Western History Essay Example | Topics and Well Written Essays - 750 words

Western History - Essay Example In this switch, there was a great deal of social upheaval as people living in these changing times began to question the status quo. Social class structures were beginning to break down as common men were able to make fortunes in industry and landowners found it more and more difficult to keep the idyllic life they’d constructed alive. Women gained greater freedoms as more job opportunities came their way. Although these weren’t the equal rights position of today, women were at least given an option when it was necessary for her to earn a living wage. The great migration from the country to the city was an expression of a people ready for a chance to better their futures, an expression of hope for a greater tomorrow for their children. This period is also characterized by widespread colonization in numerous directions. Englishmen were still relocating to America seeking their fortunes, but also had the option of retaining their English citizenship by going into Africa, India or even Australia. For many, it proved to be just so, for many others, it proved to be their destruction, for men and women. The biggest benefits the Industrial Revolution introduced to society were also the biggest setbacks. Stories existing out of the middle ages are full of epic battles, travels through sparsely populated country, images of vast forests and romantic tales of knights and ladies. This is contrasted sharply against the images of Victorian Industrialization with its smokestacks, grimy streets and poisonous air. Machines began taking on more importance than people as the massive in-pouring of country citizens all seeking their fortunes provided ample replacements for those who could not keep up. Societal norms were breaking down as accountability began declining. This was because younger sons and daughters, as well as those with more disreputable reputations, found a means of escaping their sometimes

How does the TV show ( The Simpsons) effect my live Essay

How does the TV show ( The Simpsons) effect my live - Essay Example the following analysis will seek to engage the reader with an understanding of the impact that The Simpsons have had upon my own life and the manner by which I will ultimately intend on raising my own family. Essentially, The Simpsons served as a comedy program that could be enjoyed by each and every member of our family; when I was growing up. As such, my parents were not overly conservative and did not attempt to restrict the overall access the television in our home or otherwise only approved certain programs for viewing. The understanding that was unspoken, at least in our home, was the fact that when situations arose and were represented on television that did not abide by the rules of our family or the point of view that children were expected to respect, these did not need to be restricted; they merely needed to be dismissed. As such, I have distinct memories of several occasions in which my parents would laugh along with the plot of The Simpsons; turning to me and saying something to the effect of â€Å"you had best never do anything like that†. As such, even though this particular show was comedy, I was fully aware of the fact, even from an early age, that not everything represented within it should be taken exemplification from model behavior. In terms of The Simpsons and its overall impact upon how I will raise my own family, it should be noted that I will follow a very similar approach to that which my parents ascribe to during my own childhood. Rather than restricting access to certain television shows and ultimately attempting censor the media, the best approach is to enjoy these television shows with the appropriate age groups in question and merely categorized at certain times that specific behavior is not allowable and when not be tolerated. As such, the parent child relationship does not become one that is domineering; it becomes one that is beneficial and utilizes media as a way of helping to further define the roles that individuals within the

Enterprise Resource planning system

Enterprise Resource planning system In the business world organization, business software plays a very important role to support the business and its activities to increase the productivity and efficiency of the business. Although business software give benefits but it also brings major implication for a company when the firm decide to implement different type of business software. This paper will discuss the role and purpose of Enterprise Resource Planning (ERP) system and Accounting Information System (AIS). Moreover the paper will discuss the benefits and drawbacks for companies of implementing ERP system. Specifically the paper will discuss how AIS system maybe be affected by implementation of a full ERP system and the identification of major implication for AIS system. Role and Purpose of Enterprise Resource planning system Enterprise Resource Planning Systems (ERP) is specifically designed for distributors, retailers and professional services firms according for their needs. The company named SAP Aktiengesellschaft is currently a world market and technology leader in providing ERP systems (Benjamin B. Bae, Paul Ashcroft, 2004). ERP system implemented to provide more accurate information and safe time, reduce asset costs and financial cycles, increase customer satisfaction, and globally integrate information across the enterprise supply chain. ERP system use relational database technology to integrate various units of an organizations information system which integrated all business process and sub-processes linked and unified into a single system. It has been designed to focus on four main areas in a company that is financial, human resources, marketing and supply chain management. ERP system is essential to the needed by all organizations and can use in any kind of organization, irrespective of the business objectives, size, and area of operations. Using this system not only economizes but it also improves the efficiency of the average existing work process. Without ERP system on modern day, firms cant run competitively between organizations because leak of proper drafted and formulated which ERP system can provide it. The main reason for this is because it can be configured to accommodate a large variety of different work processes and this lead it became popular not only in large corporations but midsized companies also frequently used it because it provides a seamless integration of their business. During the 1990s, Global 2000 companies spent billions of dollars on ERP systems (Dan Everett, 2003). The attractive of ERP systems seem likely to be the promise of centrally coordinating and controlling many aspect of corporate planning. As such, ERP systems can be seen to represent a far reaching technological promise and even the ultimate manifestation. Benefits and Drawbacks for Implementing ERP System The benefits for implementing ERP system is it helps organization to reduce costs and cycle time, increase the productivity and quality and improve the customer service by automating basic and repetitive operations. ERP systems automatically estimate the demand for a product, order the raw materials, provide production schedules, track down the entire inventory, allocate costs, and keep historical customer. The ERP systems repeat all the operation and keep the information so that the organization can analysts the impact of changes in product mix and volumes in order to maximize corporate profit margins. Besides that, ERP systems also provide informational benefits to management. ERP system helps an organization to achieve better resource management for example in workforce management improved the manpower allocation, in inventory management improved inventory turn and stock allocation, in production management optimized supply chain and production schedules. It also improved decision making by increase the market responsiveness, has a better profit and also controls the cost and also flexible the customer services, increase the service adjustment and response customer demand. Integrated information systems bring a new opportunity to directly support a tight link with customers (Vitale, 1986; Malone and Yates, 1987; Clemons and McFarlan, 1986). ERP systems provide E-business by using the web integration capability it helps get closer to customers. It provides benefit to business to business (B2B) interactive customer service, for example through customer directs feedback it can improved product design, and most importantly provide real time and reliable data enquiries. In addition with the integrated and standard application architecture in ERP system it provide infrastructure support business flexibility for future changes by response to internal and external changes and provide range of options in react, reduced IT costs and increased capability for quick and economic implementation of new applications (Shari Shang and Peter B. Seddon, 2000). The drawback for implementing ERP system is the cost of installing is expensive. The cost of installing an ERP system can cost up to US$100 million. In1999, most organizations spent between US$90-180 billion on ERP system (Shari Shang Peter B. Seddon, 2000). The ERP system be installed by ERP consulting organization and the fees to modify the system is also expensive with average at about US$150-$225 per hour (Benjamin B. Bae, Paul Ashcroft, 2004). Besides that, installing an ERP systems take a longer period. ERP system is a complicated system and it often takes 12-18 months to be installed and operating (Benjamin B. Bae, Paul Ashcroft, 2004). It take longer period is because a detailed study must be made and reviewed before installing ERP system to match the business requirement. Any mistake in planning of ERP systems will affect the entire performance system. The implementation of the ERP systems will hurting the companys process, break customer relations and will required a longer time from achieving its long-term financial goals (Dr. Yan Wang, 2005). In addition, implementation ERP system required a new procedures to manage the system, train employee the procedures of system, and managerial and technical support and this cause many companies found the change takeover to ERP systems a slow and painful process. Role and Purpose of Accounting Information System (AIS) Accounting Information System (AIS) play as a backbone in accounting transaction processing system. It combine the accounting format, implementation, and monitoring of information systems. It uses the modern information technology resources with the traditional accounting methods to provide financial information to organizations. AIS analyze and record all the business transactions to prepare financial statements and provide accounting data to manage the organization. Constantinos J. Stefanou state that, AIS is a process of collecting and recording the data and information based on the events that have an economic effect upon organizations and keep the maintenance, processing and communication of information to stakeholders. All the information will be use for decision making purpose and evaluate the financial position of the organization. Andreas I. Nicolaou, 2000 claim that a critical research issue in the fields of accounting and management decision-making concerns the fit of the AIS with the organizational requirements for information communication and control. This means that AIS is a computer-based system which process all the financial information and supports the decision tasks in the context of coordination and control of organizational activities. Besides that, Ed ODonnell, Julie Smith David, 2000 state that AIS provide input for decision making. AIS summarized the important business events for example production, purchasing and selling activities for internal decision making. AIS make business accounting activities easier, quicker, and more accurate. It safe accountant time and avoid mistake since the system is analyzed accounting records and prepare financial statements. Since AIS gather all accounting and transaction information by electronically and automatically so it avoids data entry and computing errors. The Major Implication for AISs of the Implementation of a Full ERP System Basically, a successful implementation of ERP system will increases competitiveness by increase the quality and customer satisfaction, speed up the processes, and reduce all the lead times. There is few reason AIS system may be affected by the implementation of a full ERP system. Steve G. Sutton 2006 claim that ERP system change the financial accounting environment significantly as the processes used to record, assimilate and distribute such information all radically change. ERP system can be traced back all the transaction recording process for example it will captures the data when a cashier scanning a bar code or warehouse worker at a receiving dock and automatically update the processes in the AIS records. Besides that, ERP system also supports two major processes in AIS that is order-to-cash and purchase-to-pay. In order-to-cash process, the ERP system can analyze to determine the goods being requested by customers. At this process, ERP system links together customer, inventory, purchasing, and vendor data to determine whether the customer is in good standing and likely to pay the bill. After that, ERP system schedules the order for delivery, if the goods are not available within the organization, a purchase order is sent to a vendor. ERP system will ensure that the invoice contains correct quantities, prices, terms, and addresses and then it will record cash receipts and updating cash and accounts receivable amounts in the general. For the purchase-to-pay process, ERP system will automatically generate the purchase requisition on the basis of data and reviews purchases requests to determine that organization is authorized and within budget. ERP system will assists the buyer in identifying sources of supply for the requested item, analyzing vendor quotations, and selecting vendors by comparing vendor prices, terms and past performance. ERP system will automatically links purchasing order, receipt and vendor invoice and record in accounts payable in the general ledger. Further, ERP systems also improved decision-making process and used financial ratio analysis. It act as facilitators of business processes and also support management decision-making. It has enabled to use financial ratio analysis and production of budgets which also including cash budgets, absorption costing and profitability analysis per customer. In addition ERP systems have introduced sophisticated accounting techniques in their accounting processes which is activity based costing (ABC) and target costing. It also lesser extent involves time reductions for accounts closure and preparation of financial statements. The last reason through the implementation of a full ERP system is it not just a business process of electronic procedures but it is also with the scientific management idea. This system can always thinking of integrating the organization internal control throughout the entire business cycle, through a set of pre-designed and improved cover the plans, prices, supply, production, sales, and inventory management and automatically update. In summary, the ERP systems appear to have fulfilled its purpose as demonstrated in the changes in the accounting practices brought in. Overall, the benefits achieved by ERP systems strongly influence accounting information and practices and also organizational planning at a strategic level. Booth et al. (2000) states that ERP users perceptions of the quality of accounting IS in financial and management accounting as adequate in terms of reporting and decision support and good in terms of transaction reporting. Therefore, results between the two systems are comparable, indicating that there are still benefits accrued for ERP adopters. Stefanou (2002) also reports that the integration of accounting applications, information exchange and reporting capabilities as notable advantages and strengths of ERP systems. Conclusion In conclusion,

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. Last accessed 16 April 2011. 2.  Ã‚  Ã‚   Fanales-Belsio.E, Raimondo.M, Suligoi.B and Butto.S. (2010). HIV virology and pathogenetic mechanisms of infection: a brief overview.HIV virus and pathogenicity,46(1): 5-14 3.  Ã‚   Pillay.S, Shephard.E, Meyers.A, Williamson.A, Rybicki.E. (2010). HIV-1 sub-type C chimaeric VLPs boost cellular immune response in mice .Journal of Immune based therapies and vaccines, 8 (7). 4.  Ã‚   Hook, E. W., III, R. O. Cannon, A.J. Nahmias, F.F. Lee, C. H. Campbell, Jr., D. Glasser, and T.C. Quinn. (1992). Herpes simplex virus infection for human immunodeficiency virus infection in heterosexuals. J. Infec. Dis. 165:251-255 5.  Ã‚  Ã‚  Ã‚  Ã‚   Gabali, A.M., J.J. Anzinger, G.T. Spear, and L.L Thomas. (2004). Activation by inflammatory stimuli increases neutrophil binding of human immunodeficiency virus type 1 and subsequent infection of lymphocytes. J. Virol. 78:10833-10836 6.  Ã‚  Ã‚  Ã‚  Ã‚   Bomsel, M., Heyman. M, Hocini. H, Lagaye. S, Belec. L, Dupont. C, and Desgranges. C.   (1998). Intracellular neutralization of HIV transcytosis across tight epithelial barriers by anti-HIV envelope protein dIgA or IgM. Immunity: 9, 277-287. 7.  Ã‚  Ã‚  Ã‚  Ã‚   Hocini.h., Becquart. P, Bouhlal. H, Chomont. N, Ancuta. P, Kazatchkine. M, and   Belec. I (2001). Active and selective transcytosis of cell-free human immunodeficiency virus through a tight polarized monolayer of human endometrial cells. J. virol: 75, 5370-5374 8.  Ã‚  Ã‚  Ã‚  Ã‚   Moses. S,   Plummer. F,   Ngugi. E, Nagelkerke. N, Anzala. A, and Ndinya-anchola. J. (1991). controlling HIV in Africa: effectiveness and cost of an intervention in a high frequency STD transmitter core group. AIDS: 5, 407-411 9.  Ã‚  Ã‚  Ã‚  Ã‚   Robinson. H. (2002). New hope for an AIDS vaccine. Immun: 2, 239-250 10.  Ã‚  Ã‚   National Institute of Allergy Infectious Diseases. (2004).  How HIV causes AIDS.  Available: niaid.nih.gov/TOPICS/HIVAIDS/UNDERSTANDING/HOWHIVCAUSESAIDS/Pages/howhiv.aspx. Last accessed 16 April 2011. 11.  Ã‚  Ã‚   Yuan.w, Bazick.J, Sodroski.J. (2006). Characterization of the multiple conformational states of free monomeric and trimeric Human Immunodeficiency Virus envelope glycoproteins after fixation by cross-linker.  Journal of virology:80 (14), 6725-6737. 12.  Ã‚  Ã‚   Huber.M and Trkola.A. (2007). Humoral Immunity to HIV-1: neutralization and beyond.  Journal of INTERNAL MEDICINE: 10 (1111), 1365-2796. 13.   Freed, E.O. (2001). HIV-1 replication. Somat Cell Mol Genet:   26(1-6), 13-33 14.  Ã‚  Ã‚   Turner, B.G. and M.F. Summers. (1999). Structural biology of HIV. J. Mol Biol: 285(1), 1-32 15.  Ã‚  Ã‚   Quinn. T, Wawer. M,   Sewankambo. N,   Serwadda. D, Li. C, Wabwire-mangen. F, Meehan. M, Lutalo. T, Gray. R. et al (2000) viral load and heterosexual transmission of human immunodeficiency virus type 1. n.engl.j.med:   342, 921-929 16.  Ã‚  Ã‚   Quinn.T. C.( 1996). Global burden of the HIV pandemic. Lancet: 348, 99-106 17.  Ã‚  Ã‚   Cooper. D, Gold. J, Mclean. P,   Donovan. B, Finlayson. R, Barnes. T, Michelmore. H,   Brooke. P, and Penny. R. (1985). acute AIDS retrovirus infection: definition of a clinical illness associated with seroconversion. lancet mi:537-540 18.  Ã‚  Ã‚   Jonassen. T,   Stene-johansen. K, Berg. E, Hungnes. O, Lindboe. C, Froland. S, and Grinde. B. (1997). sequence analysis of hiv-1 group o from Norwegian patients infected in the 1960s. virology 231:43-47 19.  Ã‚  Ã‚   Mcmillan. A, Bishop. P, Aw. D, and Peutherer. J. (1989) Immunohistology of the skin rash associated with acute HIV infection. AIDs: 3, 309-312 20.  Ã‚  Ã‚   Daar. E, Little. S, Pitt. J, Santangelo. J, Ho. P,   Harawa. N, Kerndt. P, Glorgi. J, Bai. J, Gaut. P, Richman. D, Mandel. S,   Nochols. S, and the los angeles county primary HIV infection recruitment network. (2001 ). diagnosis of primary HIV-1 infection. Ann. intern. Med: 134,25-29 21.  Ã‚  Ã‚   Stenger.M, Dr Lane. (2010). Pathogenesis of HIV infection: total CD4+T-cell pool, Immune activation, and inflammmation.  Topics in HIV medicine: 18 (1), 2-6. 22.  Ã‚  Ã‚   Soto-ramirez. L, Renjifo. E, Mclane. M, Arlink. R,   O’hara. C, Sutthent. R, Wasi. C, Vithayasai. P, Vithayasai. V,   Apichartpiyakul. C, Auewarakul. P, Pena cruz. V, Chui. D, Osanthanondah. R, Mayer. K, Lee. T, and Essex. M. (1996). HIV-1 langerhans’ cell tropism associated with hetero-sexual transmission of HIV. Science :271, 1291-1293 23.  Ã‚  Ã‚   Hussain. L, and Lehner. T. (1995). copmaritive investigation of langerhans’ cells and potential receptors for HIV in oral, genitourinary and rectal epithelia. Immunology :85, 475-484 24.  Ã‚  Ã‚   Knight. S. C. (1996). bone-marow derived dendritic cells and the pathogenesis of AIDS. AIDS: 10,807-817 25.  Ã‚  Ã‚   Anderson. J, and Akkina. R. (2007). Complete knockdown of CCR5 by lentiviral vector-expressed siRNAs and protection of transgenic macrophages against HIV-1 infection. Gene therapy: 14, 1287-1297 26.  Ã‚  Ã‚   Mines.M.A, Goodwin.J.S, Limbird.L.E, Cui.F, and Fan.G-H. (2009). Deubiqitination of CXCR4 by USP14 Is critical for both CXCL12-induced CXCR4 degradation and chemotaxis but not ERK activation. J. Biol. Chem: 284 (9),   5742-5752 27.  Ã‚  Ã‚   Moriuchi. M, Moriuchi. H, Turner. W, and Fauci. A.S. (1997). Cloning and analysis of the promoter region of CXCR4, a coreceptor for HIV-1 entry.   J.Immunol: 159 (9), 4322-4329 28.  Ã‚  Ã‚   Curran.R, Donnelly.L, Morrow.R, Fraser.C, Andrews.G, Cranage.M, Malcolm.K, Shattock.R, Woolfson.D. (2009). Vaginal delivery of the recombinant HIV-1 clade-C trimeric gp140 envelope protein CN54gp140 within novel rheologically structured vehicles elicits specific immune responsese.  Vaccine: 27 (48), 6791-6798. 29.   Conant.M,   Hardy. D, Sernattinger. J, Spicer. D, and Levy. J. (1986). condoms prevent transmission of the AIDS-associated retrovirus by oral-genital contact. JAMA: 225, 1706 30.  Ã‚  Ã‚   Ngugi. E, Plummer. F, Simonsen. J, Cameron. D, Bosire. M, Waiyaki. P, Ronald. A, and Ndinya-achola. J. (1988). prevention of transmission of human immunodeficiency virus in Africa: effectiveness of condom promotion and health education amongst prostitutes. Lancet: ii, 887-890 31.  Ã‚  Ã‚   van der Straten. A, Kang. M, Posner. S, Kamba. K, Chipato. T, and Padian. N. (2005). predictors of diaphragm use as a potential sexually transmitted disease/HIV prevention method in Zimbabwe.sex.transm.dis:32, 64-71 32.  Ã‚  Ã‚   Nag. P, Kim. J,   Sapiega. V, Landay. A, Bremer. J, Mestecky. J, Reichelderfer. P, Kovacs. A, Cohn. J, Weiser. B, and Baum. L. (2004). women with cervicovaginal antibody-dependent cell-mediated cytotoxicity have lower genital HIV-1 RNA loads.j.infect.dis: 190, 1970-1978 33.   Cameron. D, Simonsen. J. N, D’Costa. L. J, Ronald. A. R, Maitha. G. M, Gakinya. M. N, Cheang. M, Ndinya-Achola. J. O, Piot. P, Brunham. R. C, and Plummer. F. A. (1989). Female to male transmission of human immunodeficiency virus type 1: risk factors for seroconversion in men.   Lancet. ii: 403-407 34.  Ã‚  Ã‚   Levy. J. A. (2004). prospects for an AIDS vaccine: encourage innate immunity. AIDS: 18,2085-2086 35.  Ã‚  Ã‚   Levy.J. A,   Scott. I, and Mackewicz. C. (2003). protection from HIV/AIDS: the importance of innate immunity. clin.immunol: 108, 167-174 36.  Ã‚  Ã‚   Pashine. A, Valiant. N, and Ulmer. J. (2005). Targeting the innate immune response with improved vaccine adjuvants. nat.med: 11, s63-s68. 37.  Ã‚  Ã‚   Miller. C. J, Li. Q, Abel. K, Kim. E. Y, Ma. Z. M, Wietgrefe. S, Franco-Scheuch. L. La, Compton. L, Duan. L, Shore. M. D, Zupancic. M, Busch. M, Carlis. J, Wolinsky. S, and Haase. A. T. (2005). Propagation and dissemination of infection after vaginal transmission of simian immunodeficiency virus. J. Virol: 79, 9217-9227 38.  Ã‚  Ã‚   Karim. Q, Karim. S, Frohlich. J, Grobler. A, Baxter. C, Mansoor.L, Kharsany. A, Sibeko. S, Mlisana. K, Omar. Z, Gengiah. T, Maarschalk. S, Arulappan. N, Mlotshwa. M, Mprris. L, and Taylor. D. 2010. Effectiveness and safety of tenofovir Gel, an antiretroviral Microbicide, for the prevention of HIV infection in women. Science: 329 (5996), 1168-1174 39.  Ã‚  Ã‚   Pitisuttithum. P, Gilbert. P, Gurwith. M, Heyward. W, Martin. M, van Griensven. F, Hu. D, Tappero. J, and Bangkok Vaccine evaluation group. 2001. Randomized, double-blind, placebo- controlled efficacy trial of a bivalent recombinant glycoprotein 120 HIV-1 vaccine among injection users in Bangkok, Thailand. J. Infec. Dis: 194(12), 661-1671 40.  Ã‚  Ã‚   Nitayaphan. S, Pitisuttithum. P, Karnasuta. C, Eamsila. C, de Souza. M, Morgan. P, Polonis. V, Benenson. M, VanCott. T, Ratto-Kim. S, Kim. J, Thapinta. D, Garner. R, Bussatid. V, Singharaj. P, el Habib. R, Gurunathan. S, Heyward. W, Birx. D, McNeil.J, and Brown. A. (2004). Safety and Immunogenicity of an HIV subtype B and E prime-boost Vaccine combination in HIV-negative Thai adults. J. Infec. Dis: 190, 702-706 41.  Ã‚  Ã‚   Jones. N, DeCamp. A, Gilbert. P, Peterson. M, Gurwith. M, and Cao. H. 2008. AIDSVAX immunization induces HIV-specific CD8+ T-cell responses in high- risk HIV-negative volunteers who subsequently acquire HIV infection. Vac. 27: 1136-1140 42.  Ã‚  Ã‚   Pillay.S, Shephard.E, Meyers.A, Williamson.A, Rybicki.E. (2010). HIV-1 sub-type C chimaeric VLPs boost cellular immune response in mice.Journal of Immune based therapies and vaccines: 8 (7) 43.  Ã‚  Ã‚   Graham.B. S. (2009). What does the report of the USMHRP phase III study in Thailand mean for HIV and for vaccine developers.  The journal of Transitional Immunoligy: 158, 257-259. 44.  Ã‚  Ã‚   Montefiori. D and Mascola. J. (2009). Neutralizing antibodies against HIV-1: can we elicit them with vaccines and how much do we need.  Cuur Opin HIV AIDS: 4 (5), 347-351. 45.  Ã‚  Ã‚   Gnanakaran.S, Daniels.M, Bhattacharya.T, Lapedes.A, Sethi.A, Li.M, Tang.H, Gao.H, Haynes.B, Cohen.M, Shaw.G, Seaman.M, Kumar.A, Gao.F, Montefoiri.D, Korber.B. (2010). Genetic signatures in the Envelope Glycoprotein of HIV-1 that associate with broadly neutralizing antibodies.  PLoS COMPUTATIONAL BIOLOGY, 6 (10). 46.  Ã‚  Ã‚   Garg. A, Nuttall. J, and Romano. J. (2008). The future of HIV microbicides: challenges and opportunities. Antiv. Chem Chemo:   19, 143-150 47.  Ã‚  Ã‚   Nuttall. J, Romano. J, Douville. K, Galbreath. C, Nel. A, Heyward. W, Mitchnick. M, Walker. S, Rosenberg. Z. (2007). The future of HIV prevention: Prospects for an effective anti-HIV microbicide. Infect. Dis. Clin. N. Am: 21, 219-239