans had facilities such as mess halls, bath houses, laundry buildings and recreational areas on the primacies. The detainees could specialize and join the work force to support themselves and spend their wages in the camp store. Children attended school and most families attended church on Sundays. Restrictions to which they had to abide included leaving the premises, criminal activities, worship of the state Shinto, food and water rations, and others. Canadian camps provided extremely limited resources to the interned, sometimes providing only 10 toilets for 1,500 women, while shortages of food were common. As sickness spread so did a hatred of both American and Canadian Caucasian citizens whether they were responsible or not for the Japanese’s incarceration. Approximately 60 years later, the US felt a moral obligation to redress about 550 Japanese citizens that were associated with internment camps. About 12 million US dollars were distributed to the few remaining victimized families. This is embarrassing and tragic at the very least, admitting our injustice publically. However they took responsibility for their actions which explains how Japanese immersion and social acceptance in America’s society developed Americans’ humility and honor. Through such compensation of moneys and in some cases property Americans regained the faith of the Japanese to some degree. Canada on the other hand showed very little mercy to the delicate minority and interned every Japanese immigrant. Families were torn apart without hesitation, separating husbands from wives and children from mothers, leaving families with absolutely nothing but bitterness and sour remorse after being subjects to the government’s lethal power. Prior to installing the Japanese internment camps Canada and America were immersed in a state of fear after the attack on Pearl Harbor. The two regions had agreed to inform the other of changes in policies within the internment camps when they were built. One country fed off the other, trying to find some justification for their dreadful treatment of the interned Japanese. As the United States confiscated land Canada dispossessed boats for economic profit; while the standard of living in American internment camps was low, the Canadians took it one step further, providing them with little supplies and a socially cold shoulder. The United States maintained the Japanese internment camps better than the Canadians, providing them with scarce necessities but humane conditions. Works Cited Challenge to Democracy, A (1944). U. S. War Relocation Authority. March 3rd, 2010 http://www. archive. org/details/Challeng1944 The Politics of Racism . Ann Sunahara. March 3rd, 2010 http://www. japanesecanadianhistory. ca/

Wal-Mart Essay Example | Topics and Well Written Essays - 500 words

Wal-Mart - Essay Example The website is very well organized in terms of links and images. The left hand side of the e-store has a list of categories of consumer items for sale. The check-out function of the website is very easy to use. Wal-Mart can be benefit a lot from a more aggressive e-commerce strategy. The global market for online sales is huge. In 2010 e-commerce sales were $153 billion (Plunkett Research, 2011). Wal-Mart should look to increase its market share of this multi-billion dollar marketplace. The beauty of e-commerce is that it has the potential to reach a hundred percent of the global population of 6.9 billion. Wal-Mart has to look for ways to increase the traffic of its website. Simple solutions such as offering a discount coupon with every Wal-Mart receipt redeemable at its online stores can help increase the revenue of the store. The firm should advertize the site in TV commercials, radio, and in the press. An untapped market that could help the company increase its ecommerce sales is c ellular video advertizing. The assistance of G4 networks is enabling people to use the internet more through smart phones such s the I-phone.

Relationship Analysis Paper Essay Example | Topics and Well Written Essays - 1500 words

Relationship Analysis Paper - Essay Example s an intelligent man, highly educated and emotive at times due to loss of his twin brother and use of one kidney which makes him have a poor health status. To support him, I encourage him on most occasions, offering emotional support which I know he needs considering the fact that those close to him, like his parents are far off. This paper is a reflection of my relationship with Tony, my boyfriend and it analyzes it using the concepts of mediated communication, the self-concept and emotions. Due to the nature of our relationship, we had to use different media of communication just to convey our concerns and emotions towards each other. We have mostly used the internet via twitter, facebook and skype and as outlined in different section of my paper. These agents of mediated communication have their pros and cons which directly affected the scope of our relationship. A brief definition of the concept of mediated communication is that it uses a medium to transmit a message (Wright et al, 4). The concept of self outlines my esteem and how my boyfriend managed to make me feel confident of myself. Another aspect involves emotions, and it depicts the weaker side of my boyfriend and me. This paper reflects on how through collaborations, we managed to take care of each other’s emotions and resulting to a stro nger bond between us. The relationship with my boyfriend can be termed as a long distance relationship by all definition, we are constantly communicating with each other. Thanks to the agents of mediated communications, namely the internet (Wright et al, 5). The internet as a medium has twitter, facebook and skype, which enables us to communicate constantly and effectively with my boyfriend. With the help of skype, we manage to chat and use video calls to communicate and express our emotions and share our daily events. On most occasions and I tweet my boyfriend just to express my love to him, and he responds back. We are able to follow each other’s daily

Problem and Solution paper Assignment Example | Topics and Well Written Essays - 750 words

Problem and Solution paper - Assignment Example Mobile phones have formed an integral part in the current telecommunications as well s electricity and have since formed part of the day to day living of people. The exposure of a person to a mobile phone is usually measured in terms of Specific Absorption Rate. Mobile phones use radio frequency signals whose exposures to body issues have adverse health effects. Even though the exposures to these signals from phones are low and within EU limits, they could have long term effects on humans. The RF fields normally penetrate the tissues of the body to varied depths depending on their frequency. When energy from RF is absorbed into the body, it is changed into heat which is then carried away by body tissues. It is this heat that has health effects. Recent research has shown that the exposure of RF to body tissues do not cause cancerous infections because of its low levels. Issues that raise concerns over the safety of mobile phones on health are whether continued exposure to these signals could cause infectious diseases. Mobile phones have not been embraced for a period long enough to ascertain their long term effects. For this reason, it is sti ll unclear the extent of long term use of these forms of technology in relation to the occurrence of the disease acoustic neuroma. (Burgess 188) On the other hand, those who live around power lines are at a risk of contracting cancerous infections though the probability is extremely low. These lines provide Extremely Low Frequency fields. A recent research conducted by the International Agency for Research on Cancer there is a possibility that these magnetic fields are a human carcinogen. However, it was stated categorically that this is not a direct insinuation that the magnetic fields are actually carcinogenic but was classified as a possibility. (McCall 120) A 1979 research raised concerns of increased incidences of leukemia realized in children who lived in residential places that were close to

Market Orientation in Business Essay Example | Topics and Well Written Essays - 2250 words

Market Orientation in Business - Essay Example Considering any business term requires a clear understanding of what is meant by its name. Thus, it is necessary to give an explanation of the term ‘market orientation’ before going to its detailed analysis. According to a definition provided by the Business Dictionary, market orientation is ‘a business approach or philosophy that focuses on identifying and meeting the stated or hidden needs or wants of customers’. As stated in the Cambridge Dictionary, by the term ‘market orientation’ one should understand ‘management of a company, product, brand, etc. so that it satisfies the needs of customers’. In the book ‘Changing Market Relationships’ written by Jean-Jacques Lambin, market orientation is defined as ‘a set of capabilities, activities and behaviors needed to implement a strong marketing orientation’ (Lambin 2008).Despite the fact that all the definitions mentioned above reflect the main idea behind mar ket orientation quite clearly, the most comprehensive definition of the term is presented by Morgan and Strong in their work named ‘Market Orientation and dimensions of strategic orientation’. In this work, the two authors define perceive the concept of market orientation as a certain mode of corporate management that is founded on encouraging a permanent gathering and sharing of statistical information regarding customers, appreciation of customer needs as a fundamental aspect of the business.

Marketing management Essay Example | Topics and Well Written Essays - 2000 words - 4

Marketing management - Essay Example Since the last 10 years, HCC has grown swiftly, principally throughout an insistent policy of efficient management. However in the wake of the economic crisis and the following recession that has also hit the company steering its sales down by almost 50%, the company has been forced to lay down 50% of its employees, and the company is motivated to look towards new options and destinations. Russia and South America have been identified as new selling destinations for the company along with trying to profit margins in the highly competitive environment. However, the company is also trying to promote its products in the local Chinese markets. The next section presents a detailed analysis of the market conditions for the company, the opportunities and threats, the current position and management strategy of the company and the forces of change that are required to build a more efficient and successful organization. I choose to select the method of various analysis tools like SWOT, Porter analysis to study the above requirements and conditions for the company. 1. The company commands significant lead in many segments in the export sector of the Chinese industry for entertainment goods. It is also a well known international company and its products have been able to set their own brands in the international market up till now 2. The company has a flexible and customer oriented product history with a considerable development in the export sector as they have increased the market share by 3% to over 6% within the last two years. Porter’s five forces analyze the strong points of a company. Traditionally, the analysis is used to take notice of new product launches, upgrading of business services, and appropriate balance of power. 1. Provider control: in this case, the power and control of the suppliers of HCC to drive the prices of the final product or

The Major Old Testament Eras Essay Example for Free

The Major Old Testament Eras Essay The Old Testament, the first section of the Christian bible has been reputed to date back to around 4000 years. Different scholars divide the old testament period into different epochs depending on the major events that occurred during this time. The occurrences may have happened in the world at that time or in the history as related by the bible. Some of the old most categorization divides the Old Testament history into primeval period/creation, patriarch era, exodus, Canaan conquest, the era of the judges, monarchial era, exile, return and restoration era. The dates of the eras are believed to have started from the beginning (4000BC). After this unique era of time, then came: the Patriarch Era around (1800-1290BC),Exodus era dated1447-1406,conquest of Canaan Era(1406-1390),The Era of Judges(1290-1050BC),the era monarchy(1050-750BC),Era of division and Exile(605-538BC)and finally the era of return and restoration (538-323BC). It is noteworthy that these given dates may not be precise and accurate. However they are archaeologically accepted. In the history of the Old Testament many outstanding events are recorded not only biblically but also secularly. This period of the existence of the old Old Testament could therefore be termed as one of the most outstanding historically active period in the history of world existence. In the primeval Era it is believed the creation took place. Heaven and earth with their contents remain inclusive were created as designed by God. In the Patriarchal Era recorded in Genesis 12-50, God in abide to reconcile with man ets requirements and laws to govern the existing generation of humans. During the Exodus, Moses led the Israelites who had been slavery in Egypt for 400yearsout of Egypt to go to the Promised Land as promised to Abraham their main descendant . This is the very period when the children of Israel of bondage stayed in the wilderness on their way to Canaan for 40 years. During this period world’s process of civilization continues in the powerful empires like Egypt. In the Era of conquest, Israelites led by Joshua after Moses death are conquering and entering Canaan hence focusing more on Joshua’s leadership. In other parts of the world the Iron Age commences, the 19th Dynasty is on and the powerful Chinese Chou Dynasty is in being. In era of Judges, there is no concrete leadership in Israel after their settlement the 19th Dynasty of Egypt is on and also the reign of raameser 2 perceived mistakenly to be spear led of the oppression to non- believers of alliterate translation by the liberal scholars of the time. In the Monarchial Era Israel comes up with Monarchy establishing a kingdom and doing away with the Judges. The king of Israel in this era was Saul, David and Solomon. Subsequently some world religions are being established secularly, it’s the period of time when a solar eclipse is recorded in Nineveh and Jerusalem on June 15th. In addition to that Mayan civilization commences in South America. After proper establishment and being of the kingdom there is division and exile of inhabitants and occupants of some portions of the kingdom go to Exile after capture by the Babylonians. Subsequently Rome was conquered by Etruscans and the existence of Buddha of India in rule hence development and establishment of Budhism. This is also time of some major prophets like Jeremiah of service to Gods people. Finally in the Era of Return and Restoration Israelites who were definitely of a generation were now set free from captivity and were to return to their original land. This is the very period that would when in the coming of the Messiah hence the New Testement. Majorly in the world, Mexander becomes the king of Macedonia and the statues of Zeus, Artenis temple Tomb of Mausollos are identified as 3rd, 4th, and 5th wonders of the ancient world respectively. In this era great scholars like: Alexander the great and Aristotle lived making there inventions remembered update. This is also the time of Confucius of China. Conclusively, these era of history are clearly cited in biblically as follows: The creation Era is found in Genesis 1-12, the Patriarch Era in Genesis 12-50, Exodus era in Exodus, Leviticus, Numbers and part of Deutronomy, conquest Era in the book of Joshua and even Deutronomy, the Era of Judges in Judges and era of Monarchy is found in Samuel and chronicles. The era of Division and Exile is found in 2kings, 2chronical, Jeremiah etc while the era of Return and restoration is chronologically cited in Jeremiah, Ezra, Nehemiah and Esther. After all these events the New Testament is ushered in with the coming of the Messiah (Jesus Christ) fulfilling the prophecies made in the Old Testament in during the existence of various prophets in various occasions and under different inspirational cases and circumstances. References NLJV (2007). Life application study bible. NY: Tyndale House Publishers. Merill. (2000). Settle for more. Alabama: Wisconsin.

Human Carbonic Anhydrase II

Human Carbonic Anhydrase II Human carbonic anhydrase II is one of the fastest studied enzymes known with a variety of roles in reaction catalysis. Its primary function is to catalyze the reversible hydration reaction of carbon dioxide. In addition to carbon dioxide hydration, it is also capable of other latent skills, such as catalyzing esterase activity. The ability of human carbonic anhydrase II to function as a catalyst derives from key residues in and around the active site that play crucial roles in the mechanism. Substitutions to two of those particular key amino acids were performed via Quick-change site directed mutagenesis: H64A and V142D, to investigate the particular role they have in the catalytic active site. Various kinetic experiments and structural analyses were performed on wild-type carbonic anhydrase and the mutants to discern and compare their activity to each other and to literature, including Michaelis-Menten parameters for PNPA hydrolysis, CO2 hydration, and inferring function molecular m odelling. Though the same trends can be seen as the literature, individual values were found to be much lower owing to errors in measurement and equipment. Trends were found to coincide with the mutants known roles in the active site: His64 is the proton shuttle that facilitates proton transfer during the rate limiting step and Val142 participates in the hydrophobic pocket to bind and recruit substrates to interact with the active site. Mutations to both of these sites show that enzyme efficiency and activity strongly decreases. Introduction Human carbonic anhydrase II (hCAII) is a zinc metalloenzyme that catalyzes the following reversible reaction: . The enzyme commonly functions to help shuttle carbon dioxide in red blood cells to rid the body of metabolic waste, and catalyzes the hydrolysis of many aromatic esters [1, 2]. Structurally, a zinc ion is located in the active site, coordinated to 3 histidine residues (H94, H96, H119) and usually a hydroxide ion or water molecule [2]. The mechanism of hCAII proceeds through two major steps: 1) the conversion of carbon dioxide to bicarbonate, and 2) the regeneration of Zn-OH by proton transfer. The active hydroxide that is bound to zinc nucleophilically attacks a nearby carbon dioxide molecule, resulting in a bicarbonate ion binding to zinc [3]. The zinc-oxygen bond breaks to subsequently release a bicarbonate ion, which is replaced with water [3]. The Znà ¢Ã¢â€š ¬Ã¢â‚¬â„¢OH bond is regenerated by a proton transfer to the external buffer, which is facilitated by the His64 residue [3]. The proton transfer step is the rate limiting step of the reaction [3]. The diazole side chain on the histidine residue is what gives it the ability to be a proton acceptor and donor. Mutations in that position (His64) usually result in decreased enzyme activity due to a lack of proton transfer; however the reaction does proceed to a lesser degree without an active His residue, possibly due to its extensive water network in the activ e site forming secondary proton wires [4]. Carbonic anhydrase catalyzes one of the most rapid reactions; it is one of the fastest enzymes studied [1]. Its reaction speed is due, in part, by the amphiphilic nature of the active site [1]. The hydrophobic side is used to bind carbon dioxide, while the hydrophilic patch functions to optimally orient the carbon dioxide molecule for the reaction [1]. The hydrophobic wall forms a well-defined pocket near the zinc-hydroxide and is composed of the following amino acids: Val142, Val121, Leu197 and Trp208. The hydrophilic patch consists of Thr198 and Glu106, which form a hydrogen bond network with the Znà ¢Ã¢â€š ¬Ã¢â‚¬â„¢OH to stabilize and orient it for nucleophilic attack on CO2 [2]. Therefore, any modifications to the hydrophobic pocket would change its structure, and consequently, its catalytic efficiency [1]. In this study, the importance and role of His64 and Val142 to the structure and mechanism of hCAII are determined through site-mutagenesis and subsequent characterization of the new mutants, H64A (His64 Æ’Â   Ala) and V142D (Val142 Æ’Â   Asp) via kinetic and structural analysis. The changes that arise from the substitutions may prove to be applicable to drug synthesis because hCAII is known to be involved in a variety of diseases, for example, Marble brain disease, where mutations in the hCAII gene leads to a deficiency in the enzyme which is an autosomal recessive disease [5]. Studies in hCAII mutations can be used to design folding modulators to suppress misfolding which frequently occurs due to hCAII destabilization [5]. Another major disease involved with hCAII gene is osteopetrosis. The hCAII genes inactivation decreases osteoclast function in bone, and knowledge of hCAII mutations that inactivate the enzyme may lead to better understanding of bone remodelling [6]. Some carbonic anhydrase diseases use inhibitors (CAI) to suppress the hCAII as a therapeutic treatment. Inhibitors prevent hCAII activity by inhibiting either of the reaction steps: the conversion of CO2 which involves V142 in the hydrophobic pocket, or the rate limiting step, proton transfer, in which His64 is crucial. Experimental Procedure Site directed mutagenesis via the PCR-based Quick-change method was performed on hCAII as cited in Woolley (2011) for 10 ng and 20 ng wild-type plasmids (hCA2pET24b from Novagen) [7]. Table shows the sequence of the primers used in the PCR reactions. Products of PCR mutagenesis reactions were run on 0.7% agrose gels to determine size. The gels were run at 150 V in 1X TAE buffer. Red safe dye from Intron Biotechnology was used in the agrose gel instead of ethidium bromide for safety reasons [7]. The standard molecular weight ruler used was a 1 kB DNA ladder from Fermentas. Table : Primer sequences used in mutagenesis of hCAII in the forward and reverse direction for mutants H64A and V142D Mutant Direction Sequence MW (Da) %GC TM ( °C) H64A Forward GGATCCTCAACAATGGTgcTGCTTTCAACGTGGAG 10778 51 67 Reverse CTCCACGTTGAAAGCAgcACCATTGTTGAGGATCC 10709 V142D Forward CTGATGGACTGGCCGaTCTAGGTATTTTTTTG 9868 44 62 Reverse CAAAAAAATACCTAGAtCGGCCAGTCCATCAG 9779 The enzyme, DpnI, was then used to digest methylated DNA (the parent template DNA). The DNA vector that contained the mutation was transformed into supercompetent E.coli turbo cells from New England Biolabs by heat shock [7]. LB-agar plates were prepared to grow the transformed cells containing mutant genes (i.e. H64A and V142D hCAII gene) [7]. Both were injected with Kanamycin to ensure that the culture that grows will have the desired mutation [7]. A miniprep culture was set-up from the LB-agar plate into LB medium to grow one colony for DNA analysis [7]. Restriction enzyme mapping was prepared and XhoI and BglII were chosen, they were used under buffer 3 for optimal efficiency. Plasmid putification was performed using the QIAprep Spin Miniprep Kit, and then the chosen restriction enzymes were carried out and were run on 1% agrose gel [7]. A sample of the purified DNA was sent to an external company (ACGT) for commercial sequencing (Sanger dideoxy type) to verify if the mutagenesis occurred correctly. The sequence was analyzed using the program BioEdit. To determine the level of confidence of the sequencing results, the purified DNA was quantified using UV/Vis absorption via a spectrometer [7]. The concentration was calculated using Ecà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1(260) as 50 ÃŽÂ ¼g/mL. Purified plasmid DNA was transformed into E.coli BL2(DE3) cells to initiate protein expression by heat shock, similar to the transformation into turbo cells [7]. The cells were cultured and a single colony was grown. Once sufficiently grown, ITPG and ZnSO4 were added to induce protein expression [7]. SDS-PAGE was used to confirm protein expression and was analyzed against an unstained protein molecular weight marker by Fermentas. The protein and ladder was stained with coomassie blue [7]. Affinity chromatography was used to purify the mutant hCAII proteins [7]. The matrix used was agrose linked to p-(aminomethyl)benzenesulfonamide, exploiting the tight binding that occurs between hCAII and sulphonamides. Once purified, the protein was dialyzed using a 6000-8000 Da dialysis membrane to replace the elution buffer with protein buffer and removes the matrix from the protein [7]. SDS-PAGE is again used to confirm the protein is still present after purification and to check its approximate molecular weight. It was run for two different amounts of protein, 2 ÃŽÂ ¼g and 10 ÃŽÂ ¼g, and also ran 10ÃŽÂ ¼L of wash fractions from affinity chromatography [7]. Protein concentration was determined by UV absorption at 280 nm in a final concentration of 6M guanidine hydrochloride. From the calculated concentrations, purity of the protein could be assessed via SDS-PAGE. To characterize this purified hCAII protein, a variety of analyses were done. Two types of mass spectrometry (MS) were performed: electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) [7]. The MS analysis was used to confirm the presence of the mutation in hCAII with intact and digested protein. Protein samples (H64A and V142D hCAII) were not diluted for either of the MS analyses as cited in Woolley (2010). Samples of 10 ÃŽÂ ¼L of stock protein concentrations (37.6 ÃŽÂ ¼M H64A and 3.2 ÃŽÂ ¼M V142D hCAII) were used for analysis of the molecular weight of the intact protein by ESI-MS. Both mutants were then digested by Trypsin Gold (MS grade) from Promega and the resulting fragments were evaluated by ESI-MS as well [7]. A 50 ÃŽÂ ¼L sample was used for each mutant, 40 ÃŽÂ ¼L of the mutant at stock concentration and 10 ÃŽÂ ¼L of the Trypsin Gold. A couple ÃŽÂ ¼L of the digested mutants were saved for MALDI-MS and the rest was used for ESI-MS. Once the molecular weights for each of the digested fragments were determined by ESI-MS, the products were run through a protein database to confirm the identity of the protein and mutations [7]. The 1 ÃŽÂ ¼L of the tryptically digested mutants prepared for ESI-MS, subsequently underwent MALDI-MS. The 1 ÃŽÂ ¼L samples were mixed with a matrix consisting of 1 ÃŽÂ ¼L ÃŽÂ ±-cyano-4-hydroxycinnamic acid (CHCA) and 1 ÃŽÂ ¼L of 0.1% trifluoroacetic acid (TFA) [7]. The entire mixture was pipette onto a MALDI well and was inserted into the mass spectrometer and a MALDI-MS spectra was obtained. Michaelis-Menten kinetics was used to determine the KM and kcat of the p-nitrophenyl (PNPA) hydrolysis reaction [7]. The ionized product from the hydrolysis, p-nitrophenol (PNP) produces a bright yellow colour that was used to follow the rate of the reaction via the Perkin Elmer Lambda UV/Vis spectrophotometer [7]. Various sample concentrations of PNPA were set up to have a final enzyme concentration of 0.2 ÃŽÂ ¼M in protein buffer [7]. The initial rate measurements of each PNPA concentration were taken for wild-type enzyme, H64A mutant, V142D mutant, and a blank with no additional enzyme added (refer to data tables in Enzyme Kinetics I [7]). PNP has a molar absorption coefficient (ÃŽÂ µ) of 1.73ÃÆ'-104 Mà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1cmà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1. This was used to calculate Michaelis-Menten values: Vmax, KM, kcat, and kcat/KM [7]. The ability of hCAII mutants (H64A and V142D) and wild-type hCAII to catalyze the hydration of CO2 was measured. The pH of the solution was measured to track the progress of the reaction because the reaction generates protons. Enzyme solutions were prepared according to table 2 in [7]. The buffer used in the table was 50 mM TRIS buffer (pH 7.8). Additional enzyme samples were prepared for 25 nM of wild-type hCAII and 100 nM of H64A mutant in a final concentration of 22.5 and 29.92 mM imidazole buffer (pH 7.8) respectively to determine chemical rescue of mutant H64A. The pH of the CO2 hydration assay was measured using a pH probe and pH meter at 5 second increments for a total of 90 seconds starting at the beginning of the reaction [7]. The slope of the initial changes in the first 2 points was considered to be the V0 for each enzyme concentration. From the initial velocity, a kcat value can be calculated for each enzyme using the assumption that [S] >> KM, the Michaelis-Menten equati on simplifies to kcat=V0/[E]. The third kinetics experiment used fluorescence to determine the binding constant of dansyl amide (DNSA) and acetazolamide (AZ) (from Sigma-Aldrich) to H64A and wild-type hCAII was performed using the Perkin Elmer Fluorometer [7]. Stocks of 1 mM and 200 ÃŽÂ ¼M of DNSA were prepared from a 21.6 mM DNSA stock by dilution with DMSO. Enzyme stocks were diluted to 0.25 ÃŽÂ ¼M with TRIS buffer to make a 10 mL solution. A 1 mL sample of H64A from stock made was titrated with DNSA in small increments [7]. The fluorometer emissions were taken at 470 nm. AZ titration in competition with DNSA was not able to be completed. The last characterization experiment done was molecularly modelling the hCAII wild-type enzyme, as well as the mutants H64A and V142D. The molecular model of hCAII analyzed was derived by x-ray crystallography and found in the Protein Data Bank (PDB) repository. The wild-type and H64A hCAII structures examined had a PDB code of 1CA2 and 1MOO respectively. At present, no crystal structure has been found for V142D hCAII. The Swiss PDB Viewer program was used to visualize the protein structures. Secondary structures of the proteins were able to be observed. Residues around the metal active site and the Ramachandran plot were explored. Homology between hCAII and other carbonic anhydrase isozymes, hCAIV (PDB code 1ZNC) and hCAI (PBD code H1CB), were also studied by performing an iterative magic fit on the ÃŽÂ ±-carbons and structure alignment for each pair. The root mean square (RMS) between hCAII and the other isozymes were also analyzed to determine conserved and deviated regions in the structures. The binding of cobalt in the hCAII active site was also investigated (PDB code 3KOI). The structural inhibition of hCAII by AZ was also gleaned by structural analysis (PDB code 3HS4). Its mode of inhibition and binding sites were shown through the crystal structure. Lastly, the Swiss PDB Viewer program was used as a tool to theoretically synthesize mutations and compare it to the actual structure as determined by other scientists, for example, by aligning the virtual and crystallized mutations to determine deviations in structure by performing RMS. Results Site-directed mutagenesis PCR. Products from the PCR mutagenesis reactions were examined using 0.7% agrose gel electrophoresis. Two samples of differing amounts of template DNA (10 ng and 20 ng) were used for each mutant (Figure ). Bands were only observed for samples containing 20 ng of the hCA2pET24b DNA template plasmid (Figure ). The size of the bands observed coincides with the size of the plasmid used, 6018 bp. Heat shock transformation and isolation of plasmid. Several colonies were observed after plasmid transformation for both mutants, and 1 colony from each mutant was chosen for restriction enzyme digest with BglII and XhoI. Figure : Electrophoretic run on 0.7% agrose gel of DNA of hCAII mutants from PCR mutagenesis reactions. Lane 1 is the GeneRuler ladder by Fermentas and lanes 10-13 are the following: V142D (10 ng), V142D (20 ng), H64A (10 ng), and H64A (20 ng). As suggested from the gel, the mutants in the 20 ng plasmid was more successful than the 10 ng plasmids in determining relative molecular weights. Both mutants in the 20 ng plasmid show a band at approximately the 6000 base pair mark, which coincides with the number of base pairs in the hCA2pET24b plasmid that was used (6018 base pairs). Quantification of pure plasmid DNA. A 1/20th dilution was carried out on the purified DNA with elution buffer (EB; 0.1 M Tris, 0.4 M KSCN, pH 7). The absorption of the diluted DNA at 260 nm and 280 nm was taken by a UV/Vis spectrophotometer and the relative DNA purity was determined (Table ). The assumption that Ecà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1 260 = 50 ÃŽÂ ¼g/mL for DNA was applied in the calculation of concentrated and diluted concentrations of purified DNA (Table ). Table : Relative DNA purity for mutants V142D and H64A determined by UV/Vis spectrophotometer absorbance at 260 and 280 nm. Calculated concentrations of mutants from absorbance data, where Ecà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1 260 = 50 ÃŽÂ ¼g/mL. Mutant Wavelength, ÃŽÂ » Absorbance Units Relative DNA Purity (A260/A280) Concentrated (ÃŽÂ ¼g/mL) Diluted (ÃŽÂ ¼g/mL) V142D 260 nm 0.3117 1.7852 311.70 15.59 280 nm 0.1746 H64A 260 nm 0.2653 1.7581 265.30 13.27 280 nm 0.1509 Enzyme restriction digest. Purified plasmid DNA of mutants were digested with XhoI and BglII, separately and together in a single and double digest for both mutants. The digested and undigested samples were run on 1% agrose gel, and 2 bands were observed around the 6000 and 7000 bp marker for all 8 samples (Figure , Figure ). The expected length of the bands in the double digest should be 892 bp and 5126 bp (Figure ). Figure : Electrophoresis performed in 1% agrose gel of digested V142D hCAII in lane 1-4. The (1 kB) GeneRuler DNA ladder is shown in lane 5. Lane 1-4 contain the following: V142D plasmid, V142D + XhoI, V142D + BglII + XhoI, and V142D BglII. Double bands are shown at the 6000 and 7000 bp marker for all 4 V142D samples. Figure : Electrophoresis performed in 1% agrose gel of digested H64A hCAII in lane 1-4. The (1 kB) GeneRuler DNA ladder is shown in lane 5. Lane 1-4 contain the following: H64A plasmid, H64A + XhoI, H64A + BglII + XhoI, and H64A BglII. Double bands are shown at the 6000 and75000 bp marker for all 4 H64A samples. Figure : Restriction enzyme cut sites and position of hCAII gene (5072-5854) on the hCAI2pET24b plasmid DNA Sequencing. The mutations for both V142D and H64A in the hCAII gene were successful according to the sequenced DNA result obtained from ACGT. Other mutations in the DNA sequence were observed in both mutants, but since the aligned protein sequence was the same, mutations were likely to be silent mutations due to amino acid redundancies. When sequenced in the forward direction by T7 polymerase, a protein mutation was found (K153N) other than the desired mutation of V142D; however, when sequenced in the reverse direction by T7 polymerase terminator (T7TER), K153N was not observed. Plasmid DNA transformation into E.Coli BL21(DE3) cells. Following transformation into BL21(DE3) cells, colonies were observed for both hCAII mutants (V142D and H64A). A random colony was chosen to be cultured and then was induced to express protein with 270 ÃŽÂ ¼M IPTG and 0.1 mM ZnSO4. SDS-PAGE for protein expression. Protein expression was tested with SDS-PAGE. The expected molecular weight of V142D hCAII is approximately 29.2 kDa and the expected molecular weight of H64A hCAII is approximately 29.1 kDa. SDS-PAGE bands are observed between the ladder markers 25.0 kDa and 35.0 kDa for both mutant proteins (Figure , Figure ). Figure : SDS-PAGE loaded with V142D hCAII proteins to examine protein expression. Samples were loaded in different volumes of protein to ensure gel visualization. Lane 15 contains the Fermentas protein molecular ladder and lane 1-4 contain the following: 1 ÃŽÂ ¼L à ¢Ã¢â€š ¬Ã¢â‚¬â„¢IPTG, 4 ÃŽÂ ¼L à ¢Ã¢â€š ¬Ã¢â‚¬â„¢IPTG, 1 ÃŽÂ ¼L+IPTG, 4 ÃŽÂ ¼L +IPTG. All 4 samples had some form of protein expression between 25.0 to 35.0 kDa. Figure : SDS-PAGE loaded with H64A hCAII protein to examine protein expression. One sample was loaded with 4 ÃŽÂ ¼L of H64A protein and +IPTG in lane 10. Lane 6 contains the Fermentas protein molecular ladder. The one H64A sample loaded showed an expression between 25.0 and 35.0 kDa. Calculation of pure protein concentration and extinction coefficient. Following affinity purification and dialysis, pure protein concentration was calculated from UV absorption measurements at 280 nm and the known extinction coefficient of hCAII as 50070 Mà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1cmà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1 (Table ). The final concentration of the samples of V142D and H64A hCAII were 3.2 ÃŽÂ ¼M and 37.6 ÃŽÂ ¼M respectively. Table : UV absorption measurements at 280 nm of purified protein and the resulting final concentration Mutant Average A280 Protein concentration (ÃŽÂ ¼M) V142D 0.01583 3.2 H64A 0.1884 37.6 SDS-PAGE to assay purity and check approximate molecular weight. Several samples were loaded into the SDS-PAGE for each mutant protein: lysate and wash fractions (collected from affinity chromatography), 2 ÃŽÂ ¼g protein, and 10 ÃŽÂ ¼g protein. For H64A, a visible band was only observed for the 10 ÃŽÂ ¼g sample (Figure ). The band was located between the 35 kDa and 25 kDa markers on the ladder. For V142D, none of the 4 samples resulted in a band on the gel (Figure ). Figure : SDS-PAGE shown for H64A mutant protein. Lane 1 contains the Fermentas protein molecular weight marker. Lane 11-14 contains H64A samples of the following (in order): lysate, wash fraction, 2 ÃŽÂ ¼g protein, and 10 ÃŽÂ ¼g protein. Only the 10 ÃŽÂ ¼g protein had (faint) observable bands located between the 25 and 35 kDa markers. Figure : SDS-PAGE shown for V142D mutant protein. Lane 4 contains the Fermentas protein molecular weight marker. Lane 12-15 contains V142D samples of the following (in order): lysate, wash fraction, 2 ÃŽÂ ¼g protein, and 10 ÃŽÂ ¼g protein. No observable bands are seen for any of the samples. Mass spectrometry. ESI-MS was not successful in analyzing the molecular weight of intact and digested protein of both mutants. A MALDI spectrum was able to be generated for the digested proteins; however, without the digested ESI spectrum to compare to, the peaks from the MALDI spectrum can only be speculatively assigned. Kinetics: Hydrolysis of PNPA. Using the molar absorption coefficient of PNP (1.73ÃÆ'-104 Mà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1cmà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1), the rate of each reaction was determined. The predicted rate was calculated using the Michaelis-Menten kinetics: . The plot of predicted rates and actual initial rates vs. PNPA concentration can be seen in Figure , Figure , Figure for wild-type, H64A, and V142D hCAII respectively. The Vmax and KM values for each enzyme were calculated by minimizing the square difference between the predicted and actual reaction rates, and the kcat was calculated using the equation: (Table ). Table : Calculated Michaelis-Menten parameters for wild-type, H64A, and V142D hCAII catalyzing the hydrolysis of PNPA. Wild-type hCAII H64A hCAII V142D hCAII Vmax (ÃŽÂ ¼M/sec) 1.202 0.812 0.218 KM (mM) 1.280 1.957 8.362 kcat (sà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1) 5.141 ÃÆ'- 10à ¢Ã¢â€š ¬Ã¢â‚¬â„¢3 2.159 ÃÆ'- 10à ¢Ã¢â€š ¬Ã¢â‚¬â„¢2 6.825 ÃÆ'- 10à ¢Ã¢â€š ¬Ã¢â‚¬â„¢2 kcat/KM (Mà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1sà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1) 4.02 11.032 8.162 Figure : Michaelis-Menten plot of initial rate vs. concentration of PNPA added for wild-type hCAII enzyme. Figure : Michaelis-Menten plot of initial rate vs. concentration of PNPA added for H64A hCAII enzyme. Figure : Michaelis-Menten plot of initial rate vs. concentration of PNPA added for V142D hCAII enzyme. Kinetics: CO2 hydration. Initial velocity (V0) values were calculated by measuring the progression of the reaction (via concentration of protons) with time (Table , Table , and Table ). kcat values were then calculated using the same equation as in the hydration of PNPA and averaged for the individual enzymes (wildtype, H64A, and V142D hCAII) in a particular buffer (i.e. TRIS or imidazole). Table : Initial velocity (V0) and kcat values calculated for the hydration of CO2 by wild-type hCAII in TRIS buffer and imidazole buffer. Wild-type concentration (nM) V0 for WT+TRIS (M/s) V0 for WT+Imidazole (M/s) 0 1.3E-08 6.05778E-08 1.5 1.1E-08 N/A 2.5 1.1E-08 5.63E-08 5 2.1E-08 5.16E-08 12.5 5.9E-08 5.63E-08 Average kcat (sà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1) 5.3 ±1.62 12.44 ±9.19 Table : Initial velocity (V0) and kcat values calculated for the hydration of CO2 by H64A hCAII in TRIS buffer and imidazole buffer. H64A concentration (nM) V0 for H64A+TRIS (M/s) V0 for H64A+Imidazole (M/s) 12.5 1.4E-08 6.57E-08 25 1.4E-08 5.8E-08 50 1.7E-08 7.53E-08 Average kcat (sà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1) 0.67 ±0.39 3.03 ±1.97 Table : Initial velocity (V0) and kcat values calculated for the hydration of CO2 by V142D hCAII in TRIS buffer. V142D concentration (nM) V0 for V142D+TRIS (M/s) 12.5 6.2E-09 25 5.4E-09 50 5.5E-09 Average kcat (sà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1) 0.27 ±0.19 Fluorescence detection of ligand binding. DNSA was titrated with H64A hCAII to determine its affinity for the enzyme. The dissociation constant, KD, for DNSA was determined to be 0.086 ÃŽÂ ¼M when protein concentration was 0.25 ÃŽÂ ¼M. Competitive titration of H64A-DNSA hCAII with AZ was attempted, but was not successful as DNSA binding was too tight, making it difficult to be displaced by AZ. Molecular modeling. Literature models of wild-type (PDB code 1CA2) and H64A (PDB code 1MOO) hCAII were analyzed. There is no available structure of V142D hCAII at present. The secondary structure of wild-type is composed of 18 ÃŽÂ ²-sheets (77 residues) and 10 ÃŽÂ ±-helices (42 residues), with the majority of the ÃŽÂ ±-helices falling in the domain of right-handed helices, while very few show left-handed helical properties according to the Ramachadran plot. It also seems that the active site is solely composed of ÃŽÂ ²-sheets, and no ÃŽÂ ±-helices (Figure ). Analyzing PDB structure 3HS4 (AZ bound hCAII), the mechanism as to how AZ inhibits hCAII function can be seen. AZ has 3 binding sites, 2 are novel binding sites and the other provides a mechanism of inhibition. AZ binds the zinc directly at the active site, displacing crucial ligands needed for catalysis. There were some discrepancies found between the crystal structure of H64A [1MOO] as cited on PDB and virtually mutated H64A from wild-type hCAII, resulting in a RMSD (root mean square deviation) of 0.29 Ã… (Figure ). Since no literature structure of V142D is available, no comparison between virtual and crystal structures could be made. Figure : Secondary structure of wild-type hCAII overlain with ribbon to visualize the higher arrangement. Figure : RMSD between H64A hCAII virtually mutated and literature crystal structure. Blues denote the same or similar residues, while reds and oranges indicate completely different amino acids. Discussion Agrose gel results were only visible for samples that contained 20 ng of the plasmid template DNA, rather than the 10 ng plasmid. This may be a result of more amplification during PCR with the 20 ng plasmid, and so would intensely be more visible. Though the 20 ng samples showed bands at the appropriate 6000 bp mark, there was also a faint band that can be seen near the end of the gel. This may be due to non-specific primer annealing. Quantification of DNA purity was done by exploiting the peak absorbances of protein and DNA. DNA maximally absorbs at 260 nm, while protein dominantly absorbs at 280 nm. The purity ratio reports the relative amount of DNA compared to protein present in the sample. The purity of both mutants were approximately 1.8, which is regarded as a relatively pure sample; however, a purity ratio of more than 2.0 would have been ideal. The restriction enzyme digest showed 2 bands (7000, 6000 bp) for all samples, which may have been a sign of poor mixing/ pipetting since the volumes of restriction enzyme were extremely small amounts. If this is the case, only some of the DNA was nicked and some were not, which would result in 2 bands. It was expected that the plasmid sample would have a high band (supercoiled), each of the singly digested samples would have a slightly lower band (nicked), and the doubly digested would show 2 bands that indicated the fragment size of 892 and 5126 bp. Sequencing results showed that a protein mutation occurred when the sample was sequenced in the forward direction by the T7 polymerase. A lysine at position 153 had mutated to glutamine (K153N). However, this mutation was not observed when the T7 polymerase terminator was used to sequence the sample in the reverse direction. A mutation that occurs in one sequencing direction and not the other is usually attributed to sequencing errors, which may be the reason in this case. The SDS-PAGE bands for protein expression coincided with the expected molecular weight for both mutants, which could suggest that the correct proteins were expressed; however, there is a possibility that the proteins expressed could be of similar weight, but completely different. Interestingly, the V142D samples that did not include the protein inducer, IPTG, had a more intense band than the faint ones found for the samples that did include IPTG. This may just be a result of mislabelling. The SDS-PAGE performed to assess purity after the purification process. Mutant V142D had low protein expression as evidenced by its concentration of 3.2 ÃŽÂ ¼M. The V142D mutant should have very low protein expression according to Fierke et al. (1991) because valine at position 142 is uniquely required for maximal expression in E.Coli. It is suggested that by altering position 142, protein stability decreases [2]. Therefore, the protein that was expressed in the previous SDS-PAGE gel may not be V142D hCAII at all. The sample may have been small fragmented contaminant proteins that would have completely run off the gel altogether. However, the low concentration of V142D after purification may also be a major factor in the lack of gel bands observed as well. Unlike V142D, H64A hCAII concentration should not have affected its lack of bands because it was calculated to have had a reasonable concentration of 37.6 ÃŽÂ ¼M. There were some problems loading the samples into the wells; t his could be an explanation as to no observable gel bands. ESI-MS is dependent on concentration because it affects the size of primary droplets [8]. The unsuccessful determination of molecular weight of V142D hCAII may be attributed to its low concentration. The H64 hCAII mutant was also not able to be successfully analyzed with ESI-MS. A possible reason for the failure was that it was not kept on ice while it was not being used. The enzyme may have become inactive and degraded into smaller fragments. This would explain the ESI-MS output obtained for H64A. No definite molecular mass was determined, but the spectrometer did detect a lot of small protein fragments in the sample, all under 1000 amu. The kinetic values obtained from PNPA hydrolysis do not follow similar trends found in literature [2]. The kcat/KM for wild-type hCAII (2500 ±200 Mà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1sà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1) was found to be significantly larger than V142D hCAII (3 ±0.3 Mà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1sà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1) in literature, more than 800ÃÆ'- larger [2]. Experimental calculations yielded kcat/KM for V142D (8.16 Mà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1sà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1) to be about 2ÃÆ'- larger than wild-type (4.02 Mà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1sà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1), which did not follow literature patterns. The literature trends make more biolog