The City Council s Radar - 929 Words

After a final swig of Bacardi a figure stumbles down an empty street in a drunken stupor. He settles down on the steps of the infamous Mckell housing commission in Redfern just two blocks from Sydney’s Central Station. His name is Tyson, he is 13 years old. Below the skylines located in the heart of Sydney City lies the once proud McKell Housing commission, a collection of flats that once hosted a vibrant living environment for poor families in need. However it is now an epicenter of drug abuse and violence with its most vulnerable victims being the next generation of Australia, its children. Tyson and Jai are amongst the many children aged between 10 and 16 who live in and around the area of Redfern. In the lives of these youths, fleeing the police is an everyday occurrence. Could this be part of an overarching social issue that has flown under the City Council’s radar? Or rather blatant neglect by the system in place? With crime being rampant and methamphetamine abuse in the area said to be multiple times higher than the state of NSW itself. Many children were forced to bypass their adolescence, moving forth into an extremely rough life often filled with drugs and other illicit activities. Studies have linked over 70% of youth crime with alcohol intoxication and is a problem in lower income areas. Alcoholism amongst the youth, however, is product of a larger social issue. â€Å"There ain’t much to do in dis area bruv, schools boring but liquor keeps me alive hey† TysonShow MoreRelatedHistory Of The United Arab Emirates897 Words   |  4 Pagesunique Arabian country, UAE’s society has always been an open minded and a welcoming host. With UAE’s harsh deserts covering mass amount of the country, most of their population is located near the coast and in the mountains in the far eastern region(s) of the country. The coast has been key to their survival for many generations; they rely on fishing as a main source of food as well as trading routes with neighboring countries across the Persian Gulf and the Arabian Sea. Now, 88% of UAE’s populationRead MoreBtk Killer Dennis Radar1752 Words   |  8 PagesBTK KILLER DENNIS RADAR Dennis Radar also known as the BTK Killer was born on March 9, 1945 to William Elvin Radar and Dorothea Mae Cook he was the oldest of their four children he was born in Pittsburg Kansas he grew up in Wichita and attended Riverview School and later graduated from Wichita Heights High School, according to several reports for him as a child made some confessions that he tortured animals, he also had a sexual fetish for women’s underwear he would later start stealing pantiesRead MoreThe Evolution Of The International Civil Aeronautics Organization1586 Words   |  7 Pagesgovernment. They are the assembly, the council, and the secretariat led by the Secretary General. 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Clara Luper, a black girl who wanted to be a part of change, said in an interview â€Å"‘We were having an NAACP Youth Council meeting, and I was eight years old at that time. That’s how I can remember that I was not ten years old. And I – we wereRead MoreCareer And College Research Paper1504 Words   |  7 Pagesthings with my career, and that is to travel and to meet as many new people I can. In becoming a Pharmaceutical Sales Representative, those cravings can transform into my own reality. Becoming a Pharmaceutical Sales Representative (PSR) has been on my radar for quite sometime. Furthermore, It is going to be vital for me to advance my education and receive a college degree. It is an absolute necessity to comprehend the learning and training requirements, skills or talents required, salary and benefitsRead MoreChina Culture1091 Words   |  5 Pagesmost notably being the, â€Å"Chang Jiang (Yangtze River) literally translated as â€Å"Long River,† the Chang Jiang, at 6,300 km (3,915 mi), is Asia’s longest river and the third longest in the world† (Defense, 2010). China has 20 of the 30 most polluted cities in the world, according to the World Bank (Defense, 2010). Consequently, the Chinese government has made pollution control a high priority and even made efforts to cut carbon intensity levels in half, but according to the Defense Language InstituteRead MoreSweatshops : A Factory Or Workshop1264 Words   |  6 Pagesare employed at very low wages for long hours and under poor conditions.† Sweatshops typically produce product like shoes, clothes, and toys. These factories are usually located in developing countries mainly because it’s easier to slip under the radar. It is said that â€Å"Women sewing NBA jerseys make 24 cents per garment that will eventual ly sell for $140.† Women predominantly work in Sweatshops and often endure physical and sexually abuse. Despite the harsh condition and the negative grotesque imageRead MoreTraffic Cameras Should be Forbidden Essay examples2032 Words   |  9 Pageswhether the cameras are effectively functioning to keep drivers safe or whether they are just another source of revenue for cites. Facts have proven that the purpose for the traffic cameras is simply generating more money for the cities. The estimated amount of money that the city of Denver will be making in 2011 from these cameras (Kaminsky)—excluding the ones recently put up—a grand estimation total of seven million dollars†¦ â€Å"According to the Insurance Institute for Highway Safety more than 550 communities

The 20th Century - The Most Violent Century of All Time....

The progress made in the 20th century is staggering. Advancements in science, medicine and technology alone have brought incalculable benefits to human beings. Yet on the darker side, the 20th century was also the most violent time of human history. Two world wars, the massacres of Stalin, the Holocaust of Hitler, and many other such events killed over hundreds of millions of people and inflicted extreme suffering on hundreds of millions more that will make this period in time and period that will be remembered forever. The century had a trend toward weapon improvements. It wasnt until the 20th century that weapons became common in war and on the streets. The 20th century brought automatic firearms, missiles, and nuclear warheads.†¦show more content†¦Some were left to die in concentration camps and many others were killed in a line fashion in concentration camps. This hideous time in human history became known as the Holocaust. This was a horrible type of violence that should outrage us. Hitler, in my thought and also be seen in the book â€Å"Physicists†. The doctor spies on Mobies, to figure out his knowledge and she wants to take over the world with it. She always says how much power she has and how much money she has. This represents militarism and she is going to establish a dictatorship and dominate the earth and the noble people walk right into the trap. Violence has influenced lives because violence isnt as what it used to be. As a race we have become more tolerant of violence. People own guns because they feared somebody else with a gun will try to rob or kill them so they defend themselves. We deal with hearing and reading about war or violence every single day, even if we dont pay attention or notice it, its still there. People in the 20th century had to choose whether to run or face the violence and these voices didn’t go unheard. There were many arts and literature that were taken from the war that showed the true cruelty behind all this. One of the most well known survivors of the Holocaust was Elie Wiesel who wrote the book â€Å"Night† where she described her traumatic experiences in a graphic and terrifying way. A philosopher known as Sartre greatly influenced existentialism in thisShow MoreRelatedThe Rise Of The 19th Century1441 Words   |  6 PagesThe 19th century contributed to Europe’s history in the form of ideologies. In contrast to the 20th century, there were no wars or acts of violence used to support these ideologies; instead the forces of capitalism drove the history of the 19th century. By this notion, the last time Europe had experienced extreme forms of violence was during the French Revolution, therefore the decades before the 20th century were relatively peaceful. In the summer of 1914, Europe crossed the point of no return withRead MoreHumanity: A Moral History of the 20th Century by Jonathan Glover1325 Words   |  6 PagesAs most people would agree, the 20th century contained some of the bloodiest and most gruesome events ever recorded in history. Why do words such as Hiroshima, Rwanda, The Final Solution, A Great Leap Forward, The Great Purge and so many more sp ark such vivid images of blood, torture and murder in our minds? And despite those horrific images, what is it that causes us humans time and time again to commit such crimes against humanity? Those are the kinds of questions Jonathan Glover, a criticallyRead MoreDeath Of The 19th Century947 Words   |  4 Pages1850 in the north of Melbourne, housed Victoria’s most infamous criminals, which included; Russell Street Bomber Craig Minogue, 1920’s gangster Squizzy Taylor, Hoddle Street gunman Julian Knight and Mark ‘Chopper’ Reid. Pentridge Prisons history emphasizes the changes in crime, offending and punishment over its period of operation. By the end of the 20th century, Australia was less violent in comparison to the end of the 18th and 19th centuries. Crimes that have reoccurred during this period includeRead MoreSatyagraha, By Mahatma Gandhi1410 Words   |  6 PagesIn 1869, one of the most prominent and influential men to ever live was born. Mohandas Karamchand Gandhi was born in Gujarat, India. Known for leading India to its eventual freedom and inspiring movements across the world, Gandhi became India’s ‘Father of the Nation’. His non-violent and non-cooperative civil disobedience provided a sense of truth and firmness. Better known as Satyagraha, as coined by Gandhi, became one of the best methods to fight injustices. Around the world, Satyagraha has provenRead MoreEssay on Modernism in Literature1532 Words   |  7 Pagesturn of the 20th century conveyed revolution in psychological, social, and philosophical thought. It was time for something neoteric. It was time to break out of the mund ane tradition. This time of revolution conceded men, such as Charles Darwin, Karl Marx, and Sigmund Freud, to rise to fame with their radical and cutting edge theories. Also, women were exasperated of their modeled roles in society. They sought to be independent, they longed to have the ability to vote, and most of all, they wantedRead MoreThe First Midterm Assignment During American History1500 Words   |  6 Pageslate-19th century was the most prolonged time period in American history and also what the bloodiest. Many violent events occurred that gave the 19th century its name, such as, profound labor conflicts between boss and workers, the Indian wars that was an extended battle for peace and a benefit in their lifestyles, the Jim Crow law which was established to separate races throughout the city, and also the fight for the American Empires power. These were the major issues that made the 19th century an importantRead More##eau, Gandhi, And King And Henry David Thoreaus Civil Disobedience1317 Words   |  6 PagesAmerica. The methods of protesting have been changing. Instead of using violent tactics, such as war and destruction, reformers such as Thoreau, Gandhi, and King have brought about a more impactful way of protesting. Nonviolent protests are one of the most successful ways to bring about change, and it is through this method that we achieve our goals while at the same time keeping the peace. Setting a precedent even in the 21st century, Thoreau’s Civil Disobedience underlines the basis of nonviolent protestsRead MoreThe Rise Of The Late And Early 20th Century1693 Words   |  7 Pages The period surrounding the late and early 20th century was the most dramatic era in modern history. It was a period where Western ideals were pushed by motivated empires with ambitions of wealth and expanding territory and, while impossible to distinguish the most influencing factor that led to the Westernization of nations on a global scale, it is easy to determine key points that played important roles in world history. With Western cultures emerging with both technological and military advantageRead MoreThe Impact Of The Civil Rights Movement1018 Words   |  5 PagesAfrican Americans to gain equal rights to their white counterparts. This civil rights push lasted the better part of a century, lasting all the way into the 1960s. It was a hotly contested issue that was the cause of many major events, including riots and protests. Important figures such as Malcolm X and Dr. Martin Luther King Jr. led the Civil Rights movement in the 20th century. The movement was not without opposition, and it had faced many setbacks. Throughout its duration, the civil rights movementRead MoreDescartes, Hobbes, and Pascal Essay examples1033 Words   |  5 Pagesand Pascal During the 17th and 18th century, religion, religious beliefs and most of all the religious leaders played a very influential role in the direction of politics. This was also a time when religion and politics played a large role in the direction of what was accepted as a result of the new discoveries in the natural sciences and in a time when there were some very influential writers, philosophers, scientists and mathematicians. At the time, these people were considered to be very

Culture in a Global Economy Free Essays

Culture in a global economy is a critical factor in international business. While many business transactions make economic sense, the ability to successfully fulfill profitable relationships often depends on being able to reconcile international differences arising from separate cultures. Understanding cultural differences is an initial step, but managers also need to engage in learning processes to develop international cultural competence. We will write a custom essay sample on Culture in a Global Economy or any similar topic only for you Order Now Cross-cultural training enables managers to acquire both knowledge and skills to fulfill the role of cultural agents. Advancing cultural intelligence and international cultural competence is critical to the future success of managers and leaders working in a global context. Culture, as defined in Kroeber and Kluckhohn’s classic, Culture: A Critical Review of Concepts and Definitions, is the â€Å"patterned ways of thinking, feeling, and reacting, acquired and transmitted mainly by symbols, constituting the distinctive achievements of human groups, including their embodiments in artifacts; the essential core of culture consists of traditional (i. e. , historically derived and selected) ideas and especially their attached values† (1952). In international management research, Hofstede defined culture as â€Å"†¦the collective programming of the mind which distinguishes the members of one group or category of people from those of another† (1991). Many other definitions of culture are available. Common elements in the definitions are the shared and dynamic nature revolving around norms, values, and beliefs that are expressed in different behaviors, artifacts, and interactions. Within the context of international business, culture involves multiple levels that span from broad to narrow and different dimensions. On a broad level, supranational culture differences span multiple countries and include regional, ethnic, religious, and linguistic dimensions. On a national level, governments create sovereign boundaries to distinguish different nations with political and legal regulatory systems. In the business literature, most research on culture uses the nation-state as a proxy for culture. Other levels of analysis for culture include subcultures, as well as professional and organizational groups. In addition to various levels, culture also involves different dimensions. Four major classifications schemes provide frame-works for identifying international differences in culture. First, anthropologist Edward T. Hall (b. 1914) classified cultural differences along five different dimensions: time, space, things, friendships, and agreements. Second, Kluckhohn and Strodtbeck developed a cultural orientations framework that identified six issues, with variations in each one: relation to nature, relationships among people, mode of human activity, belief about basic human nature, orientation to time, and use of space. Third, Hofstede’s framework is one of the most prominent one in international management. He identified four major dimensions of cultural values—individualism-collectivism, power distance, uncertainty avoidance, and masculinity-femininity—along with a fifth dimension subsequently identified as Confucian Dynamism, or long-term orientation. Finally, Trompenaars and Hampden-Turner extended Hofstede’s classification with seven dimensions that include universalism versus particularism, collectivism versus individualism, affective versus neutral relationships, specificity versus diffuseness, achievement versus ascription, orientation toward time, and internal versus external control. The four different classifications provide different and overlapping approaches to organize the many complex dimensions that make up culture. A major premise underlying the need for organizing different cultural dimensions is a means to avoid costly mistakes in conducting international business. The different classifications provide a map to make sense of the complex nature of culture. Important caveats to keep in mind are that each classification is not exhaustive and each one originates from a particular cultural perspective. Managers have to engage in learning processes with cross-cultural training to develop both cultural intelligence and international cultural competence. Cross-cultural training for international assignments encompasses a broad range of methods that may include area briefings, readings, lecture/discussions, language lessons, films, self-assessment exercises, role plays, field trips, sensitivity training, and cross-cultural simulations. Cross-cultural training also needs to be coordinated in multiple phases to maximize the learning effectiveness for individual managers and organizational performance. The three phases are predeparture orientation, in-country socialization, and country exit debriefing. The exit debriefing is important for organizational learning, and a knowledge management system can support the capture of the cultural lessons that are learned. Kim and Ofori-Dankwa described four major delivery methods for cross-cultural training: the intellectual model, the area simulation model, the self-awareness model, and the cultural awareness model. The intellectual model involves the traditional classroom approach of general readings and lecture. The area simulation model incorporates culture-specific activities (e. . , working in Japan or Mexico) with games and exercises. The self-awareness training method focuses on having participants identify their strengths and weaknesses in dealing with different cultures, especially taken-for-granted assumptions about intercultural situations. The cultural awareness model focuses on the theoretical foundation for behavioral differenc es across cultures. The key to effective cross-cultural training is the integration of multiple methods that allow a participant to move from simple to complex levels of learning with increasing levels of training rigor. The purpose of using multiple methods in cross-cultural training is to advance the learning process through the learning stages to develop cultural intelligence and international cultural competence. Cultural intelligence integrates the three interrelated elements of knowledge, mindfulness, and behavioral skills. International cultural competence goes a step further with a more complex skill set that integrates cognitive, affective, and behavioral learning to effectively engage in successful cross-cultural relationships. International cultural competence is very similar to intercultural communication competence, which integrates three components: culture-specific understanding of the other, culture-general understanding, and positive regard of the other. Increasing one’s ability to work effectively across cultures also provides positive support to address a range of adjustment issues for expatriates who often face culture shock in the acculturation process. Overall, the most important key of cultural intelligence and intercultural competence is the integration of multiple spheres of cross-cultural learning to effectively engage in international business situations. Effectiveness in reconciling cross-cultural differences often leads to creativity, innovation, and synergy for productive workplace performances. Although cross-cultural training supports global managers’ ability to be effective, the learning process often moves through different stages of development. The different development stages of cultural intelligence are: (1) reactivity to external stimuli, (2) recognition of other cultural norms and motivation to learn more about them, (3) accommodation of other cultural norms and rules, (4) assimilation of diverse cultural norms into alternative behaviors, and (5) proactiveness in cultural behavior based on recognition of change cues that others do not perceive. Global managers with high levels of cultural intelligence and competence play important strategic roles as cultural agents (c-agents), helping their organizations to span international boundaries. C-agents require both the ability to navigate different cultures and the legitimacy from different cultural perspectives, including organizational and within the local community. Organizations have increasing needs for global managers to fill the role of c-agents because demands of globalization increasingly depend on successful relationships with strategic alliance partners, international vendors, and global customers. Within the global arena, national borders often form the defining entity for a culture. However, analysis of cultural differences needs to account for a range of diversity within a national culture. On a continuum of cultural diversity that ranges from homogenous to heterogeneous, Japan, Norway, and Poland are relatively more homogeneous when compared to India, Papua New Guinea, Australia, Britain, and Canada. The more heterogeneous societies encompass more distinctions between subcultures within the national borders. However, it is important to account for the fact that â€Å"almost no country is entirely homogeneous. The world’s nearly 200 countries contain some 5000 ethnic groups. Two-thirds have at least one substantial minority—an ethnic or religious group that makes up at least 10 percent of the population. † In many ways, how a society addresses issues of multiculturalism creates an orientation that enables its citizens to live and work together in a global community. Cultural norms shaped by national government policies will need to avoid and dismantle policies for separation (keeping different cultural identities but not integrated) or assimilation (forced rejection of traditional cultural identity to integrate into dominant identity) in order to adopt new approaches of multiculturalism. Important principles for multicultural policies center on promoting tolerance and cultural understanding to respect diversity, recognize multiple identities, and build common bonds of membership to the local community. In the future globalization will continue to increase the flow and interactions of people across cultures, which surfaces even more international differences. Understanding the different dimensions of culture provides an initial knowledge base to develop cultural intelligence and competence for effective international business relationships. However, global managers require cross-cultural training to advance their learning and growth in cultural intelligence and competence as they take on international assignments. More importantly, organizations will have an increasing need for global managers to become c-agents to develop effective international relationships. In addition, government leaders have opportunities to shape their national culture and support international competitiveness with new multiculturalism policies that promote both the inclusion of multiple cultural identities and the development of local communities in an era of globalization. How to cite Culture in a Global Economy, Papers

Inspiring Entrepreneurs Essay Sample free essay sample

Filipinos do hold a genius for concern. judgment from informations from the Department of Trade and Industry. Harmonizing to the DTI. 99. 6 per centum of registered concerns in the Philippines are micro. little and average endeavors ( MSMEs ) and these provide 63. 2 per centum of entire occupations in the state. â€Å"Our Filipino entrepreneurs contribute so much to the country’s economic system and supply the support of most of our work force. This is why our micro. little and medium enterprisers need and merit all the support they can acquire. † says Manny Aligada. Head of Corporate and SME Segments. Globe Business. Harmonizing to Aligada. Globe Business went through a batch of research in order to orient its solutions to the demands of the Filipino enterpriser. We will write a custom essay sample on Inspiring Entrepreneurs Essay Sample or any similar topic specifically for you Do Not WasteYour Time HIRE WRITER Only 13.90 / page In the class of that research. the group discovered some traits and qualities that make Filipinos good enterprisers. Here are some of Globe Business’ penetrations into the character of the Filipino enterpriser. Practicality and common sense. The most successful Filipino enterprisers are non needfully those who have the best instruction. or have an impressive academic record. Some of them. in fact. neer even finished grade school. So how come they are now successful business communities? â€Å"Filipino enterprisers have what we might name street-smarts and a great trade of common sense. They are practical minds. They make determinations based on what they know about human nature from experience. They are non merely able to come up with good merchandises but. more significantly. they know how to associate to their clients and give what their clients want. You truly can non larn such accomplishments from a book or in a schoolroom. â€Å"This is practical cognition that you gain by associating to existent clients and seeking your ain manus at a concern. In other words. enterprisers develop their street-smarts and sharpen their common sense through experience. They learn from both their successes and their errors. That’s what makes them effectual enterprisers. † Aligada says. Filipino enterprisers are besides practical when it comes to money. They make certain that they get maximal net income for th e lowest possible cost. Passionate about concern. When a Filipino enterpriser decides on what concern he will set up. normally it will be related to something he or she is passionate about. This passion drives him to larn everything possible about the merchandises or services he is offering to clients. The same passion besides gives the Filipino entrepreneur a strong committedness and engagement in his endeavor. â€Å"Filipino enterprisers are all in concern to gain for themselves and their households. However. the most successful 1s have the passion that fuels their heads. emotions and organic structures to do the concern grow. This passion reaches the point where the concern in no longer merely about the bottomline ; the concern itself brings fulfilment and felicity to the enterpriser. This proves that if one is passionate about one’s endeavor. so one will happen the resources to do it successful for its ain interest. † says Aligada. Confident and autonomous. The Filipino enterpriser is ever hands-on and autonomous. When he is merely get downing out his concern. he is practically able to make everything involved in running it-from happening the best providers to leasing the topographic point of concern. to the selling. merchandising and client dealingss. and even ac counting. All this would non be possible without the entrepreneur’s supreme assurance in himself or herself. Filipino entrepreneurs trust their ain judgement and their ain capacity to run their concerns. They are able to take calculated or even make bolding. apparently unwise. hazards. They know that while they can acquire the best advice. the best research. finally. the success of the concern depends on their ain judgement. Hard-working and goal-oriented. No 1 can doubt how hardworking the Filipino enterpriser is. His engagement in his concern is about absolute. to the point that his personal clip for himself and his loved 1s is compromised. He is besides goal-oriented. and has the subject to put smaller ends that he will carry through in order to run into his concluding aim. A leader and an pioneer. The most successful Filipino enterprisers have first-class leading accomplishments. He is able to convert and act upon his employees and his clients positively. He is besides able to innovate-to come up with fresh solutions when a ambitious state of affairs arises. Value-oriented. Finally. the Filipino enterpriser has a set of values that guides him in making concern. The greatest value for a Filipino enterpriser is his household. â€Å"In general. a Filipino enterpriser. no affair how successful he has become. neer loses sight of why he is endeavoring to do his concern thrive and grow—he is making it for the wellbeing of his household. † says Aligada.

Technology and negative effects

10 College Essay Topics on the Importance of Education

10 College Essay Topics on the Importance of Education Education has been termed a basic human right due to its impact on society and its ability to provide anyone regardless of race, location or social status, the knowledge to becoming successful in today’s modern world. Its importance spans across various aspects of our lives including its ability to inspire development and break the chains of poverty experienced in the most vulnerable environments where humans reside. Societies understanding of the importance of education have led to the creation of numerous governmental and non-governmental programs designed to provide everyone with the basic educational requirements needed to function positively in any community.   Also, in educational circles its importance is understood and this is why financial aid is usually made available for gifted students from less-privileged backgrounds to help their intellectual growth. Now, for college students who have been saddled with the enviable opportunity to discuss the importance of education through essays, this is for you. Here, you will find some accurate and timely facts on education which will be accompanied by two other articles providing you with topics on today’s subject matter, a sample essay and some direction on how you should proceed when writing your own.   Here are useful Facts on the Importance of Education: Education helps reduce hunger. The expansion of the means to receive an education by impoverished nations- in Africa and Asia- has had a positive effect in reducing poverty ratings and malnutrition in these continents. A study by the International Food Policy Research Institute (IFPRI) between 1970 and 1995, covering 63 countries showed that education accounted for 55% of the decline in malnutrition achieved within that period. Female education also played its part and accounted for 45% of the decline in malnutrition between the same periods. Education plays an important role in improving maternal health and child survival rate. Statistics show that children born to an educated mother are twice more likely to survive to age five than kids born to an uneducated mother. This can be attributed to the fact that educated mothers are 50% more likely to seek all the necessary immunizations for their kids than their uneducated counterparts. This goes to show the important role female education plays in maternal and child care. Education paves the way for economic growth. Education has been outlined as one of the most important factors that determine the economic status of a population as these stats shows. Statistics show that no country without a minimum of 40% of its adult population having the ability to read or write has achieved continuous economic growth. An individual’s earning potential increases by approximately 10% for each year of schooling received and this in turn increases the GDP of nations that offer quality education opportunities to its citizens. Lastly, the education of girls leads to high return rates when compared to other forms of investment thereby making education for the girl-child a prerequisite for development. Education encourages peace. Education plays an important role in developing the building blocks that ensure peace reigns within local communities which in turn ensures the peaceful conduct of a nation’s entire society. It is a fact that every year a male-child spends in school decreases his propensity for violence by 20%. Education empowers women and the girls. One very important role education plays in today’s society is providing women with the resources to be independent as well as make good choices as regards their health and finances. Educating women has drastically reduced the impact of HIV/Aids and child mortality rates while also alleviating poverty in many communities. A household with an educated wife also increases its earning potential by approximately 50% and this makes for better living of the family unit and society as a whole. Education affects the Environment. Educating the world on the need to be more aware of its environment has been identified by UNICEF as one of the important subject matters of the 21st The UNICEF includes the need for protecting the ecosystem from pollution in its Sustainable Development Goals and Environmental Education (EE) has begun to bear fruits. Research showed that students with attention-deficit disorder (ADD) have benefited from exposure to nature and this drastically increases their motor-skills and ability to learn. While education at the adult level makes individuals more sensitive to nature thereby increasing recycling and creating sustainable cities. Education affects globalization. Education has led to better communication among individuals from every nation and culture in the world. Thereby leading to more collaboration and understanding of fellow culture. Research shows that student who are taught about the global issues facing the world and about other cultures are twice more likely to take social action than their colleagues who have no knowledge about the world. Global education has made it possible for professionals to be found in every nook and cranny of the world. Statistics show that the number of multinational corporations worldwide rose from 7,000 in the 1990’s to 65,000 in 2013 and this promotes development across every facet of the world. Education and educated individuals make good citizens. Education is viewed as a prerequisite to good citizenship for it provides individuals with the ability to make good decisions and understand the need for civility in all their dealings. Statistics show that educated citizens are 8% more likely to file complaints on misconduct or report incidents than uneducated individuals. This ensures that government institutions operate at a high level of accountability. Education affects equality. Education creates an environment that fosters equality between minority populations and the west which makes it important for global economic growth. Statistics show that providing quality education for students from all backgrounds- low income and high income- reduces the achievement gap among students from diverse backgrounds. In some European countries such as- Germany, Ireland and Switzerland- the literacy rate is approximately 92% due to the provision of affordable quality education. Education affects extremism. Education has also been hailed as a solution to religious extremism and according to Former US Ambassador- Bill Richardson- supporting public education in the Muslim world will drastically reduce terrorism by making the populace less easy to exploit by extremist leaders. The 9/11 commission report also stresses the importance of education in promoting life over death and why the Arab world should be educated. It states that the increase in economic growth and intellectual knowledge across the Muslim population will drastically reduce extremism. Note that these are 10 accurate facts on the importance of education you can use when writing a college essay on this subject matter. As stated earlier, this is simply part one of three pieces that include our guide on how to write about the importance of education for college students and the 20 topics covering the importance of education. References: Lawrence, S. (2009). Investing in all the People: Educating Women in Developing Countries campaignforeducation.org/docs/reports/IMF%20paper2_low%20res.pdf Littledyke, M. Eames, C. (2009). Education for Sustainability in the Primary Curriculum 43-47. Barbara, H Gene, S. (2004). What Works in Girl Education cfr.org/publication/6947/what_works_in_girls_education.html The Equity and Excellence Commission. (2013). A Strategy for Education, Equity and Excellence for Each and Every Child http://www2.ed.gov/about/bdscomm/list/eec/equity-excellence-commission-report.pdf Brian, F. Peter, N. (2012). Educational Equality for Gypsy, Roma and Traveller Children and Young People in the UK. equalrightstrust.org/ertdocumentbank/ERR8_Brian_Foster_and_Peter_Norton.pdf Newman, F. Scurry, J. (2004). The Future of Higher Education 35-40.

U04d1 Case Study Essay Example | Topics and Well Written Essays - 500 words

U04d1 Case Study - Essay Example This will definitely cause numerous issues and problems, however, knowing the family, I believe that the children will come to an unanimous decision to appoint a full time nurse / medical help and to chip in and pay for the medicals and care. However there will be a complete opposition from Carl, since he loves his wife a lot and is willing to give up his life for her. With age the memory power also reduces. Also age brings about numerous other issues like irritability, and reducing skills sets. In the case of Carl, an appointment will be taken with the doctors to get immediate attention and care for him as well. The main idea of the appointment will be to get a complete body health check for Carl to assess his issues (Perls, Silver & Lauerman, 1999). I realize that the family goes through a lot missing their lost child, and lost sibling. Hence the topic will be brought out to assist them in any possible manner, emotionally. Hence to help them get through this issue, firstly we will talk of the individual concerns and the remembrance of the sibling (Kirkwood, 1999). Also a decision will be made to ensure children come to stay with parents over the

Housing Declared Bottoming in U.S Essay Example | Topics and Well Written Essays - 250 words

Housing Declared Bottoming in U.S - Essay Example The central issue in the article is the state of the U.S housing market this year, prior to past trends and expected outcomes in future. The market’s collapse hits a bottom position in the 20th and 21st centuries, but different stakeholders show their optimism towards recovery and sustainability of favorable business in the market. Confidence of consumers, home seizure trends, supply of foreclosure and market healing processes in 2012 (Keith & Short 89) are central to the interest of the article. Different views are aired but one thing is for sure; the housing market’s performance is poor, with unpromising months to come due to recovery allowance period. The housing market’s recent performance is the issue at hand. Past recorded prices and house demands are not comparable to the current situation. The tortoise speed increase in demand is shedding some light to improved sales, but the optimal functionality of the market is far from being realized. The author’s point of view is not different from those of contributing stakeholders, evident that the housing market is on the verge of collapsing if not already collapsed. However, the author is one of the optimistic stakeholders, arguing that the market is taking a stable aspect and that its recovery is or will be sustainable in the next few months. The author’s opinion is substantiated by drawing evidence from numerous experts in housing matters. The housing market in U.S is actually bottoming. Hitting an all-time low since 1930s means that the trough has already been realized. This means that the article is right, and this is because the claims of the article are actually backed by evidence from various housing variables in the market. The claims presented by various parties including the author are right. The performance of the market is poor, with low response to recovery strategies (Olivier 150). Data has also been employed to reinforce the claims. All interviewees

Second paper Essay Example | Topics and Well Written Essays - 1000 words

Second paper - Essay Example t France lack sufficient outlets since Germany is situating obstacles of trade, the U.S have become protectionists, huge markets are attenuating causing intricacy in access them as well as the large states empty alien goods into the French markets. He states that currently, competition, demand and supply commandment, liberty of trade, and consequences of conjecture, all exude in a vast circle and this makes it an immense economic intricacy and tremendously grave predicament. Jules argues that it is necessary to take safety measures against the South American market being uncertain and possibly occupied by goods from North America. He maintains that greater races have the right since they have an obligation to advance substandard races. In addition, those European states exonerate themselves with munificence, with sumptuousness and genuineness of the responsibility of advanced civilizing. He affirms that the French colonial development policy has put them below the empire that has led them to Tunisia then to Madagascar. That the policy was stimulated by the verity that a navy like one they have cannot be exclusive of docks that are secure, defenses, as well as delivery centers on the soaring seas. According to Jules, these contemplations advantage the complete interests of nationalists. The naval warfare settings have changed significantly. Refuge, areas of supply, docks for security and provisioning are deeply required and thus the need of Tunisia, Indochina and Saigon, as well as Madagascar. The strengths of the ideas developed by Jules rely majorly on the economic factors brought about by the period. This is in form of exports and trade ties that the two regions benefitted from each other. Since countries like France were weak in form of trade, it would benefit from trade carried out by its neighbors (Ooi 2004). The weakness is in form of policies being implemented by the superior nations. This point can be used by other regions to implement slavery and

Water Crisis: Causes, Impact and History

Water Crisis: Causes, Impact and History Fresh water crisis The damages people made to get the water supplies higher has harmed A lot of people and their population has lowered glaciers continue to shrink worldwide, and conflicts are likely to erupt. Others are with no access to water people refuse to sanitize correctly to better the freshwater crisis less than 1% of the population have access to water. The freshwater crisis in the world today is that people are still without access to water sanitation is very low. To help people we can purifying methods The freshwater crisis in todays time The population has changed in many different ways united nations water use has grown more than twice the population. Two thirds of the worlds population is living without water some regions seem relatively flush with fresh water. The water is harder to get than it was twenty years ago it needs work or significant currency to obtain not enough rain is coming in around the places that really needs the water and people are trying to help africans but they can only try so hard without enough water people could pass or suffer. Why fresh water shortage will cause the next global crisis The consequences are providing to be profound reservoirs and aquifers dry its reduced to desert overuse water. Global nature of the crisis in the underlined pumped groundwater farmers rely on it massive loss of ground water. Glaciers continue to shrink worldwide it has increased since the 20th century they have more conflicts to erupt because there dehydrating and its causing them to freak out on others without thinking or carrying of the consequences of their actions and wrong ways they have treated everyone they will do anything at this point to get some water in their houses. Causes and effects and solutions to water crisis Water has been luxury for people living in droughts they have fought with water borne disease but people also take the water they do have for granted.once water becomes polluted it becomes very harmful to humans and animals. It could lead to sickness, infections its polluted dangerous chemicals. People in bigger cities has not complained this reason is because they have more chance of surviving they have a lot more water access than most states these places are around africa and india they get sicker easier than people who have proper drinking water. Causes of water scarcity Human waste is directly dumped in lakes and rivers without proper cleaning so this is causing a health issue and causing people to get very sick and possible death. The leaks from oil spills and landfill leaks are having a effect on humans not being able to get proper drinking water and sanitizing correctly. They are going hungry the farmers dont have enough water to feed their crops so their not getting enough money on the farm causing their business to go down. The more water farmers have the less problems their is to face the crops and animals will not face death and be dehydrated. Threats to water supply side and demand- side problems One supply-side threat arises from instances in which we are withdrawing freshwater from surface water sources and groundwater aquifers at rates faster than replenishment or recharge (T boone pickens). The rates on freshwater crisis have lowered since the 20th century not many humans have much access to water and if they do its not very sanitized its filled with many diseases and bacteria. The population has lowered in small states than in large this has been an issue and we havent found a solution to solve this The population on death and diseases The cities have lowered with their population in small states. While nations so far have found more reasons to cooperate than go to war over water, pressures are mounting rapidly with rising population and absolute scarcity levels(STEVEN SOLOMON). The greater, imminent risk today is failed states, and all the fall-out they will spread.So many families have lost many important family members and knowing that in a few days that will be them lying there begging for forgiveness. To many deaths for something that could be fixed or the people suffering be saved and moved to someone that can help them have a future. Works Cited The Crisis. Britannica School, Encyclopà ¦dia Britannica, 3 Feb. 2017. school.eb.com/levels/high/article/The-Crisis/2977. Accessed 10 Feb. 2017. Henry, Tom. Water Crisis Grips Hundreds of Thousands in Toledo Area, State of Emergency Declared. Blade, The (OH) 03 Aug. 2014: Newspaper Source. Web. 8 Feb. 2017. Inland Water Ecosystem. Britannica School, Encyclopà ¦dia Britannica, 18 Sep. 2016. school.eb.com/levels/high/article/inland-water-ecosystem/117268. Accessed 7 Feb. 2017. Interlandi, Jeneen, and Ryan Tracy. Fresh Water Is Becoming a Privatized Commodity Instead of a Public Trust. Will the World Run Out of Fresh Water? Ed. David M. Haugen and Susan Musser. Detroit: Greenhaven, 2012. At Issue. Rpt. of The New Oil. Newsweek 18 Oct. 2010: n. pag. Opposing Viewpoints in Context. Web. 8 Feb. 2017. Water. Britannica School, Encyclopà ¦dia Britannica, 15 Apr. 2016. school.eb.com/levels/high/article/water/76210. Accessed 7 Feb. 2017.

Comparing A Thousand Acres and King Lear :: comparison compare contrast essays

A Thousand Acres and King Lear: A New Twist When Jane Smiley wrote A Thousand Acres, she consciously made the story parallel to Shakespeare's King Lear for several reasons. The novel's characters and basic storyline are almost direct parallels to King Lear, but Smiley's dissatisfaction with the traditional interpretation of King Lear is showcased in her modern day version (Berne 236). The story of the Cook family is almost a carbon copy of the saga of Lear's family. The ruler, or father, possesses so much power that he is driven to insanity. Both divide up their kingdoms and land, giving the largest portion to the most "loving" daughter: "In spite of that inner clang, I tried to sound agreeable. 'It's a good idea.' Rose said, 'It's a great idea.' Caroline said, 'I don't know.'" (Smiley 19). In each family, one daughter, the youngest, rebels against her father's wishes and is not given any land. Cordelia: Nothing, my lord. Lear: Nothing? Cordelia: Nothing. Lear: Nothing will come of nothing: speak again. Cordelia: Unhappy that I am, I cannot heave My heart into my mouth. I love your majesty/ According to my bond; nor more nor less. (Shakespeare I. i. 89-94) Shortly after giving up his power, the father realizes that he is nothing without it and appears to be slowly becoming insane. In both instances, the father, in a crazed moment, wanders off and puts himself in a life-threatening situation. In the end the youngest daughter comes to the fathers' rescue. With so many basic plot similarities, Smiley manages to convey a new take on an old-fashioned story. At the end of King Lear, Lear traditionally is believed to be a changed man. Smiley doesn't buy into this common belief; therefore Larry Cook remains a static character throughout the novel. He never changes his attitude towards his possessions, his daughters and his land. Another difference that contributes to Smiley's new interpretation is the point of view from which the story is told. King Lear is told from a strictly male point of view. A Thousand Acres is told through the viewpoint of Ginny, Smiley's parallel to Goneril. Through Ginny's self-revelation, the reader is made aware of many circumstances that would cause a daughter to hate her father. Smiley believes that Lear's daughters must have had some reason for hating him. This is why, in her novel, she includes a childhood of incest.

Urban deprivation is one of the characteristics of large cities in all parts of the world

The inner city areas of many Global cities have an image of decay with poverty, pollution, crime, overcrowding, poor housing conditions and unemployment. Such problems are more prevalent in inner-city areas than in other areas of the city. Deprivation has been caused by old industries closing down and increasing the unemployment levels which are not tackled due to the old workers not being skilled enough to work in these new factories or line of jobs. This happens more in MEDC's compared to LEDC's where overpopulation and urbanization have cause the problems in the inner city. Counterurbanization has been another problem within MEDC's as it has left houses derelict and the people would rather commute than live in the inner city. This has then led to out of town shopping centers being set up. In MEDC's the inner city initiatives for reversing the decline of the inner city started back in 1945 with comprehensive redevelopment. This program involved large-scale clearance of old terraces in order to provide space for new housing and inner city environmental features. Over twenty years 1. 5 million properties wee knocked down in the inner city. Elswick and Kenton in Newcastle were two areas embarked for comprehensive redevelopment. Existing residents were moved either into new towns of Cramlington or to extensive council houses estates built in areas such as, Byker. Many local authorities followed identical planning and soon the landscape of the inner city was transformed with huge concrete and glass tower blocks separated by flat expanses of grass. At the time these high-rise flats were a great success architecturally however the policy failed due to redevelopment underachieving demolition. This gave a housing shortage and vast spaces of derelict land. This policy that lasted till 1967 also failed to tackle the social and economic problems. 1968 saw another scheme come into action; the Urban Aid programme gave grants to local authorities to expand services in deprived areas and to establish community development projects using self help. This scheme was a great deal more localized and it was unfortunate that the economic downturn limited the funds and therefore by 1977 the scheme had finished. The next year the new towns policy was abandoned in an effort to stop decentralization of people and businesses. For the first time inner cities were officially declared problem areas. In 1988 Margaret Thatcher introduced the â€Å"Action for cities† policy. From 1991 onwards-Local authorities were able to bid for funds for specific urban projects. An example is Sunderland; the money was used to redesign parts of the city center with a new shopping precinct. And bus station. A single government department, the end of the 1990's had created the Urban Regeneration Agency. In Greater Manchester 4. 5 hectares of the city were destroyed with 30,000 homes left damaged form the bombing of World War II. By the end of the war 70,000 homes were deemed unfit for living mostly in the high density Victorian inner center. The plan for Manchester was launched in 1945 with the aim of clearing all Victorian housing. Following the repair of the war the Manchester Slum Clearance Programme restarted in 1954. Over five years 7500 properties were demolished mostly in the Miles Platting area. In 1961 the policy of comprehensive development took place with the clearance programme expanding in four main areas: Hulme, Beswick, Longsight and Harpurhey. Over 55,00 new houses, a mixture of low and high rise were built to replaced the cleared terraces reducing the housing density and population by up to 50% in some areas. The Hulme area was a typical Victorian area of Manchester and was tightly packed with terraces. Conditions were overcrowded and polluted with few housing having toilets. After the demolition of the terraces, shopping facilities were introduced in three areas. By 1972 the redevelopment of Hulme was completed with 5,000 new houses being built. Problems did arise with new properties leaking and then the heating bills were too high for the residents and many found the accommodation inappropriate. This area fell into a spiral of decline with growing unemployment, drugs and violence along with eh deteriorating environment. The Hulme city challenge was launched in 1992. This plan involved building of 3000 new homes, shops, roads, offices and community facilities to replace existing properties in a 60-hectare area. The funds of i200 million came from the government, local authority and private finance. Manchester faced other problems form the closure of the nineteenth century industries that left 24,000 jobs unavailable between 1974 and 1984. Plans included 2000 new houses and 375,000 square meters of industrial and commercial floor space to provide 10,000 jobs. In 1988 central Manchester was given n UDC to regenerate 200 hectares of land and buildings in the southern part of the city center. This area included six conservation areas, over ninety listed buildings, three universities, the Granada Studios Tour and the Museum of Science and Industry. However these were the areas of contaminated land, derelict warehouses, mills and canals. The IDC ended in 1996 and in the eight years of operation invested i420 million. Urban deprivation in the LEDC's have been tackled in many ways however there have been schemes that have proven to be a lot more successful than the others. In Chennai there has been a rapid increase in population due to the rural to urban migration and the high birth rates. About one third of the population lives in the slums, mostly shantytowns. The planning solutions began with the building of four to six storey blocks however these largely failed due to high maintenance and lack of uptake as the tenants would be unable to afford the rent. If the rent were reduced the scheme would lose money. After this initial failure The Board took up a new idea of upgrading the slums. The aims set were providing one bath and one toilet per ten families; one public fountain per twenty families; one street light per forty meters of road and one pre school per two hundred families. Other initiatives required self-help financing after an initial investment had been made either by the World Bank or welfare organizations. These schemes encouraged greater community involvement. Some of the start up loans were gave to families to build their own homes. Site and service schemes were implemented with finance provided for the acquisition of land; purchase of building materials, road building and the provision of basic services such as, water and sewage. New owners were then responsible for building the property on their allocated land. The upgrading after that often led to the sale of homes to higher income groups. The generated some money for the poor families and allowed the Board to re-invest in new schemes. An area in the southern outskirts of Chennai was the location for a site and services scheme known as Velacheri. It provided fourteen hectares of land to house 2,640 families many of whom were being forced out of Chennai. Waiting for the new residents building their homes would have caused a delayed the rail building so contractors were used to begin the building of properties. Along with the roads, water supply, streetlights and many other services. These services did face problems with many being left unfinished and extra floors being added without regulation. Some families sold their home for profit and the poorest were unable to afford these houses. These are some example of the initiatives being taken in order to solve the problem of urban deprivation. However there have been many other schemes and one of the most successful was that of the Favelas in Brazil redevelopment that won several prizes such as, the famous Habitat Award from the United Nations. These have been more successful as it didn't break up the families and kept the community spirit and the families could continue to access their place of employment. The similarities between the initiatives of the MEDC and LEDC worlds that have been undertaken for there reduction of urban deprivation are not all that similar. This is due to the fact that the MEDC's have more finance so there are able to use other schemes to tackle their problems. Also the fact that the problems they face are of a different cause. In the LEDC's it tends to be shanty towns are therefore have to look to house these people unlike the MEDC it not so overcrowding but unemployment due to the decline of the Industrial Revolution and recently Counterurbanization has left old Victorian buildings derelict and an eye-saw.

Why Beowulf is a True Epic Hero Essays

Why Beowulf is a True Epic Hero Essays Why Beowulf is a True Epic Hero Paper Why Beowulf is a True Epic Hero Paper It seems as though humanity is fixed on one form of what and how a hero ought to be. The epic hero is the central figure in a long narrative that reflects the values and heroic ideals of a particular society. Beowulf is an illustration of this in most aspects because he fought for the common good, had a superhuman trait of strength in his hands, and showed tremendous courage and bravery when entering a battle despite death might win in the end. Beowulf shows throughout the poem that his actions are in no way selfish. John D. Niles states in The Fatal Contradiction in Beowulf (94), Before the fight he declares himself willing to accept whatever outcome the lord see fit to grant. This shows that Beowulf does not fight to win personal glory; he did it for his people. A quality an epic hero should acquire. Beowulf was proud to know he pleased his people by defeating Greened and later on Grenades mother. He goes on to show throughout the poem that he will defend the Gears at all costs. Bernard F. Happen suggests that Beowulf is in fact far from a true hero. He states In The Failure of the Heroic Ideal (82), Beowulf behavior from beginning to end Is governed by the ethic of vengeance and the desire for glory. Its apparent that this Is not exact because its proven that Beowulf chose to fight alone so the fight would be equal. He knew the potential outcome, death. Epic heroes dont naturally fight with an army or side by side a friend, they take on their challenger alone. Beowulf chose to conquer a number of villains by his own hand to ensure the Seats would remain In peace. When we think of heroes, we automatically think of superpowers and extravagant Holon suits with capes and gadgets. Like Superman and Spenserian, modern day examples of heroes, Beowulf contained an almost superhuman Like quality. He had an ultimate strength In his hands. In his battle with Greened, Beowulf fought with no weapons to equal the fight and to show the meaning of true defeat without It being all for his glory. He killed Greened and put Warthogs and his people at ease. They saw Beowulf as a true hero as well. Beowulf Is definitely an epic hero. He fought for his people to the death and defeated numerous monsters. He was aware of his responsibility as a king to do whatever It took to protect the Seats. Beowulf Is a ferocious warrior with no tragic flaws who died for his people. Like John D. Niles states, If the behavior of anyone In the poem Is to be considered Irresponsible and Impractical, It Is that of the Gears, whoso failure to live by the heroic Ideal proves to be Impractical In the extreme, even to the point of leading to their ruin as soon as they no longer have a hero to protect them. This Is a Ideal example of why Beowulf Is a true epic hero. BY earner 991 be. The epic hero is the central figure in a long narrative that reflects the values and Niles states in The Fatal Contradiction in Beowulf (94), Before the fight he declares Beowulf does not fight to win personal glory; he did it for his people. A quality an epic that he will defend the Seats at all costs. Bernard F. Happen suggests that Beowulf is in fact far f rom a true hero. He states in The Failure of the Heroic Ideal (82), Beowulf behavior from beginning to end is governed by the ethic of vengeance and the desire for glory. Its apparent that this is equal. He knew the potential outcome, death. Epic heroes dont naturally fight with an conquer a number of villains by his own hand to ensure the Seats would remain in examples of heroes, Beowulf contained an almost superhuman like quality. He had an ultimate strength in his hands. In his battle with Greened, Beowulf fought with no weapons to equal the fight and to show the meaning of true defeat without it being Beowulf is definitely an epic hero. He fought for his people to the death and defeated numerous monsters. He was aware of his responsibility as a king to do whatever it took to protect the Seats. Beowulf is a ferocious warrior with no tragic flaws who died for his people. Like John D. Niles states, If the behavior of anyone in the poem is to be considered irresponsible and impractical, it is that of the Seats, whos failure to live by the heroic ideal proves to be impractical in the extreme, even to the point of leading to their ruin as soon as they no longer have a hero to protect them. This is a ideal example of why Beowulf is a true epic hero.

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. 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