Computer Technology and the Physically Challenged :: Exploratory Essays Research Papers

Computer Technology and the Physically Challenged Computer technology has positively affected the physically challenged. Computer technology brings new techniques for the blind, deaf, and people with movement disabilities. New programs are making it easier for the disabled. Some schools and states are helping the disabled by setting up computer labs and computer recycling programs. Computer technology has really helped the blind and the deaf. It has brought Braille keys to a regular keyboard. The blind can print out their documents as a Braille copy or a regular copy. If a blind person does not know Braille, then they can use speech synthesizers. Speech synthesizers can speak the words on the screen aloud. A 62-year-old professor uses a portable speech synthesizer. Computer technology has helped make life easier for the deaf because they can read the computer screen easier than before. A center called Adaptive Technology Resource Centre (ATRC) is making it easier for the disabled to access information technology. The new ATRC program, at the University of Toronto, designed to access information. Another program, called MathML, was intended to put math on the web. ATRC worked with MathML to make it accessible to people. The ATRC has also added haptics, the sense of touch and audio, to interactive programs. In an article I read, one example given was a periodic table that showed the relative elements and weight. Another example given was of a pendulum model that showed the properties of a pendulum. ATRC is also exploring the use of captioning and video description. People can look up a word they do not know using one of these methods. ATRC, along with three other companies, has come up with the smart card system. The smart card sets up the computer to the individuals’ preference. Another program that was set up to help disabled people is called Equal Access to Software and Information (EASI). EASI is like ATRC; it provides access to information and guidance to the physically challenged. Through a National Science Foundation (NSF) grant, EASI had developed several projects. At the Georgia Institute of Technology, they are trying to make science labs more accessible to students with disabilities. They also take an interest in distant learning by having two online workshops to assist in understanding the Internet and computers. Other schools are starting to help the disabled; whether it is by setting up a new computer lab or recycling used computers.

Self Respect

Self-respect means to have respect yourself. It’s a term that means something different to everyone one. But it’s something everyone wants, not many have, and few want to give out. Self respect is one of the most important values a person can have. To gain respect you have to be confident in yourself. Also you have to have respect for others to fully achieve it. Self respect goes along with self esteem. The way you see yourself means everything in order to build strong character. If you don’t see yourself in a positive light then you don’t really have respect for yourself.Many people seek attention in negative ways because they have a low self esteem. When people do this they are not respecting themselves and they are allowing people to walk all over them. For instance, girls who have poor self esteem look for attention anyway they can. An example of how they would do this is by sleeping around with numerous guys that shows them the slightest bit of intere st. They do this to make them feel loved and wanted but in reality it creates a bad name for them. Inevitably this has a bad effect causing them to feel even worse than before.So in the end you should always value yourself and be confident about whom you are because that’s what really makes someone have self respect. The way you treat other people is also a way of showing you have self respect. When you show people consideration for their problems and you actually listen to them when they talk your showing them you have respect for them and they will respect you back. This also goes along with not letting people disrespect you. Someone with self respect doesn’t let someone treat them badly and when someone is disrespecting them they do something about it.For example, when someone walks all over you and you don’t want to be say something because you don’t want to loose that person your showing a sign of weakness. You should be able to confront that person and tell them how they are treating you bad and if they don’t stop you just wont be around them anymore. When people do this it shows that you have true respect for yourself. In the end its clear that people don’t get far without self respect. Its what builds your character so you have to embody all the attributes that go along with it. You have to have a positive self esteem and you have to be able to have respect for the people around you. Self Respect Self-respect means to have respect yourself. It’s a term that means something different to everyone one. But it’s something everyone wants, not many have, and few want to give out. Self respect is one of the most important values a person can have. To gain respect you have to be confident in yourself. Also you have to have respect for others to fully achieve it. Self respect goes along with self esteem. The way you see yourself means everything in order to build strong character. If you don’t see yourself in a positive light then you don’t really have respect for yourself.Many people seek attention in negative ways because they have a low self esteem. When people do this they are not respecting themselves and they are allowing people to walk all over them. For instance, girls who have poor self esteem look for attention anyway they can. An example of how they would do this is by sleeping around with numerous guys that shows them the slightest bit of intere st. They do this to make them feel loved and wanted but in reality it creates a bad name for them. Inevitably this has a bad effect causing them to feel even worse than before.So in the end you should always value yourself and be confident about whom you are because that’s what really makes someone have self respect. The way you treat other people is also a way of showing you have self respect. When you show people consideration for their problems and you actually listen to them when they talk your showing them you have respect for them and they will respect you back. This also goes along with not letting people disrespect you. Someone with self respect doesn’t let someone treat them badly and when someone is disrespecting them they do something about it.For example, when someone walks all over you and you don’t want to be say something because you don’t want to loose that person your showing a sign of weakness. You should be able to confront that person and tell them how they are treating you bad and if they don’t stop you just wont be around them anymore. When people do this it shows that you have true respect for yourself. In the end its clear that people don’t get far without self respect. Its what builds your character so you have to embody all the attributes that go along with it. You have to have a positive self esteem and you have to be able to have respect for the people around you.

Ralph Waldo Emerson Essay

Note: Nineteenth Century American Transcendentalism is not a religion (in the traditional sense of the word); it is a pragmatic philosophy, a state of mind, and a form of spirituality. It is not a religion because it does not adhere to the three concepts common in major religions: a. a belief in a God; b. a belief in an afterlife (dualism); and c. a belief that this life has consequences on the next (if you’re good in this life, you go to heaven in the next, etc. ). Transcendentalism is monist; it does not reject an afterlife, but its emphasis is on this life. The Assumed, Presumed, or the Self-Identified Transcendentalists: Central Points of Agreement: NOTE: The Transcendentalists, in keeping with the individualistic nature of this philosophy, disagreed readily with each other. Here are four points of general agreement: Basic Assumption: The intuitive faculty, instead of the rational or sensical, became the means for a conscious union of the individual psyche (known in Sanskrit as Atman) with the world psyche also known as the Oversoul, life-force, prime mover and God (known in Sanskrit as Brahma). Basic Premises: 1. An individual is the spiritual center of the universe – and in an individual can be found the clue to nature, history and, ultimately, the cosmos itself. It is not a rejection of the existence of God, but a preference to explain an individual and the world in terms of an individual. 2. The structure of the universe literally duplicates the structure of the individual self – all knowledge, therefore, begins with self-knowledge. This is similar to Aristotle’s dictum â€Å"know thyself. † 3. Transcendentalists accepted the neo-Platonic conception of nature as a living mystery, full of signs – nature is symbolic. 4. The belief that individual virtue and happiness depend upon self-realization – this depends upon the reconciliation of two universal psychological tendencies: a. the expansive or self-transcending tendency – a desire to embrace the whole world – to know and become one with the world. b. the contracting or self-asserting tendency – the desire to withdraw, remain unique and separate – an egotistical existence. Correspondence. It is a concept which suggests that the external is united with the internal. Physical or material nature is neutral or indifferent or objective; it is neither helpful nor hurtful; it is neither beautiful nor ugly. What makes one give such attributes to nature is that individual’s imposition of her/his temperament or mood or psyche. If I’m feeling lousy, I may dismiss a gorgeous day; if I’m feeling bright and cheerful then the most dreary of days becomes tolerable. And so, the Transcendentalists believed that â€Å"knowing yourself† and â€Å"studying nature† is the same activity. Nature mirrors our psyche. If I cannot understand myself, may be understanding nature will help. Here is Darrel Abel’s â€Å"take† on this concept: â€Å"Since one divine character was immanent everywhere in nature and in man, man’s reason could discern the spiritual ideas in nature and his senses could register impressions of the material forms of nature. To man the subject, nature the object, which shared the same divine constitution as himself, presented external images to the innate ideas in his soul. † (American Literature, Vol. 2, 1963, 4-5. ) Transcendentalism and the American Past. Transcendentalism as a movement is rooted in the American past: To Puritanism it owed its pervasive morality and the â€Å"doctrine of divine light. † It is also similar to the Quaker â€Å"inner light. † However, both these concepts assume acts of God, whereas intuition is an act of an individual. In Unitarianism, deity was reduced to a kind of immanent principle in every person – an individual was the true source of moral light. To Romanticism it owed the concept of nature as a living mystery and not a clockwork universe (deism) which is fixed and permanent. A subtle chain of countless rings The next unto the farthest brings; The eye reads omens where it goes, And speaks all languages the rose; And, striving to be man, the worm Mounts through all the spires of form. – Ralph Waldo Emerson, Nature, 1836 Transcendentalism was a 1. spiritual, 2. philosophical and 3. literary movement and is located in the history of American Thought as (a). Post-Unitarian and free thinking in religious spirituality (b). Kantian and idealistic in philosophy and (c). Romantic and individualistic in literature. A Brief Chronology of Events. †¢ 1832 Emerson resigns the ministry of the Unitarian Church – unable to administer the holy communion. †¢ 1836 The annus mirabilis of the movement, during which Emerson published Nature (the â€Å"gospel† of transcendentalism); George Ripley published Discourses on the Philosophy of Religion; Orestes Brownson published New Views of Christianity, Society, and Church; Bronson Alcott published Record of Conversions in the Gospel (based on classroom discussions in his Temple School in Boston, and provoking severe criticism); the Transcendental Club, also known as Hedge’s Club, met for the first time. †¢ 1837 Emerson delivers his Phi Beta Kappa address on â€Å"The American Scholar† at Harvard, which James Russell Lowell called â€Å"an event without former parallel in our literary annals. † †¢ 1838 Emerson delivers his Divinity School Address at Harvard which touched off a great storm in religious circles. †¢ 1840 The founding of the Dial, a Transcendental magazine, which â€Å"enjoyed its obscurity,† to use Emerson’s words, for four years. †¢ 1841 The launching of George Ripley’s Brook Farm – a utopian experiment. Hawthorne was a resident there for a short time and wrote The Blithedale Romance based upon his experience there. †¢ 1842 Alcott’s utopian experiment at Fruitlands. †¢ 1845 Thoreau goes to live at Walden Pond. †¢ 1846 Thoreau is put in jail for his refusal to pay poll tax. †¢ 1850 Passage of the Fugitive Slave Act. The Transcendentalists found themselves increasingly involved in abolition of slavery. †¢ 1855 Walt Whitman publishes his Leaves of Grass. †¢ 1859 Charles Darwin’s Origin of Species is published. †¢ 1862 Henry David Thoreau dies. Basic Tenets of American Transcendentalism: Note: This list must not be considered to be a creed common to all transcendentalists. It is merely a grouping of certain important concepts shared by many of them. †¢ 1. Transcendentalism, essentially, is a form of idealism. †¢ 2. The transcendentalist â€Å"transcends† or rises above the lower animalistic impulses of life (animal drives) and moves from the rational to a spiritual realm. †¢ 3. The human soul is part of the Oversoul or universal spirit (or â€Å"float† for Whitman) to which it and other souls return at death. †¢ 4. Therefore, every individual is to be respected because everyone has a portion of that Oversoul (God). †¢ 5. This Oversoul or Life Force or God can be found everywhere – travel to holy places is, therefore, not necessary. 6. God can be found in both nature and human nature (Nature, Emerson stated, has spiritual manifestations). †¢ 7. Jesus also had part of God in himself – he was divine as everyone is divine – except in that he lived an exemplary and transcendental life and made the best use of that Power which is within each one. †¢ 8. â€Å"Miracle is monster. † The miracles of the Bible are not to be regarded as important as they were to the people of the past. Miracles are all about us – the whole world is a miracle and the smallest creature is one. â€Å"A mouse is a miracle enough to stagger quintillions of infidels. † – Whitman †¢ 9. More important than a concern about the afterlife, should be a concern for this life – â€Å"the one thing in the world of value is the active soul. † – Emerson †¢ 10. Death is never to be feared, for at death the soul merely passes to the oversoul. †¢ 11. Emphasis should be placed on the here and now. â€Å"Give me one world at a time. † – Thoreau †¢ 12. Evil is a negative – merely an absence of good. Light is more powerful than darkness because one ray of light penetrates the dark. †¢ 13. Power is to be obtained by defying fate or predestination, which seem to work against humans, by exercising one’s own spiritual and moral strength. Emphasis on self-reliance. †¢ 14. Hence, the emphasis is placed on a human thinking. †¢ 15. The transcendentalists see the necessity of examples of great leaders, writers, philosophers, and others, to show what an individual can become through thinking and action. †¢ 16. It is foolish to worry about consistency, because what an intelligent person believes tomorrow, if he/she trusts oneself, tomorrow may be completely different from what that person thinks and believes today. â€Å"A foolish consistency is the hobgoblin of little minds. † – Emerson †¢ 17. The unity of life and universe must be realized. There is a relationship between all things. †¢ 18. One must have faith in intuition, for no church or creed can communicate truth. †¢ 19. Reform must not be emphasized – true reform comes from within. Reasons for the Rise of American Transcendentalism There was no one precise â€Å"cause† for the beginning of Transcendentalism. According to Paul Boller, chance, coincidence and several independent events, thoughts and tendencies seemed to have converged in the 1830s in New England. Some of these were: †¢ 1. The steady erosion of Calvinism. †¢ 2. The progressive secularization of modern thought under the impact of science and technology. †¢ 3. The emergence of a Unitarian intelligentsia with the means, leisure, and training to pursue literature and scholarship. †¢ 4. The increasing insipidity and irrelevance of liberal religion to questing young minds – lack of involvement in women’s rights and abolitionism. †¢ 5. The intrusion of the machine into the New England garden and the disruption of the old order by the burgeoning industrialism. †¢ 6. The impact of European ideas on Americans traveling abroad. †¢ 7. The appearance of talented and energetic young people like Emerson, Fuller, and Thoreau on the scene. †¢ 8. The imperatives of logic itself for those who take ideas seriously – the impossibility, for instance, of accepting modern science without revising traditional religious views. Important ideas from: Warren, Robert Penn, Cleanth Brooks, and R. W. B. Lewis. â€Å"A National Literature and Romantic Individualism. † in Romanticism. eds. James Barbour and Thomas Quirk. NY: Garland, 1986, 3-24. 1. Transcendentalism was a philosophical, literary, social, and theological movement. 2. Its origin is traced to the relaxing of Puritan Calvinism into Unitarianism – a belief very much like Deism. From its early liberalism, Unitarianism developed, for some of the young intellectuals, into â€Å"a new orthodoxy of smug social conformity that denied the spiritual and emotional depths of experience – ‘corpse-cold Unitarianism,’ as Emerson was to call it. † (11) 3. German and English Romanticism provided some inspiration towards the search for some deeper ‘truth. ‘ 4. â€Å"Transcendentalism represented a complex response to the democratization of American life, to the rise of science and the new technology, and to the new industrialism – to the whole question, in short, of the redefinition of the relation of man to nature and to other men that was being demanded by the course of history. † (11-12) 5. Influences: a. From Plato came the idealism according to which reality subsists beyond the appearances of the world. Plato also suggests that the world is an expression of spirit, or mind, which is sheer intelligibility and therefore good. b. From Immanuel Kant came the notion of the ‘native spontaneity of the human mind’ against the passive conception of the 18th c. sensational theory (also known as the philosophy of empiricism of John Locke and David Hume; the concept that the mind begins as a tabula rasa and that all knowledge develops from sensation). c. From Coleridge came the importance of wonder, of antirationalism, and the importance of individual consciousness. d. From Puritanism came the ethical seriousness and the aspect of Jonathan Edwards that suggested that an individual can receive divine light immediately and directly. 6. â€Å"Transcendentalism was, at its core, a philosophy of naked individualism, aimed at the creation of the new American, the self-reliant man, complete and independent. † (22) 7. â€Å"The achievement of the transcendentalists has a grandeur. They did confront, and helped define, the great issues of their time, and if they did not resolve those issues, we of the late twentieth century, who have not yet resolved them, are in no position to look down our noses at their effort. † (23) Towards a Definition of Transcendentalism: A Few Comments: from Henry David Gray, Emerson: A Statement of N. E. Transcendentalism as Expressed in the Philosophy of Its Chief Exponent, 1917 1. â€Å"The spirit of the time is in every form a protest against usage and a search for principles. † – Emerson in the opening number of The Dial. 2. â€Å"I was given to understand that whatever was unintelligible would be certainly Transcendental. † – Charles Dickens in American Notes 3. â€Å"I should have told them at once that I was a transcendentalist. That would have been the shortest way of telling them that they would not understand my explanations. † – Thoreau, Journal, V:4 4. â€Å"The word Transcendentalism, as used at the present day, has two applications. One of which is popular and indefinite, the other, philosophical and precise. In the former sense it describes man, rather than opinions, since it is freely extended to those who hold opinions, not only diverse from each other, but directly opposed. † – Noah Porter, 1842 5. Transcendentalism is the recognition in man of the capacity of knowing truth intuitively, or of attaining a scientific knowledge of an order of existence transcending the reach of the senses, and of which we can have no sensible experience. † – J. A. Saxton, Dial II: 90 6. â€Å"Literally a passing beyond all media in the approach to the Deity, Transcendentalism contained an effort to establish, mainly by the discipline of the intuitive faculty, direct intercourse between the soul and God. † – Charles J. Woodbury in Talks with Ralph Waldo Emerson 7. â€Å"Transcendentalism was not †¦ speculative, but essentially practical and reformatory. † – John Orr in â€Å"The Transcendentalism of New England,† International Review, XIII: 390 8. â€Å"Transcendentalism was a distinct philosophical system. Practically it was an assertion of the inalienable worth of man; theoretically it was an assertion of the immanence of divinity in instinct, the transference of supernatural attributes to the natural constitution of mankind. †¦ Transcendentalism is usually spoken of as a philosophy. It is more justly regarded as a gospel. As a philosophy it is †¦ so far from uniform, that it may rather be considered several systems than one. †¦ Transcendentalism was †¦ an enthusiasm, a wave of sentiment, a breath of mind. † – O. B. Frothingham in Transcendentalism in New England, 1876 9. â€Å"The problem of transcendental philosophy is no less than this, to revise the experience of mankind and try its teachings by the nature of mankind, to test ethics by conscience, science by reason; to try the creeds of the churches, the constitution of the states, by the constitution of the universe. † – Theodore Parker in Works VI: 37 10. â€Å"We feel it to be a solemn duty to warn our readers, and in our measure, the public, against this German atheism, which the spirit of darkness is employing ministers of the gospel to smuggle in among us under false pretenses. † Princeton Review XII: 71 11. â€Å"Protestantism ends in Transcendentalism. † – Orestes Brownson in Works, 209 12. â€Å"The fundamentals of Transcendentalism are to be felt as sentiments, or grasped by the imagination as poetical wholes, rather than set down in propositions. † – Cabot, A Memoir of Ralph Waldo Emerson, 1887, I: 248 13. â€Å"First and foremost, it can only be rightly conceived as an intellectual, aesthetic, and spiritual ferment, not a strictly reasoned doctrine. It was a renaissance of conscious, living faith in the power of reason, in the reality of spiritual insight, in the privilege, beauty, and glory of life. † – Frances Tiffany, â€Å"Transcendentalism: The New England Renaissance,† Unitarian Review, XXXI: 111. 14. â€Å"The Transcendentalist adopts the whole connection of spiritual doctrine. †¦ If there is anything grand or daring in human thought or virtue, any reliance on the vast, the unknown; any presentiment, any extravagance of faith, the spiritualist adopts it as most in nature. The oriental mind has always tended to this largeness. Buddhism is an expression of it. The Buddhist †¦ is a Transcendentalist. †¦ Shall we say then that Transcendentalism is the Saturnalia or excess of Faith; the presentiment of a faith proper to man in his integrity, excessive only when his imperfect obedience hinders the satisfaction of his wish? † – Ralph Waldo Emerson’s lecture on â€Å"The Transcendentalist,† Works I: 317-320 15. â€Å"(Transcendentalism was) a blending of Platonic metaphysics and the Puritan spirit, of a philosophy and a character †¦ taking place at a definite time, in a specially fertilized soil, under particular conditions. † – H. C. Goddard, Studies in New England Transcendentalism, 1908. 16. â€Å"If I were a Bostonian, I think I would be a Transcendentalist. † – Charles Dickens in American Notes.

Banking Regulation around the World Patterns, Determinants, and Impact essayEssay Writing Service

Banking Regulation around the World Patterns, Determinants, and Impact essayEssay Writing Service Banking Regulation around the World: Patterns, Determinants, and Impact essay Banking Regulation around the World: Patterns, Determinants, and Impact essayThe US has historically been a state based on the principles of free market and perfect competition. However, increasing economic growth and increasing interdependence among countries resulted in regional, in the first place, and then global crisis. For example, the Great Depression was the starting point for the development of the economic theory of John Maynard Keynes deriving from the fact that the market is not able to come to equilibrium independently. Later criticism of Keynes’s paper led to the rejection of some of its provisions, but the idea of the state as a third party regulating the market has remained. As Robert Lucas stated in 2009: â€Å"I guess everyone is a Keynesian in the foxhole† (Rouse, 2013).Indeed, certain points of the program of state regulation today cause special dissatisfaction on the part of supporters of free market principles. Denying, however, the need for extern al support, although costly, would be, at least, impractical because the calculated potential losses from the crisis would exceed the most expensive plan for economic recovery.  Assessing the borderlines of government interventionInitially, liberalization of the financial sector in the early 2000’s by eliminating Glass-Steagall Act and reducing the interest rate of the Fed to 1% caused a rapid growth of the US economy for the past eight years. However, market deregulation allowed many commercial banks to buy short-term profitable but risky assets at the expense of their depositors, while need to involve more people into the system prompted banks to start issuing mortgages even to less reliable households without requiring any fee or proof of income, which in the end led to massive defaults on the mortgage and financial crisis. In the situation where the discount rate already tends to zero, the US economy was actually on the verge of a liquidity trap when further reduction o f the discount rate does not affect the behavior of market agents (Li, 2007). As a result, the Federal Reserve has not coped with the tasks of control leading to the need for greater government intervention, in particular the enactment of Dodd-Frank Wall Street Reform and Consumer Protection Act.The Dodd-Frank Act suggest activity in six areas: improving accountability and increasing transparency of financial institutions, rejection of the principle of rescuing systemically important financial institutions (â€Å"too big to fail†), search for alternatives to rescuing bankrupt companies at the expense of state aid, i.e. taxpayers, consumer protection, introduction of the Volcker Rule, and regulation of the market of precious metals. In addition to reforming the financial legislation, much has been done through direct state intervention in the financial sector (Dodd-Frank Wall Street Reform and Consumer Protection Act). The main ways to stimulate the economy at the moment are: the Fed financial programs, tests assessing the state of major banks, financial assistance to banks through TARP, Recovery Act, the Term Asset-Backed Securities Loan (TALF), organizations such as AIG, Freddie Mac and Funnie Mae, preventing foreclosure of the mortgaged property, as well as the activities of the Small Business Administration (SBA).Acting together, these measures involve a significant reduction in the risk of major default. Indeed, the presence of short-term capital debt declined from 62% in 2007 to 37% in 2013, increasing the stability of financial institutions (Rouse, 2013). At the same time, banks have increased their own capital by about $450 billion, which now serve as the airbag in the event of new economic shocks, reduce dependence on credit and can be spent on investments in long-term projects. In addition, while by March 2009, banks participating in the TARP program and receiving funding had to pay out $238 billion, 99% of this amount was already repaid in Apr il 2013 (Rouse, 2013). Moreover, investments of some programs of the Fed and TARP returned to the state budget as additional revenues, which certainly shows the effectiveness of the state support. However, it is not all as rosy as it might seem at first glance.First, Dodd-Frank Act may be regarded as a return to the past due to the use of one of the principles of the Glass-Steagall Act (Banking Act of 1933), repealed in 1999, namely the Volcker Rule which delineates the activities of banks dealing with consumer credit with government guarantees and investment banks (Dodd-Frank Wall Street Reform and Consumer Protection Act). This decision is likely to lead to further restriction of liquidity and size of the financial market. In addition, state intervention significantly limits the ability of financial institutions, especially large ones, markedly increasing their responsibility. However, such a rigid legislative regulation will complicate a quick exit from the recession and will pre vent the rapid development of the banking sector. In turn, the increase in the share of banking assets controlled by the state reduces the rate of development of financial non-banking sector and the stock market. In general, government regulation associated with rising costs, in fact, voluntarily overtakes the unnecessary long-term risks (Rouse, 2013).Moreover, analysis data from 54 countries show that there is an inverse relationship between the proportion of state involvement in banks and financial development: a large proportion of state involvement in the banking system leads to slowing of economic development, moreover, none of the limits reduces the probability of banking crisis (Li, 2007). At the same time, the most acute problem with the introduction of a new regulatory legislation is related to its procyclicality: the law hinders economic development during the recession limiting the abilities of financial institutions, and during the phase of recovery, its most rigid princ iples will be eliminated, which can lead to another liberalization of financial markets and the economy overheating.  ConclusionOn the one hand, the imposition of restrictions and strengthening of control over certain types of banking activity reduces the efficiency of the whole sector. On the other hand, the modern-day banking regulation in the US is a necessary measure in response to the weakening of market discipline, loss of caution and skepticism, which should be common to investors putting up their capital in a particular company. Regulating the banking sector, the state today operates in a versatile way: it tries to help investors, â€Å"pull† systemically important financial institutions, and stimulate small business. To a certain extent, based on the experience of the recent crisis, today it is difficult to imagine a situation of full liberalization of the financial market; and in our opinion, we should not expect weakening of state intervention in the management o f the banking system in the short term, because it is the most important tool for managing the entire economy, reflects the ownership structure of the state and its economic goals. In this case, the main task of the state is effective limiting of the growth of the financial sector at the peak stage of overall economic development, so that the smoothing of the business cycle allowed surviving the possible subsequent recession without the deep decline and without the heavy pressure on the state budget.

Human Immunodeficiency virus 1 (HIV-1) The WritePass Journal

Human Immunodeficiency virus 1 (HIV-1) 1. Introduction Human Immunodeficiency virus 1 (HIV-1) 1. Introduction1.1    Human Immunodeficiency virus 1 (HIV-1)1.1.1   HIV epidemic and methods of transmission1.1.2 HIV: The virus1.1.3 HIV entry and replication cycle1.1.4 Clinical features of the HIV-1 infection1.2   Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   Vaccines1.2.1   Ã‚  Ã‚  Ã‚   vaccine trials1.2.2   Vaccines: what is desirable2.   Materials and Methods2.1 Immunogen2.2   Adjuvants 2.4   Ã‚  Ã‚  Ã‚   Immunization groups 2.5   Ã‚  Ã‚  Ã‚   Immunization and bleed schedule2.6   Ã‚  Ã‚  Ã‚   Reagents2.6.1   Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   Reagent preparation2.9   Ã‚  Ã‚  Ã‚   Statistics3.1   Ã‚  Ã‚  Ã‚   Zeta Potential Measurements3.2.4    Grouped analysis of end point Titres4. Discussion4.1   Results overview4.2   Fixed and unfixed protein4.2.1   Zeta potential4.2.2   Ã‚  Ã‚  Ã‚   End point titres4.3   Addition of DS003  4.4   Native and wild type protein. 4.5   Conclusion5  Ã‚   ReferencesRelated 1. Introduction 1.1    Human Immunodeficiency virus 1 (HIV-1) 1.1.1   HIV epidemic and methods of transmission    Acquired Immunodeficiency Syndrome (AIDS) is caused by an infectious agent known as Human Immunodeficiency Virus (HIV). Since 1980 when HIV was first discovered, nearly 25 million people have died from AIDS and nearly   34 million were living with HIV in 2009 [1]. Over the past 25 years it has overshadowed all other forms of immunodeficiency and is currently considered a severe global pandemic. There are currently two recognised types of HIV; HIV-type 1 and HIV-type 2 with type 1 being the main agent of AIDS worldwide[2]. HIV is a virus which belongs to the lentivirus genus of the retroviridae family [2]. This is an important factor in that infections with lentiviruses tend to have a chronic course of the disease with a long period of clinical latency, persistent viral replication and infection of the central nervous system [2]. HIV is transmitted through blood and mucosal tissue via sexual intercourse, needle sharing in drug use, blood transfusion and breast feeding. Today the mai n transmission method of HIV is the exchange of bodily fluids between partners during sexual intercourse.   The transmission frequency of HIV is determined by the amount of infectious agent   present in the particular bodily fluid in relation to how much contact the other individual   has with that body fluid.   The determination of whether the infection is established is mainly based on three factors, considered as the three points of the classic epidemiological triangle. These points include: Characteristics of the infectious agent. Host related factors;   includes such as how susceptible the host is and how their body’s immune system responds. Environmental factors such as social, cultural and political status also have a part in the process[2]. Today the pandemic is fuelled predominantly by heterosexual transmission, compared to homosexual transmission in the early 1980s. The last decade has seen the majority of new HIV infection cases being established in women rather than males [2, 3]. This implies that there is increasing discrepancy between infection rates of men and women. It is possible that gender inequalities associated with socio cultural norms play a role in that condom use is generally in the control of the male partner. This situation is particularly   true of developing countries [3].  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚     Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚     Ã‚  Some early studies revealed that there is a two to fivefold- greater risk of infection from male to female transmission. There is a low incidence of infection taking place during any exposure below 1:100 during   unprotected heterosexual intercourse [4]. For HIV transmission to occur there needs to be   an entry point (i.e. unhealthy or damaged mucosa) in the vagina or anal canal because normally, a healthy mucosa is an effective barrier to transmission. Venerial diseases or lesions at these specific points would   weaken them therefore increasing   the chance of transmission [4] . Furthermore, during penetration female genitalia becomes inflamed due to small scale trauma. This is significant because activated neutrophils which migrate to these inflamed areas bind HIV-1 and can thereby transfer the virus to target cells [5]. The virions or infected cells that enter the uterus can infect CD4+ T cells and macrophages that reside within the endometrium by entering the c ells by transcytosis [6 , 7] (process by which macromolecules are transported across a cell into the circulation0. In addition interleukin 8 which happens to be the most prominent cytokine found in the female genital tract can   increase HIV replication in T cells and macrophages [8]. 1.1.2 HIV: The virus The general structure and genetic layout of an HIV particle can be seen in figure 1.   Just like other retroviruses the core structural proteins (p24,p7,p6) and matrix (p17) are encoded for by the gag gene. The viral envelope glycoproteins gp120 and gp41 whose job it is to recognise cell surface receptors and fuse membranes are encoded for by the env gene. The pol gene encodes for enzymes that are crucial for viral replication. These enzymes are reverse transcriptase, integrase and protease.   The protease enzyme cleaves large Gag and Pol protein precursors into their functional components.   Reverse transcriptase is responsible for converting viral RNA into ssDNA, while the intergrase incorporates the viral DNA   into the host cells chromosomal DNA [2]. The HIV life cycle can be summarised into six steps: Binding and entry ; uncoating;   reverse transcription; provirus integration; virus protein synthesis and assembly and budding [2].  Ã‚   HIV’s viral envelope plays an integral part concerning how the virus binds to the cell. The envelope is a trimeric complex that is made of two heterodimer proteins- gp120 and gp41 [2].  Ã‚   In addition to its fusion, it is essential for virus recognition and entry into target cells.  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   1.1.3 HIV entry and replication cycle The entry of HIV-1 into the host cell and subsequent HIV infection is mediated by the interaction of   viral envelope glycoproteins and proteins gp120 and gp41 with the cellular receptor CD4 and a co-receptor which is normally CCR5 or CXCR4   [11 ]. This combination allows the virus to fuse with the host cell membrane and enter the cell. The glycoproteins are derived by proteolytic cleavage of a trimeric, glycosylated gp160 envelope glycoprotein precursor 2   and 46   [11 12].  Ã‚   When the gp120 binds to the cellular receptors there is a change in the viral envelope conformation which leads to the exposure of gp41-a hairpin like glycoprotein.   This exposure initiates the fusion between the HIV virus and its target cell via insertion of the glycoprotein into the cell wall [13 14]. With one end of the gp4 attached to the cell wall and the other to the virus, the virus is able to pull itself close enough to the target cell membrane allowing it to fuse with the cell in a manner in which the inner part of the virion, the viral core and its associated RNA as well as the pre-integration complex enter the cells cytoplasm [13 14]. It was concluded after a study in Uganda [15] that the probability of HIV-1 heterosexual transmission is dependent on the viral load, as results showed that transmission was rare for the individuals with HIV RNA plasma levels of less than 1,500 copies per ml. This study is in agreement with the theory that a reduction in plasma viral load would decrease the chances of transmission [15]. As previously mentioned the three main principal method of transmission are via blood, sexual intercourse and mother-to-child. The risk of transmission can potentially be explained by the relative concentration of HIV in various body fluids , and studies have suggested that this high virus load can be observed in the blood during acute HIV infection or the symptomatic period [15 16]. 1.1.4 Clinical features of the HIV-1 infection    Symptoms of the HIV virus when it first enters the patient’s system can usually be observed in the first four weeks [17]. Such patients are likely to present with flu-virus-like illness known as acute retroviral syndrome (ARS, whose symptoms include: swollen lymph nodes; high and low grade fever; nonpuritic muscular erythematous rash around the trunk; oral candidias   and diarrhoea in some and headaches amongst others [ 2,18, 19, 20]. While the rash can be difficult to diagnose it is a valuable diagnostic sign in the diagnosis of HIV as its appearance can distinguish between primary HIV infection and other types of infection. The presence of a rash during diagnosis could possibly be a reflection of antigen: antibody complexes in the skin [19].The above mentioned symptoms can last up to three weeks, followed by an asymptomatic period which can last from   months to years. The HIV infection causes one’s immune system to become dysfunctional through two processes. The first process is that it causes the depletion of CD4+ T-cell causing immunodefiency, and the second is that HIV causes immune activation caused by an inflammatory response to HIV which leads to immunosuppression.   It is believed that immune activation is caused by a homeostatic response to CD4+ T cell depletion and the inflammatory response to HIV infection that occurs [21]. Many studies cited by Stenger [21] have shown that there is a correlation between the CD4+ T-cell count and the clinical manifestations   of HIV infection as well as the progression of the infection to AIDS. This CD4+ T-cell count is additionally used as a marker as to when one should begin treatment [21]. The mucosal environments such as that of the vagina, cervix and foreskin, there are specific CD4+ dendritic cells known as Langerhans cells. These specific dendritic cells can be found specifically within the superficial epithelial layers of the vagina and foreskin of men [22, 23]. According to some research, studies [22,23,24] have shown these cells to be the targets for HIV as they appear to express more chemokine receptor type-4 (CXCR4) than chemokine receptor type 5 (CCR5). HIV uses both CXCR4 and CCR5 as co-receptors in order to enter their target cells but there is a reason it is more attracted to one than the other. CCR5 has specific ligands it utilises which are RANTES, MIP-1 beta and MIP-1 alpha [25]. It was reported [25] that these ligands have the ability to suppress HIV-1 infection in vitro . Viruses that use CCR5 can be isolated in the early stage of infection. CXCR4 is an alpha chemokine receptor specific for stromal- derived -factor-1 ( SDF-1) , which is a molecule that is endowed with potent chemotactic activity for lymphocytes [26 ]. This chemotactic activity is of particular interest to HIV because the virus uses these kinds of receptor to infect CD4+ T cells. CXCR4 is readily found in the female genital tract because during the natural implantation window or hormone replacement therapy, CXCR4 is up regulated in the endometrium [26, 27]. 1.2   Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   Vaccines HIV prevention is now regarded as an umbrella term encompassing structural, behavioural and biomedical preventative strategies. These strategies include vaginal microbicides, oral pre-exposure prophylaxis, and implementation of male circumcision, highly active antiretroviral therapy, male and female condoms, and vaccine development [28]. The most common method of HIV prevention is that of condom use, which has been demonstrated to be efficacious by both vitro and epidemiological studies in preventing the passage of the virus [29, 8, 30]. The use of a diaphragm in woman is also useful because transmission occurs mainly by the virus entering through the cervical os into the uterus [31]. The authors of [31] also suggested that in the last few years a lot of attention has been on male circumcision. They found that studies between African and Asian countries where the risk factors of heterosexual transmission are similar have shown   lower HIV transmission rates are associated with high er rates of male circumcision.   The same studies also showed that uncircumcised men had a two-fold increase in the risk of contracting HIV per sexual act as compared to circumcised men [31, 15].It would appear therefore, that it has become accepted that male circumcision can partially protect against HIV. In contrast, there are rare cases in females were individuals do not contract HIV despite being exposed to the virus via genital fluids of infected partners. These individuals have antibodies in their vaginal fluids   that appear to neutralize HIV and possibly block virus infection [32,33].The suggested theory is   that Antibody- dependent cell-mediated cytotoxicity (ADCC) directed against HIV-infected cells in vaginal/cervical fluids can be associated with a reduction in levels of infectious HIV is an idea this project browses past   [32].It is widely acknowledged by researchers [28]that the ultimate strategy in the eradication of HIV/AIDS would be to develop a fully effi cacious vaccine. This is still a challenge to date due to lack of knowledge and understanding about the process by which HIV-1 evades antibody-mediated neutralisation [28]. The aim of any vaccine is to reduce and prevent infection in a given population [34]. As it stands, the biggest issue currently in the HIV vaccine world is preventing HIV infection, especially in resource limited countries. Currently, most HIV vaccine approaches are directed at inducing adaptive immune responses, including neutralizing antibodies and antigen-specific cellular immune responses. At the same time there is an appreciation for the need of an effective innate immune response [35, 36]. The need to evoke an innate response was triggered by a study   in rhesus monkeys that were intravaginaly   inoculated with Simian Immunodeficiency Virus (SIV) leading to a small focus of virus infection. It was concluded that if the virus replication at that site is suppressed, then the   infection cannot be established as the early reaction of the innate cells is able to kill incoming virus infected cells at mucosal surfaces or within lymph nodes-or better yet suppress the virus preve nting it from   spreading [36, 37]. One of the biggest problems when it comes to vaccine development is finding   an appropriate animal model of HIV infection. Generally the immunogenicity of a potential vaccine is first tested on animals like rabbits or mice where the immunogenicity is evaluated. If the results are desirable human trials of the vaccine will begin. Microbicides currently have the spotlight in the vaccine world as they have the potential to become a good prevention for the disease. These are products; namely gels, that can be applied prior to sexual intercourse to prevent the transmission of HIV. Microbicides also known as Rheologically structured vehicle (RSV) gels were designed to be a delivery system for vaginal mucosal vaccinations [28]. Recently a double-blind, randomized control trial was conducted comparing tenofovir gel with a placebo gel in sexually active HIV-negative women in South Africa. The trial was to test the effectiveness and safety of the tenofovir gel, which is an Antiretroviral microbicide designed for the prevention of HIV infection in woman[38]. At the end of the study the tenofovir gel reduced HIV acquisition by up to 39%. There was however no changes in viral load and no tenofovir resistance in HIV sero-converters were detected. Therefore tenofovir gel  Ã‚  Ã‚   could potentially fill an important HIV prevention gap [38].    1.2.1   Ã‚  Ã‚  Ã‚   vaccine trials Along with microbicides, today’s vaccine world there are several vaccines that are at the phase one and two stages. Many large trials have been conducted in Thailand, where several microbicide and intramuscular vaccines have reached phases one and two with some getting the go ahead for efficacy evaluation. The HIV epidemic in Thailand began in the 80’s; with the introduction of HIV-1 subtype B among injectable substance users followed by sexually transmitted subtype E [39]. Thailand’s first national plan for a vaccine development programme began in 1993 and since then they have been successful in controlling the heterosexual spread of HIV. The recombinant rgp120 vaccine was selected for evaluation on the basis of safety and immunogenicity profiles in humans. A phase 1 2 trial of a monovalent subtype B rgp120 vaccine among intravenous injection drug users in Bangkok was successfully conducted in 1995-1996, and was followed by a similar trial of a bivalent subtype B/E rgp120 vaccine in 1998   [39]. These trials were an important milestone as they proved that rgp120 was safe and immunogenic. In 2004 a similar phase 1/2 safety and Immunogenicity of an HIV subtype B and E Prime-Boost Vaccine Combination in HIV-negative Thai adults was conducted using the candidate vaccines ALVAC-HIV (vCP1521) and AIDSVAX B/E.   These two vaccines were developed based on the gp120 from macrophage tropic (r5) strains of CRF01_AE, in combination with antigens from subtype B HIV-1 [40]. The results of this trial showed that this vaccine combination of ALVAC-HIV (vCP1521) and either dose of AIDSVAX B/E were well tolerated and immunogenic. A dose response between AIDS-VAX B/E and antibody response was observed.   The trial also demonstrated that the vaccines developed neutralizing antibodies to Thai E HIV and/or CD8 CTL responses to ALVAC-expressed HIV antigens. This trial reached milestones that allowed for advancement to phase 3 [40]. There was a phase 3 clinical trial of an HIV vaccine (VAX004) which was conducted in the US using a candidate HIV-1 gp120 vaccine, AIDSVAX B/B (VaxGen).The h ypothesis from the US trial was that the antibodies directed against the envelope would bind ,neutralize and clear HIV particles before an infection could be established. The trial did not work as expected due to the lack of efficacy from VAX004 [41]. Subsequent vaccine trials have focused on eliciting cell responses. This is due to the presence of HIV-1-specific CD8+ T cells being correlated with the resolution of peak viremia during acute infection. The same trial found evidence of vaccine-specific CD8+ T cells in volunteers who received the vaccine regardless of behavioural risks. The CD8+ response was seen to be significantly high in participants who went on to contract HIV. This suggested that the AIDSVAX immunization may boost pre-existing immune responses-due to pre-infection exposure [41]. The future of HIV treatment is seeing a number of new experimental HIV drugs called Entry Inhibitors being designed to interfere with the interaction between CCR5 and HIV. One problem with this is that CCR5 is not the only co-receptor that can be used. 1.2.2   Vaccines: what is desirable    An effective immune response against HIV-1 would be one that is able to control and suppress viraemia during primary and chronic HIV infections. Simultaneously, you need something that would provide long lasting protection [42]. In the world of vaccines today, ideally   it is essential for an HIV-1 vaccine to be able to elicit broadly cross-reactive neutralising antibody (Nab) responses against highly neutralization- sensitive strains of HIV-1 [43]. The antibodies will be required to be neutralizing ones as it has been recognised that neutralizing ones have the ability to employ multiple mechanisms that are able to interfere with viral replication [12]. As previously mentioned receptor binding and fusion is mediated by the envelope proteins and this happens to be an essential step in the life cycle and establishment of infection. This therefore makes it a fantastic target for neutralizing antibodies to have their effect by interfering with the fusion process and/or by neutralising free virions [12]. Given that most new infections of today are established in women as suggested before, it is within reason that there is a greater need to pursue and develop female-controlled preventative strategies. These will principally involve the cervix and vagina as the predominant mucosal portal of entry in heterosexual transmission, with the aim of eliciting sterilising immunity [28]. A multi-gene vaccine appears to be the best type of vaccine as it has been proven to have the potential to elicit broad, effective responses in animal models [42]. Understandably   this kind of vaginal vaccine would need to be safe, cheap, easy to use, store and most importantly be able to induce long-lasting; high-titre protective mucosal and systemic response to diverse viral isolates through repeated and/or sustained female-controlled administration [28]. The last decade has shown that antiretroviral drugs considerably extend the lives of individuals infected with a virus, but a better solution for the epidemic would be the development of an effective and safe vaccine. So far in research, because the host immune system has not shown effective viral clearance of HIV, there is no model of protection that can be a definite emulate of a vaccine. The genomic diversity of the virus poses many barriers in the development of a good vaccine. It is therefore concluded that a good vaccine is one that would remove the virus before it can be established. Recent studies have shown that B-cells can be stimulated to generate high titres of broadly cross-reactive neutralising antibodies against multiple genetic subtypes of the HIV virus [44].   Recent evidence [44] has suggested that some of these antibodies are directed against epitopes in the CD4 binding site on monomeric gp120, compared to many others that are directed against often neutralising epitopes. An increase in the knowledge of the molecular and antigenic structure of gp120 and gp41 HIV-1envelope glycoproteins (Env) has given new insights for vaccine design. However it has been difficult to translate this information to an immunogen that elicits broadly neutralizing antibodies [44, 45]. Based on the findings and evidence identified above, this project will attempt to look at vaccine response using a clade-C recombinant trimeric envelope glycoprotein CN54 gp140 as a part of the immunization response to elicit a humoral immune response.   A broadly neutralising response is the desired outcome being attempted in this piece of work because the project ultimately aims to make the neutralising regions of the antigen more accessible. This is going to be achieved by immunization of rabbits to see if it elicits modified antibody responses on the envelope protein which will be fixed in different confirmations. If this is found to be true then a different immune response should be induced.    2.   Materials and Methods 2.1 Immunogen The recombinant trimeric envelope glycoprotein CN54gp140 was supplied by S.Jeffs (Imperial College, London).   Intramuscular immunizations of CN54 gp140 was encoded by the CN54gp140REKR HIV-1 envelope gene cassette, derived from the clade-C/B’ HIV-1 molecular clone p97CN54 of Chinese origin developed by Wolf and Wagner, University of Regensburg, Germany. 2.2   Adjuvants An adjuvant LASTS was added to the immunization to improve the immune response. The particular LASTS formulation used is an emulsion of MPLA which is monophosphoryl lipid A. DS003 a small molecule known as BMS 599793 was added to two of the group immunogen formulas. It is an HIV entry inhibitor drug. 2.3 Rabbit immunization protocol/ In Vivo procedure- 24 Rabbits split into six groups were obtained and kept at St George’s University of London, London. All the procedures were performed in accordance with the Home office standards under the Animals Scientific Procedures Act, 1986, and approved by the Schools Ethical Review Committee. Each group of rabbits was given four intramuscular immunizations at four week intervals containing 50 µg of CN54gp140 in different formulations. A total volume of 1100 µl was for each rabbit, 800 µl was used per rabbit immunization. The formulations per rabbit and groups are as follows: Group 1: 742.9  µl Wild type CN54 + 945 µl LASTS+ 4612.1  µl PBS 2.4   Ã‚  Ã‚  Ã‚   Immunization groups A table showing the immunization groups that the rabbits belong to. 2.5   Ã‚  Ã‚  Ã‚   Immunization and bleed schedule A table showing the dates when Immunizations and bleeds took place. Blood samples for serological analysis were taken before and after treatment according to the schedule above. Due to the death of several of the rabbits from undetermined causes, the final bleed and cull originally scheduled for 9th and 10th November 2010 was brought forward by four weeks to reduce the time frame for losing anymore rabbits. Blood samples were left to clot at room temperature for at least two hours. Blood samples were centrifuged at 4500rmp for 30 minutes and the sera was collected and re-centrifuged at 4500rmp for 10 minutes to remove any remaining red blood cells. The sera were recovered and stored at -80 degrees Celsius until needed.    2.6   Ã‚  Ã‚  Ã‚   Reagents The following reagents where used for the detection of IgG by ELISA: Phosphate buffered saline (PBS, 10X, BDH); Tween-20 (FISHER, Cat. No. BPE 337-500); Heat inactivated foetal bovine serum (FBS) (GIBCO, Cat. No. 10108-165); GMP HIV-1gp140 (POLYMUN); Mouse monoclonal anti-rabbit IgG  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   (Ï’ chain – specific) HRP conjugate (SIGMA A1949); Sureblue TMB 1-Component Peroxidase Substrate (KPL, 52-00-02); TMB Stop Solution (KPL, Cat. No. , 50-85-06); Standard Rabbit antiserum to HIV-1 GB8 gp120 (NIBSC, Cat No ADP440. 1/R336); Positive control- Rabbit antiserum to HIV-1 GB8 gp120 (NIBSC Cat No: ADP440. 1/R546); Negative control- Normal rabbit serum (SIGMA, R9133) 2.6.1   Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   Reagent preparation 1.  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   Coating buffer, sterile   PBS pH 7.4 1.1  Ã‚   Prepare coating buffer by adding 50ml 10X PBS to 450ml de-ionised water. 2.  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   Washing buffer, 0.1% TWEEN-20 in 1X PBS (PBST) 2.1  Ã‚   Prepare washing buffer by adding 100ml 10X PBS to 900ml of deionised water. Add 500 µ of TWEEN-20 and mixing thoroughly 3.  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   Assay buffer, 10% FBS in PBST 3.1  Ã‚   Prepare assay buffer by adding 10ml FBS to 90ml PBST 3.2  Ã‚   Filter sterilise    2.7   Ã‚  Ã‚  Ã‚   ELISA for the detection/quantification of HIV-1 gp140 IgG An Indirect ELISA was decided as the best method to detect and quantify gp140 IgG in rabbit samples because; it as a specific assay and serum antibodies to HIV can be detected by this specific type of assay within six weeks of infection; in addition in this assay recombinant envelope and core proteins of HIV are absorbed as solid phase antigens to the wells. 96-Well plates (Greiner Bio-One medium binding) were coated with 50 µl/well of HIV-1 gp140 at 5 µg/ml in Phosphate Buffered Saline (PBS) for an hour at 37 °C. The wells were washed (wash procedure was 4 washes in PBST) and blocked for one hour at 37 °C with PBST with 10% sterile Foetal Bovine serum (PBST-serum). Standards, samples and controls were diluted in PBST-serum and incubated for 1 hour at 37 °C. The wells were washed and bound antibody was detected using monoclonal anti-rabbit IgG (gamma-chain specific) Horseradish Peroxidase (HRP) conjugate (SigmaA1949) diluted 1:10 000 in PBST-serum and incubated for 1 hour a t 37 degrees Celsius. After washing, the wells were incubated with 50 µl TMB (Sureblue TMB 1-component peroxidase substrate (KPL) for five minutes in the dark. The reaction was terminated by the addition of 50 µl of TMB stop solution (KPL) after five minutes incubation in the dark. The corrected mean of the quadruple absorbance (A450) measurements of each sample was obtained and compared with those of the negative controls on a microplate ELISA reader. A450 level is used because it produces the optimal results. For the quantification of HIV-1 gp140 IgG, the first ELISA was used to screen all the serum samples. The endpoint titres of samples were obtained only when the absorbance measured at a wavelength   of 450nm (OD450)   was 0.2 or greater for samples diluted 1 in 100.Serial dilution of the samples were prepared in triplicates, and the reciprocal endpoint titres were calculated using SoftMax Pro GxP v5 software.    2.8   Ã‚  Ã‚  Ã‚   Zeta Potential Measurements of gp140 constructs The Zeta-potential, (ÃŽ ¶) of the gp140 molecules used for immunization was determined under a range of pH and salinity conditions with a Malvern ZetaSizer Nano ZS. 188  µl of the following gp140 constructs; WT, pH 4.0, pH 5.5 and pH 7.2 where all mixed individually into 10ml of 1X PBS. The sample was placed onto the zetasizer and a new cell inserted. Two titrants where also added in order to provide an acid and a base that could be added to the sample during titration in order to aid in the change of pH as the sample was titrated. The titrants were 1M HCl and 1M NaOH. The machine measures the zeta potential of the sample as the pH changes from 3 to 9, using every 0.5 interval as a target pH. The measurement for each sample was done in triplicates and each sample was measured three times. This protocol was repeated using deionised water and 154mM NaCl as the solution.    2.9   Ã‚  Ã‚  Ã‚   Statistics Data analysis was performed using GraphPad Prism; version 4.00 (GraphPad Software).One-way analysis of variance (one-way Anova) is a technic that is used to compare the means of numerical data. It requires a minimum of two samples to work. In this experiment, the one-way Anova analysis used Tukeys multiple comparison test to compare groups and immunizations, and data was considered statistically different if the p-value was less than 0.05. Furthermore, replicate data was assumed to be Gaussian distributed.    1.  Ã‚  Ã‚   Results 3.1   Ã‚  Ã‚  Ã‚   Zeta Potential Measurements The Zeta potential of the gp140 molecules used for immunization was measured in triplicates under a range of pH and salinity conditions. The conditions were- 154mM NaCL, Deionised water and PBS. The averages of the zeta potentials where calculated and plotted against the average pH reached (Figures 3 and 4). In PBS all the proteins show a strong correlation in response to change of pH. Statistically all the protein has a p-value that is less than 0.0001. The linear regression analysis shows that while the native protein has a slightly greater slope than the fixed protein there difference is not great. 1-way Anova analysis of the PBS data specifically at pH 7.5, where the graph indicates that the could be a difference, showed that there is no significant difference in the proteins. This test used Tukeys multiple comparison test, and a significant difference is considered to be when there is a p-value of less than 0.05. In 154mM Sodium Chloride (NaCl) all the proteins have a p-value less than 0.0001in a correlation analysis, which again shows a strong correlation as a result of changing pH. A linear regression shows that the native protein has a significantly greater slope than the rest of the fixed proteins whose slopes have similar gradients. 1 way Anova analysis of the proteins in 154mM NaCl reveal that there is a significant difference with a p-value less than0.05 between the native protein and that fixed at pH 4.0. There is also a significant difference between the native protein and that fixed at pH 7.2. 1-way Anova analysis at a specific pH of 6.0 revealed several significant differences between protein groups. There significant differences noted were between: the proteins between fixed and 4.0 and 5.5; the protein fixed at pH 4.0 and 7.2; the native protein showed a significant difference when compared to all three proteins fixed at 4.0, 5.5 and 7.2. In deionised water a strong correlation can again be observed in all proteins with a p-value of less than 0.0001 observed. Linear regression analysis reveals an extremely steep slope for the native protein compared to the other three fixed proteins. In addition native CN54 has the highest zeta potential measured in all the cumulative zeta potentials measures for each protein in any condition 1-way Anova analysis reveals that there is a significant difference between the native protein and that fixed at pH 5.5 and pH 7.2. 1-way Anova at the specific pH of 6.0 were it that from the graph the could be significant difference between the different proteins shows that there is a significant difference between all the groups when compared to each other. However there is no difference between the protein fixed at pH5.5 and one fixed at 7.2. Looking at native CN54 in all three conditions, a steepest slope can be observed in the water condition followed by the 154mM NaCl and then PBS respectively. It is also noted that the native CN54 has a strong correlation in all three conditions with a p-value of   less than 0.0001.1-way Anova analysis reveals a significant difference between the native protein in PBS and water, and also a significant difference between the native protein in water and 154mM NaCl. There is no difference between the protein in PBS and 154mM NaCl. The protein fixed at pH 4.0 has the steepest slope (not as steep as that of the native protein) in water   followed by PBS and then 154mM NaCl.1-way Anova analysis reveals a significant difference between the protein in PBS and in water , and a difference when in water compared to in NaCl. There is no difference between when the protein is in PBS and when it’s in NaCl. When fixed at pH 5.5 the protein still has a strong correlation when measure in all three conditions with a p-value of less than0.0001. Once again the steepest slope can be observed in the water condition followed by NaCl and then PBS. The 1-way Anova analysis shows that no significant difference is detectable for the protein at pH 5.5 in all three conditions. The protein when fixed at pH 7.2 shows strong correlation with a p-value of less than 0.0001. Regression analysis shows that the change is steepest in water followed by PBS and then NaCl. The 1way Anova analysis shows that there is a significant difference with a p-value less than 0.05 between the three conditions. There is a difference between the proteins when measure in PBS compared to in water. There is also a difference between the water and NaCl. There is however no difference between the PBS and NaCl. It has been observed that adding an adjuvant to an immunization would improve the immune response.   One potent adjuvant is the molecule Monophosphoryl Lipid A (MPLA) which is a component of bacterial cell walls, and has been used extensively in previous immunization studies because of the activation of dendritic cells through TLR4. There is also an inflammatory response, potentated through CD14 binding, which is dangerous to induce in the context of HIV, due to the increased susceptibility of infection. Modifications have therefore been made to the molecule to remove this effect, but still retain the adjuvanting properties, with the resulting formulation that is known as LASTS. This emulsion was added to each immunization. DSOO3 an entry inhibitor drug was added to a couple of the immunizations. The immunizations were administered over 12 weeks, and the end point titre of the HIV-1 gp140 IgG were tested in serum over time. No side effects were observed in the rabbits as a result o f the immunization regime. However, during the immunisation schedules and different time points, a total of seven rabbits died randomly and the cause of death was inconclusive and unrelated to the immunizations. All of the rabbits had a strong immune response against both the native antigen and the fixed antigen. The strong response in all the rabbits reached its peak at the second immunization and plateaued thereafter showing no significant increase in immune response between the second and final (fourth) immunization. Groups four and five are the two groups that seem to have yielded some of the highest end point titres when titrated against both the native antigen and he fixed antigen. Overall group three rabbits which were immunised with the native antigen combined with the envelope stabilizing HIV entry inhibitor drug DS003 produced the highest titre when titrated against the native antigen. It is also potentially important to note that this group was comprised of only three rabbits and two of them died after the second immunization, making any conclusions which can be drawn weaker than those of higher numbered groups. Group six which also had the HIV entry inhibitor drug DS003 combined with antigen fixed at pH 4.0 did not show a similar response. The peak immune response (129627.2) when measure against both the native and fixed antigen was reached after the second immunisation but it was not high as that observed in group 3 (338988.3). After the second immunisation in group three a further slight increase is observed (338988.3 to 712687.5) where as in group 6 a plateau is observed, with a possible slight decline (129627.2 to 57698.9) 3.2.4    Grouped analysis of end point Titres The immune responses for the rabbits all appeared to plateau without much increase after the second immunization. Figures seven and eight show the grouped end point titres after the second immunization, while figures nine and ten look at the grouped endpoint titres at the final bleed after the fourth and final immunization. The graphs show that there is a significant increase in the concentration of IgG from the second immunization compared to the final bleed. One-way Anova analysis of the second immunization titrations with the native and fixed antigen revealed that there was no significant difference between the groups. P values of the one-way analysis of variance were 0.3565 for the native and none was measured for the fixed antigen. The most important results to consider are those at the final bleed between the native and fixed antigen. Group one and group three were only titre against the native antigen because they were not immunized with a fixed one. From figure 9 it is evident that these two groups produced high titres than those rabbits that were immunized with a fixed antigen. That is with the exception of group 5 whose antigen was fixed at pH 4.0. This group produced high end point titre results when the serum was measure against both the native and fixed antigen. One way Anova analysis at the final bleed for both graphs in figure 10 against the native and fixed antigen revealed no significant difference in the groups. 4. Discussion 4.1   Results overview This project aimed to characterise the physical and immunological properties of CN54 gp140 trimer and the effects that chemical fixation under different conditions confers. This study assesses the ability of intramuscular immunisation of rabbits with the vaccine trimer gp140 fixed in three different conformations and in combination with DS003 to elicit a modified antibody response, measured by the immunogen specific and native CN54 trimer specific end point serum titre. Results show that the immunizations induced high serum CN54 gp140 specific IgG responses. Fixing the protein did not increase the humoral response above that observed with the wild type protein. The protein fixed at pH 5.5 was the only group to have an increased humoral response, but this did not reach statistical significance. Otherwise overall, the wild type protein induced a high immune response. The addition of DS003 to group 6 whose protein was fixed at pH 4.0 did not produce an increased response. However, addition of DS003 to group 3 whose protein is not fixed induced the highest immune response. The differences that can be detected between groups are limited due to the deaths of several of the rabbits from causes unrelated to the immunisation regime. 4.2   Fixed and unfixed protein 4.2.1   Zeta potential The physical properties of the proteins were assessed by the changes in their zeta potential. Whether the protein is fixed or not has a profound effect on resulting surface chemistry. The paraformaldehyde will reduce the flexibility and crosslink the protein in the conformation that it exist in, reducing the variability in its higher order structure, and keeping it more similar to the conditions under which it was fixed. Comparing the zeta potential in NaCl and water, the effect of fixation is it dampening the change in zeta potential while the samples where titrated. This is especially interesting because when the protein is titrated in PBS it is in a buffered system meaning there is a lot of different ions present causing the zeta potential to not be not well pronounced. The phosphate ions can act as a shielding factor, masking the alterations that fixation has induced. In NaCl however, you have a more fundamental system and other compounding factors that would be otherwise be present in the PBS have removed stripped and only sodium and chloride ions present.   In water there are no other ions present providing a highly pure environment for the zeta potential measurements. As a result when the zeta potential of the native protein was measured in water and NaCl it is observed that there is a definite change in zeta potential as a result of pH. In all cases, the zeta potential becomes more negative as the environment becomes more alkaline. This fits with the theory that the zeta potential of proteins is made up of titrating the functional groups. At low pH conditions, there is an abundance of hydrogen ions which will confer a positive charge on protein in solution. The opposite is true when the system becomes basic and the system is dominated by hydroxide ions. The difference in how the proteins react differently to the changing conditions is represented by the steepness of the native protein slope on the graphs. This zeta potential change cannot be accounted for in the PBS buffered system. While there is a change it is not a pronounced one and the native protein behaves the same as the fixed ones. A change in zeta potential as a result of change in pH is also present in the fixed protein in the water and NaCl system, but the changes are not pronounced.   The fixed proteins do not show as much of a change in the PBS system just like the native protein but it is concluded th at this is due to the buffered environment that is provided by the PBS. In water and NaCl there is much more of a change but not to the same level as the native protein. This lack of change can likely be explained by the process of fixation of the protein. Because these proteins are so strongly correlated to changes in pH it is therefore hypothesized that fixation in those pH conditions would preserve the changes. Fixation of the protein is accomplished using paraformaldehyde. As a result the formaldehyde reduces the protein’s flexibility and crosslinks the protein in the conformation that it exists in. This means that the protein will have reduced variability in its higher order structure. 4.2.2   Ã‚  Ã‚  Ã‚   End point titres Figures 5 to 8 show that all of the rabbits had a strong HIV-1 gp140-specific IgG immune response against both the native and fixed antigen. This response appears to plateau after the second immunization and holds steady up to the final immunization. This means that after the second immunization subsequent immunizations are not inducing any more of an immune stimulation -they are just boosting what is already there. One could ask what is the relevance of this result in terms of vaccine development and if you only need to administer two vaccines. The plateau does not necessarily mean that only two immunizations are required, and this experiment cannot fully answer that. To determine the complete answer, a study would need to be conducted where only two immunizations are administered and the subjects are monitored over time without giving any more immunizations to see if the immune response lasts and for how long. This would be important because ultimately for a vaccine to be considere d good you would want it to produce a response that is protective and lasts. You would also aim for a vaccine that gives you the biggest response using as little immunogen as possible.    4.3   Addition of DS003 DS003 is a small molecule also known as BMS 599793 and it was added to   the immunogens of group 3 and group 6-with group 3 being a being made up of the wild type protein and the group 6 protein are fixed at pH 4.0. DS003 is an HIV entry inhibitor drug that blocks entry of the HIV virus by interacting with gp120. Currently it is being developed as a mirobicide which is proving to be very potent [46].The interest in using it in this study though is due to its reported effects on decreasing the flexibility of gp140 molecules. Stabilization of the protein through fixation may be additive to the effect of DS003. As mentioned above, in order for infection to occur the viral envelope protein must bind to the CD4 receptor of the target cell. This binding occurs by the gp120 glycoprotein. A compound like DS003 would abrogate this process and encouragingly, it has been shown to prevent infection in vitro. Earlier studies have shown that DS003 was chosen specifically because it can bind to CN54 and gp140 trimers as well as gp120 monomer-and all these three envelope constructs bind soluble CD4. This is good because DS003 binds to the CD4 binding site on gp140.The benefit of such entry inhibitors is that they act early in the early stages of the virus cycle before infection can be established [46, 47]. Figures 7 and 8 show serum titration results for rabbits that were immunized with an immunogen that was combined with DS003. Both groups had a strong immune response to the immunogen reaching a peak after the second immunization and eventually plateauing. Group 6 however did not produce titres as high as group 3. The maximum titre produced in group 6 measured   after the second immunization was 129627.2 and subsequently decreased by the final immunization, whereas the highest titre measured in group 3 was observed after the final immunization and was measured at 712687.5. This difference could likely be as a direct effect of the protein being fixed in group 6. This could mean that DS003 is not as potent when combined with a fixed protein. It shows that fixing the protein reduces the magnitude of effect that is stimulated in the immune system. This is supported by the fact that when combined with the native protein DS003 is able to exert its effects to the maximum. Seeing as the add ition of DS003 to a wild type protein produced such high titre results, it could mean that the titre results of group 1 which were immunized with wild type protein alone could be potentially higher if DS003 were to be added, as seen in group 3. With that in mind group 1 produced higher titres especially after the second immunization and the final time point than some of the rabbits that were fixed at pH 4.0 and 7.2. These differences were small though, and not statistically significant. Overall looking at figures 9 and 10 groups 1, 3 and 5 produced the highest immune responses that were measured by titration. Group one only has the native protein; group three is a combination of the native protein with DS003 and group 5 is the only one with a fixed protein at pH 5.5. This could be interpreted to mean that with respect to the results from groups 1 and 3, the best immune response is produced when a native protein is used as an immunogen. DS003 increases the immune response best when it is in combination with a wild type protein rather than a fixed one as illustrated in figures 7 and 8. This could be due to fixation removing the binding site for DS003, and therefore leaving no opportunity for it to work. Group five shows that if an immunogen is going to be made from a fixed protein the best pH is 5.5. An experiment that could be conducted is combining DS003 with a protein fixed at pH 5.5 to see if it would have an impact on the immune response produced. This was not po ssible in the current experiment due to a limitation in the number of animals available.   4.4   Native and wild type protein. An imperative factor in the race to design inhibitors and vaccines for HIV is to gain a good understanding of the different conformational states available to the HIV-1 envelope glycoproteins [11].The CN54gp140 immunogen that was used in the rabbit immunizations was successful in eliciting a strong, specific humoral antibody response. The immunogens used in these kinds of experiments are usually protein based ones adapted to mimic HIV envelope proteins on a whole virion. Using protein based vaccines can be difficult because the proteins have a complex structure and are usually fragile [28]. CN54gp140 manufactured under GMP conditions is very comparable to the wild type protein, and is used in this study because it has been shown to be exceptionally stable in buffered solutions. This protein was manufactured to mimic the actions of the native protein gp120   and gp41 molecules [28]. Because the wild type protein was so stable it was possible to fix the material easily in different c onformations that were able to elicit a good immune response. Gp120, as previously described,   works in conjunction with gp41 to allow the virus to get close enough to the cell membrane and inject its genome into the target cell cytoplasm. The difference between the native gp140 and wild type infectious protein is that the gp140 is not near the target cell membrane. There is also a mutation on the cleavage site that would normally result in a gp41 and gp120 molecule being created instead of a single gp140-but the same external face and glycosylation patterns on both types of proteins will be the same. 4.5   Conclusion This study was successful in revealing whether vaccine response using the trimer CN54gp140 fixed in different conformations would elicit a modified antibody response. A decreased titre of antibody concentration was observed when the protein was fixed at pH 4.0 and 7.2 and there was only an increase in serum antibody in the protein fixed at pH 5.5. However the best response was seen in the immunizations with the wild type protein, especially in the group three where the protein was combined with the HIV inhibitory drug DS003. 5  Ã‚   References 1.  Ã‚  Ã‚   UNAIDS. (2010).  Worldwide HIV and AIDS statistics.  Available: avert.org/worldstats.htm. 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Overview of the Sans-culottes

Overview of the Sans-culottes The Sans-culottes were urban workers, artisans, minor landholders, and associated Parisians who took part in mass public displays during the French Revolution. They were frequently more radical than the deputies who formed the National Assembly, and their often violent demonstrations and attacks threatened and cajoled revolutionary leaders down new paths at key moments. They were named after an article of clothing ​and the fact that they didn’t wear it. Origins of the Sans-culottes In 1789, a financial crisis caused the king to call a gathering of the ‘three estates’ which led to a revolution, the declaration of a new government, and a sweeping away of the old order. But the French Revolution wasn’t simply the rich and the noble versus a unified body of middle and lower class citizens. The revolution was driven by factions across all levels and classes. One group who formed and played a massive role in the revolution, at times directing it, were the Sans-culottes. These were lower-middle-class people, craftsmen and apprentices, shopkeepers, clerks, and associated workers, who were often led by the true middle class. They were the strongest and most important group in Paris, but they appeared in provincial cities too. The French Revolution saw a remarkable amount of political education and street agitation, and this group was aware, active and willing to commit violence. In short, they were a powerful and often overwhelming street army. Meaning of the Term Sans-culottes So why ‘Sans-culottes?’ The name literally means ‘without culottes’, a culotte being a form of knee-high clothing that only the wealthier members of French society wore. By identifying themselves as ‘without culottes’ they were stressing their differences from the upper classes of French society. Together with the Bonnet Rouge and the triple colored cockade, the power of the Sans-culottes was such that this became a quasi-uniform of revolution. Wearing culottes could get you into trouble if you ran into the wrong people during the revolution; as a result, even upper-class French people sported the sans-culottes clothing to avoid potential confrontations. What Role Did the Sans-culottes Play in the French Revolution? Over the early years the Sans-culottes program, loose as it was, demanded price fixing, jobs, and crucially provided support for the implementation of the Terror (the revolutionary tribunal that condemned thousands of aristocrats to death). While the Sans-culottes agenda was originally focused on justice and equality, they quickly became pawns in the hands of experienced politicians. In the long run, the Sans-culottes became a force for violence and terror;Â  the people at the top were only ever loosely in charge. End of the Sans-culottes Robespierre, one of the leaders of the revolution, attempted to guide and control the Parisian Sans-culottes. Leaders, however, found that it was impossible to unify and direct the Parisian masses. In the long run, Robespierre being arrested and guillotined, and the Terror stopped. What they had instituted began to destroy them, and from them on the National Guard were able to defeat the Sans-culottes in contests of will and force. By the end of 1795 the Sans-culottes were broken and gone, and it is perhaps no accident France was able to bring in a form of government which managed change with far less brutality.

Reading commentary assignment Essay Example | Topics and Well Written Essays - 750 words

Reading commentary assignment - Essay Example At another level, the authors treatment of the contemporary issues of labor by conventional philosophy, and the rise of military-industrial intricate offer new insights by being clearly related to the periods of old capitalist vibrancy. On a closer analysis, it is particularly important to examine how Marx’s critiques had been anticipated in diverse ways by some of the founding fathers of economics, who were in the preference of a capitalist approach to economics; however, a critical and contemporary analysis for â€Å"A Critical Perspective† is not adequately critical especially when it comes to analyzing his arguments on Marxism and socialism, though their approach to â€Å"Economic Thought† is still appreciable given the fact that their analysis are based on range of disciplines related to Economics. (Hunt and Lautzenheise) In chapter nine, Hunt and Lautzenheiser looks critically into the contemporary subject of Marxism economics and how his arguments sought to define the modern economic viewpoints. He adopts a theoretical view point that advocates for a socialist system and differs with the capitalists on wide range of economic principles. These classical approaches to viewing economic institutions have been largely considered as pre-templates for industrial revolution. Though Marx by nature was a product of the Western economics, his contributions that combined classical German Economics and scientific technology brought to the fore a new force in viewing economic systems leading up to the Industrial Revolution. Hunt and Lautzenheiser re-affirms that the radical viewpoints as pioneered by Marx represent Marx’s interpretation of historical events and his due prediction of what was to be later a struggle between employees and management. At the same time, Hunt Lautzenheiser concurs that Marxism theories provided