Phoenix Software Project Management

Question: Depict about the Phoenix Software Project Management? Answer: Presentation: The phoenix venture is a novel made on business systems utilizing IT. It is about the DevOps, IT. The epic is considered as a cutting edge approach to see the objective. This Novel portrays those issues, which are looked by each IT association. It demonstrates the best approach to practice and culture the tests to take care of the issues, making improvement to the lives of IT workers. This Novel demonstrates a community oriented approach to deal with the work between the business and IT. The epic discloses to Bills story. IT director of parts boundless was new IT activity, who code named phoenix venture. President needed bill to fix the mass inside 90 days to forestall the redistributing of the whole division. With the assistance of the of Dr. Reid bill perceive the three potential approaches to make sense of the entire IT works. Bill came to comprehend that an assembling plant shares more for all intents and purpose with IT office. Rundown of suggestion: The suggestion from phoenix venture is to make sense of these under referenced realities Instructions to control the work discharge into IT activity. The most obliged assets are should have been utilized uniquely for the objective situated undertaking. In the wake of making sense of the most obliged assets, there are three way understanding: First path assists with understanding the way toward making quick progression of work to move from improvement to IT activity. Second way demonstrates the procedure to intensify and abbreviate input circle to keep away from modify and to fix quality at the assets. Third way depicts the formation of culture, so as to deal with making tests (Pais, 2015). Explanation for relieved fiasco: Some inadequate guarantees are given to the market. Again the deferral with in phoenix dispatch by multi week to maintain a strategic distance from the disappointment. In spite of the fact that postponing by multi week could cause dissatisfaction of clients. Underlying drivers behind disappointment: The underlying driver behind disappointment lies in the issue in tasks. The steady firefighting expected to keep lights on, which causes the finance framework disappointment. The issues in activity are brought about by the absence of procedure in various significant territories: Problem the executives, episode the board and the change the executives. The purpose for the disappointment of the change the executives is caused because of the choice to actualize an untested change, which harmed the entire finance framework at a vital purpose of time. The disappointment behind the issue the board due to not having a productive a powerful procedure for the distinguishing proof of the underlying driver The episode the executives procedure didn't have an effective plan on business-centered occurrence prioritization (Patterson, 2000). Need of a war space to oversee: The phoenix colleagues entertain themselves with their task so profoundly, that they even need to secure themselves in the war space to maintain a strategic distance from interruption. Job of Brent: In the IT anticipate of Phoenix Brent Geller is a Lead Engineer. He worked for the Wes. He was consistently there in IT anticipates. Significance of Brents job in the task/organization/story: To the extent the significance of Brents job is concerned, he expected to work with the engineers to fix the issues at the source. As a lead Engineer he used to tell designers on how things are done in reality and what those things are, which permits breaking underway (cynic, 2013). Changes in Brents job to make the undertaking effective: To make the undertaking effective, Brent was pulled off from phoenix occupation and expected to take a shot at SAN issue. Consequence of these changes: By the adjustments in Brents job, Brent Started taking a shot at the on SAN issue. In the wake of taking a shot at the SAN issue, Brent arrive at a resolution that, redesigning SAN caused finance run disappointment. At the point when Brent was helping one of the SAN architects to redesign the firmware, it took quite a while and wen nothing appropriately tech note (InfoQ, 2015). Hypothesis of Constrains: Hypothesis of imperatives depends on the setting of goalachievement, whichis restricted by at any rate one requirement. In the event that there is nothing forestalls to accomplish objective or higher throughput, at that point framework is made for creating limitless throughput, which is beyond the realm of imagination, in actuality, framework. In the task phoenix dr. Reid said about the basic component in the board of a plant is arrival of material and occupation. Without these two factors, nobody can control work in progress (WIP) (Kim, Behr and Spafford, n.d.). Job of this hypothesis to deal with the phoenix venture: This hypothesis guided those connected with individuals in this task by making them understood about the deception, which is happened when the upgrades are made alongside the bottleneck. It clarified that any improvement, which is made after the bottleneck, has no utilization in light of the harm brought about by the starvation. Bills path to the prioritization of ventures as parts boundless: To have power over arrival of the undertaking into IT activity Bill has made sense of the task prioritization into three different ways. Right off the bat, Bill saw how to make out snappy work stream, to move from the advancement to IT activities. The subsequent way portrays the procedure to enhance and abbreviate the input circles to maintain a strategic distance from modify and fix quality at source. The third way legitimizes the gaining from disappointment and understanding the training and redundancy are required as an earlier condition to apply for authority. Most proficient standard about IT the board gained from perusing this book: In IT the board, Virtualization assumed a significant job. With the idea of virtualization, there is no compelling reason to oversee loads of physical server any more. Because of the virtualization, there are the nearness of consistent examples inside the server or might be dwelling in the cloud. In this task Erick used to show the reason behind Brents over utilization of time. He was taking week to finish the straightforward brief changes. The explanation was the bottleneck of the assignments. Brent was continually above usage of 100%, consequently at whatever point Brent is required for any assignment, the undertaking continue holding up in a line. The holding up time is the level of active time (Kim, Behr and Spafford, 2013). Here a diagram is appeared: on the Y-pivot, it is the estimated holding up time and on the X-hub it is demonstrating the % to be occupied for a given asset at work textual style. In the phoenix venture, Bill understood the results. He understood that their straightforward brief undertaking needs seven hand offs (for example database group, systems administration and virtualization group). Handiness of this venture past MS training: Past the MS instruction, this phoenix venture encouraged on the most proficient method to perceive the issues which occur in an IT association. How these IT association related issues danger each dedication, for example, Information security, IT activity and improvement made in business. How issues are getting fixed utilizing DevOps strategies. DevOps represents Development and Operation. DevOps is a technique to create programming, which stresses coordinated effort, correspondence, robotization, participation estimation and incorporation between the IT experts and the product designers. End: In by and large the Phoenix venture brought many related strides of current IT and the executives rehearses together. All the things in a general sense appears to be unmistakably about the Theory of limitations, lean, Kanban, Demings arrangement of profound information, even those persuading suggestion by dr. Reid-all are the means of including individuals into work all the more successfully. This Novel demonstrates a community oriented approach to deal with the work between the business and IT. This undertaking study shows the main driver behind the disappointment, for example, the issues in activity are brought about by the absence of procedure in various significant regions: Problem the board, occurrence the executives and the change the board. Again the reason behind the relieved catastrophe is deficient guarantees, which are given to the market and the postponement in the task dispatch. Undoubtedly, in the wake of making sense of the most compelled assets, there are three way un derstanding: First path assists with understanding the way toward making quick progression of work to move from advancement to IT activity. Second way demonstrates the procedure to enhance and abbreviate input circle to maintain a strategic distance from modify and to fix quality at the assets. Third way depicts the formation of culture, so as to deal with making tests. References: Anbari, F., Dutton, A., Holt, E., King, L., Wilson, L. also, Zacharko, M. (n.d.). Undertaking Phoenix. Kim, G., Behr, K. also, Spafford, G. (2013). The phoenix venture. Portland, OR: IT Revolution Press. Kim, G., Behr, K. also, Spafford, G. (n.d.). The Phoenix venture. Pais, M. (2015). Meeting Book Review: The Phoenix Project, A Novel About IT, DevOps Helping Your Business Win⠝. infoQueue. [online] Available at: https://www.infoq.com/articles/phoenix-venture book-survey [Accessed 2 Mar. 2015]. Patterson, D. (2000). Task Phoenix. San Jose, Calif.: Writers Club Press. doubter, (2013). Book audit: the Phoenix Project. [online] Available at: https://www.itskeptic.org/content/book-survey phoenix-venture [Accessed 2 Mar. 2015].

Reaction Paper on SONA 2014 Essay

There were numerous features on the ongoing SONA President Benigno S. Aquino expressed last July 28, 2014 and these are the 3 things that captivated me the most: Under the dubious DAP, PNoy said that around P1.6 billion store was given to Training-for-Work Scholarship Program of TESDA. An aggregate of 223,615 recipients have graduated with a likeness 146,731 alumni as of now working. I would state I truly extolled him for his bits of knowledge on instruction. Valid, Education is significant in each nation. A nation comprises of individuals, presently if those individuals are for the most part ignorant what do you think would the nation become of? Furthermore, truly, in the wake of putting in a few pesos to those understudies through grants, what amount do you figure those understudies would pay for their charges when the will graduate and have their own occupations? see more:sona 2017 response paper The monstrous side of this however is the response of COA and governmental issues association. Did you realize that as per COA these sums were not completely used by the organization, providing reason to feel ambiguous about Aquino’s figures. COA said that out of the P1.1 billion assets from DAP acquired by TESDA in 2011 for TWSP, just 92.47% was used. Presently what about that?! Individuals and even ventures are not great. When will they acknowledge that?! I am as yet grateful however on the grounds that in any event he is indicating positive numbers and realities. President Benigno S. Aquino III, during his fifth State of the Nation Address (SONA) on Monday, refered to achievements in the country’s flying industry as having added to the country’s consistent financial development the previous year. First on the rundown of references was the point at which the International Civil Aviation Organization (ICAO) last March 2013 lifted the critical wellbeing concerns it had recently given for the Philippines.In that year, the European Union lifted its prohibition on Philippine Airlines, permitting it to fly indeed to Europe. Yet, with the improved aeronautics framework, President Aquino stated, Filipinos would now be able to fly legitimately from Manila to London. President Aquino additionally invited the FAA’s choice to update the Philippines’ flying status back to Category 1. He said that due to this overhaul, all things considered, thereâ will likewise be an expansion in courses heading off to the United States â€Å"The increment in trips of our neighborhood carriers to the United States and partaking nations in the EU is a major assistance to both the travel industry and business,† the President said in his discourse. I am additionally grateful for this one. Philippines being an available nation overall is a Go ! what I implied is its an unmistakable in addition to for us on the off chance that we don’t spend a lot on corresponding flights only for us to get to our goal. I not certain if at any time you encountered corresponding flights previously and I truly trust youd never do . Since, Oh my! it is such an issue with time and cash. Envision you being energized however you need visits, dozing in the air terminal simply get the soonest trip to your goal and go to GOD the climate will coordinate or, in all likelihood you’re stuc k! In the interim, President Aquino additionally called attention to how the country’s facilitating of the World Economic Forum on East Asia this year was a tremendous achievement, demonstrating the world the country’s limit in business.â€Å"†¦With the APEC (Asia-Pacific Economic Cooperation) Summit the Philippines will have one year from now, we will have the option to advise considerably more individuals of our advancement, and the open doors this has opened up for all,† he said. This is my greatest worry of all. I trust he will apply more exertion on this one. As a normal buyer I don’t see myself having a positive sides on this one. Truly I realize we are inevitably encountering Mother Nature’s updates on how we should deal with the assets she has given. In any case, by God! indeed, even the flavors that is normal and required in each family (ex: GARLIC †my preferred zest with chicken) is excessively expensive. Counting our staple food wh ich is rice. This one should be lightened! Recollect County comprises of People, People needs Education however above all it needs to FOOD so as to endure!

Artificial Intelligence Ethical Dilemma

Question: Talk about the Artificial Intelligencefor Ethical Dilemma. Answer: Presentation The ethics of mechanized thinking and AI are the principle parts of morals in new innovations, especially, in apply autonomy, self-ruling vehicles and in other misleadingly smart frameworks. It is regularly segregated into apply autonomy, the significant worry with the moral direct of individuals is that concerning how they design, fabricate, use and treat a misleadingly smart framework. This investigation is done to comprehend the moral predicament looked by an AI advisor of the firm of electric vehicles. This examination incorporates a few qualities just as moral choices and ways to deal with settle the moral predicament. The ACS code of morals are talked about in this investigation to conquer the issues in a moral difficulty. Moral Analysis Moral Dilemma The electric vehicle association dont have the ability to effectively shield themselves from open harms from advances, and moreover, they are likewise incapable to urge the customers to purchase their automobiles. As a matter of fact, John feels that the talk should be there in a general way required for all independently directed administrators inside the firm and it is crucial to give them a possibility and settle on them allowed to settle on decisions for the two customers and the association. The association for which John works is viably building up every year. The good or moral trouble here is that, Can electric cars easily learnempathy? Could electric carseffectively rehearse assorted moral decisions? Will an association give electric automobiles a complete understanding of a danger? John, an AI master insistently feels that the electric auto association's things should not to be discharged until the depiction and exhibiting of cars have been appropriately executed, as the liv es of customers and people might be at high danger. John has endeavored to instruct about each such danger to the CEO of the association. John, in like manner, feels that the association will be not able to fulfill its objectives just as be wonderful in an algorithmic significant nature of the AI. Computer based intelligence Consultant Consequences A moral reaction to the moral situation which the AI advisor of the vehicle organization John is confronting is that there is a need of streamlining of coordinations, making out of electric vehicles, regulating of an examination which gives interpretations to a canny electric vehicles structure to change the lives of the vehicle purchasers. Different endeavors are required to beat the issues of the contention. Subsequently, the AI advisor needs to set up the frameworks which are progressively proficient for the clients and furthermore for the organization. Therefore, the individuals who have assets in the AI, incited organization and will have the option to get a lot of cash-flow. Or maybe, self-administering vehicles are chosen various decisions, for example, speed, atmosphere, states of street and other data amassed from a combination of sensors, including cameras and radars. The self-governing auto will process a course of action regarding how rapidly it is going likewise with the speed of the vehicle. The guideline test is to set up the fundamental data which is sufficient to avoid the risky conditions regardless. Vehicle Customers Consequences Due to this ethical circumstance in the electric vehicle firm, the AI master has developed the most beneficial electric cars to diminish the setbacks and for saving the lives of the individuals. Extraordinary anomaly of items over the rowdiness. Issues just as issues with the utilitarianism. Simulated intelligence Consultant Duties Simulated intelligence advisor needs to sort out a gathering just as call up the CEO and some staff individuals, for example, venture pioneers, specialists, managers and examine about the whole situation with them so as to dodge the future disarrays. At the point when each and every disarray identified with the demonstrating venture is appropriately characterized, at that point the AI advisor must allot various undertakings to various staff individuals and to each worker according to their claim to fame and capacity. The AI specialist must allot the exact timeframe to finish the specific undertaking, so the venture ought to be finished on schedule and help association to accomplish the future advantages. The AI advisor, John need to empower their representatives, group pioneer, just as undertaking chief to build their effectiveness and functionality. The AI specialist likewise need to part his work moves with the goal that work will without a doubt be proceeded until the entire under taking isn't finished. Reaction of the Caring Person Typically the center morals endeavors to keep up the associations by contextualizing and propelling the thriving of guardians just as care-recipients in an arrangement of social relations (Nordenstam, 2013). Consistently portrayed as a training or perfect rather than a speculation, care incorporates the keeping up of the trust of the clients, and giving electric vehicles that doubtlessly moderate the street mishaps. Electric vehicles which firm proposals to their clients must address all necessities of the association just as their clients (Nagenborg, Capurro, Weber, Pingel, 2007). John must form an inspiration program for the CEO and other staff of the firm to give care to those people and clients who are helpless just as needy. ACS Code The significance of open enthusiasm: According to this ACS code, each contention must be illuminated inside an electric vehicle organization in the inclusion of open tendency. The enthusiasm of open ought to be thought about to join all the issues identified with the wellbeing of open and vehicle clients. The improvement of individual fulfillment: The ICT progression had the great impact on society and this impact has been invaluable to the electric vehicle organization. The firm ought to lift the comparable access to the occasion of the ICT by all overall population people (Lenk, 2016). Genuineness: This code is a key portion of trust. Without complete trust, an electric firm can't work sufficiently. The real figuring capable ought not make a deliberately bogus and deluding case about the electric vehicles or their system arrangement, be that as it may, it ought to rather give the full disclosure of all corresponded structure limitations and issues. Fitness: The electric firm ought to recognize that the displaying methodology of vehicles in which they trust are skilled to proceed just as not stop for a second to get the additional mastery reasonably qualified for the individuals where fitting (ICT Integrity: bringing the ACS code of morals cutting-edge, 2016). Proficient turn of events: The electric vehicle firm consistently keep themselves refreshed with the new advances and strategies which are material or accommodating in their work, besides, they have to support their accomplices and staff people to do a comparable thing (Tripathi, 2010). Polished skill: The ICT business is modestly depicted just as evolved by the brisk changes. The code of ACS can moreover assist everybody with determining or recover the ethical difficulty, and it gives the help to make the best possible activities against the issue (Gogoll, Muller, 2016). Prerequisite letter: The requests of this examination according to the ACS may be: Open intrigue: The electric firm should decide the potential wedged through firms fill in just as unequivocally imagine their advantage. Ability: Happily assess the duty of their own work. Trustworthiness: Never wittingly delude the client as per the reasonableness of the organizations administrations. Polished methodology: Abstain from the movement into the company's master job that may stain the image of the business (Torrance, 2012). Protection for AI Consultant There are a few different ways to protection the John on moral grounds, as, on the off chance that he runs the computerized reasoning components of the vehicles and illuminate the CEO regarding the electric vehicle organization that later on, the models of vehicles will cause a few ramifications for the association just as for their clients, for example, mishaps out and about are less a result of the self-ruling vehicles (Reisch, 2011). John can give a task report to the CEO of the firm that the venture is surrendered with heaps of blunders and bugs without completely fulfilling the displaying venture in the wake of testing just as it in like manner can make an incredible picture of the firm according to the clients (Chau, 2010). John must use the ACS leads as they dont grant the firm to convey the deserted items and administrations for their clients and mislead the client according to their administrations so John likewise need to attempt recommendations to understand moral issue. Guidance for AI Consultant As demonstrated by the ACS ethics, an association must need to hold their validity, capacity, polished methodology just as open eagerness for their work. According to the John, the time being spent on the displaying method for the mishap premise is a smart thought before the electric vehicles are released on the streets. Securing the convictions of other qualified pros can help with the plan of the issue. Partners can give their decisions and allude to other relative issues they had stood up to. In the wake of guiding various planners, the accompanying step would suggest different codes of ethics and see concerning how they would relate to the situation close by (Bellekom, Benders, Pelgrm, Moll, 2012). NSPE records numerous codes of ethics that a specialist engineer could fall in a condition where the individual would defy out in the business. ASME, gives a summary of certain standards, especially for master mechanical draftsmen. End This investigation reasons that John as an Artificial Intelligence expert for the electric vehicle firm needs to maintain and modernize the pride, trust and respect of his calling. It has additionally been presumed that the morals are existing together in the electric vehicle firm for better venturesome practices just as for holding the clients trust on the firm by giving them administrations which are beneficial for them in an ethical manner. Morals are utilized in the associations to survive

3D ProJet 5500x firmy 3D Systems Essay Example | Topics and Well Written Essays - 500 words

3D ProJet 5500x firmy 3D Systems - Essay Example The speed is twofold regarding extent thought about those of past printer variants vi. The printouts have fluctuating surfaces and appearances. The distinction in surface and appearance is because of the various materials that are blended to make the items. For example, elastic and plastic materials have various surfaces and appearances (3D Systems Polska, 2014) I. Rapid preparing power. The printer produces gatherings proficiently per unit time. Accordingly, it can join multi-materials rapid guaranteeing accommodation in consistent and work escalated mechanical exercises ii. Past adaptations may have been parting printing tasks into various stages. Nonetheless, the 5500x innovation based printer consolidates a few activities into one procedure, a viewpoint that further upgrades effectiveness iii. The printer has an appended multi year guarantee that ensures the client of the nature of the item. In this way, buyers are satisfied with the item since they can generally advance accruable whines to the concerned makers The printer can deliver extensively enormous printouts by utilizing the progressed 5500x printing innovation. Contrasted with the results of past printers, the 5500x congregations are 60 percent bigger. Multi-materials are delivered by excellence of the multi-fly printing innovation that makes quality composite materials of various hues and fixings. The innovation is equipped for delivering machines with various hues and materials. Speed is additionally a basic useful segment of the printer’s capacities. Sound system lithography is the premise of activity showed by most printers. PC Aided Design utilizes laser shafts to make or print to deliver the ideal 3D gatherings. The ProJet 5500x utilizations a similar rule of activity. Nonetheless, the new 3D printing innovation is of a higher preparing speed when contrasted with different printers. Its center working

The Essay Expert Presents How to Write a Killer LinkedIn Profile - Wednesday April 6, 1200pm CST

The Essay Expert Presents How to Write a Killer LinkedIn Profile - Wednesday April 6, 1200pm CST Some of you have been on my LinkedIn webinars before. At least one of you got a call from a recruiter one week after making the changes I suggested just from listening to the webinar!   Want to hear the recording of my last webinar?   Click on   How to Write a Killer LinkedIn Profile. Your next opportunity to hear me present LIVE is this Wednesday, April 6, 2011 at 12:00pm Central time.     Why would you want to attend this webinar? If you are a professional in any industry, you simply cant afford to have a mediocre LinkedIn profile. Your potential employers will all be scrutinizing your profile, and you want to make a great impression. In this webinar, I will take you through the most important sections of your profile and the most important strategies for getting found. Topics covered: • Why care about LinkedIn? • Whats the point of 500+ connections? • What should I write in my headline to get found? • What should I write in my Summary? • What are LinkedIn applications and how do I use them? • How do I add LinkedIns new sections to my profile? • How can I make the most of LinkedIn groups? Join me and Robert Shindell of ilostmyjob.com to learn the answers to these questions and make a Killer impression on the people who find you on LinkedIn. Wednesday April 6th 12:00pm CST

Hiring an Independent College Counselor

10 Tips for Choosing an Independent College Counselor Previously, we gave you a list of ways that you can work with your high school college counselor during the college admissions process. For families seeking additional guidance, it may be helpful to also use an independent college counselor. An independent counselor can supplement your school’s guidance efforts by providing personalized guidance throughout the college admissions process including creating a balanced college list, compiling and formatting the resume, preparing for interviews and college visits, explaining the admissions process, and brainstorming and editing components of the application including the essays and personal statement. At, we guide students and their families through every step of the school admissions process, from identifying the schools that will be the best academic and social fits for them to creating applications that portray the best and most accurate representation of who they are to the admissions committees at their targeted schools. If you are interested in hiring an independent counselor, look for the following criteria: Full membership with the IECA (Independent Education Consultants Association), and ideally a member of NACAC (National Association of College Admissions Counselors) A certificate in college counseling, a master’s degree in education, or a certified education planner Experience working in a selective college’s admissions office or for a competitive high school’s college counseling office Ongoing professional development such as attendance (and participation/presenting) at college admissions conferences Contributes to the profession regularly via articles, books, online tools, blogs, podcasts, webinars, television, social media, newsletters, etc. Has visited at least 100 colleges and universities and continues to visit about 10 per year to stay up-to-date with the latest college information Tip: Ask him or her which colleges he or she has visited in the last 3 years Ability to see their history of success and get testimonials easily What counseling methodology does he or she use? Counseling sessions should be followed up with comprehensive notes and clear next steps. Parents should be kept in the loop and the counselor should be accessible during after-work hours and on weekends, especially during the busy application season Follows standards of ethics and good practice – does he or she know about these guidelines and follow them? These standards should be posted in their office Willingness to work in collaboration with your school guidance counselor An independent counselor should not: Contact an admissions office on behalf of the student Promise admission to a particular school Write or overly-edit essays for the student Receive any compensation from educational institutions One of the most important parts of the college admissions process is learning about yourself and identifying what you want from your education. Whether or not you decide to work with an independent college counselor, surround yourself with those who encourage you to believe in yourself, both academically and personally and you will ultimately go wherever your hard work and dedication take you.

Buds Big Blue Manufacturing Case - Free Essay Example

Sample details Pages: 4 Words: 1208 Downloads: 7 Date added: 2017/09/18 Category Management Essay Type Argumentative essay Tags: Manufacturing Essay Did you like this example? 1. ) After performing any alternative procedures, the auditor should evaluate the combined evidence provided by the confirmations and the alternative procedures to determine whether sufficient evidence has been obtained about all the applicable financial statement assertions. In performing that evaluation, the auditor should consider (a) the reliability of the confirmations and alternative procedures; (b) the nature of any exceptions, including the implications, both quantitative and qualitative, of those exceptions; (c) the evidence provided by other procedures; and (d) whether additional evidence is needed. If the combined evidence provided by the confirmations, alternative procedures, and other procedures is not sufficient, the auditor should request additional confirmations or extend other tests, such as tests of details or analytical procedures. (AU 330) Paragraph . 09 of section 326, Audit Evidence, states that the auditor should consider the reliability of the infor mation to be used as audit evidence. Confirmations obtained electronically can be considered to be reliable audit evidence if the auditor is satisfied that (a) the electronic confirmation process is secure and properly controlled, (b) the information obtained is a direct communication in response to a request, and (c) the information is obtained from a third party who is the intended respondent. 2. ) During the performance of confirmation procedures, the auditor should maintain control over the confirmation requests and responses. Maintaining control means establishing direct communication between the intended recipient and the auditor to minimize the possibility that the results will be biased because the interception and alteration of the confirmation requests or responses. The possibility that results will be biased based on interception and alteration of the confirmation requests and responses, is a prospect of what can go wrong if the auditor does not maintain control ove r the confirmation process. 3. ) Confirmation #Date SentDate ReceivedDifference Noted By Customer? Is the noted difference a misstatement Has sufficient evidence been obtained that confirms the client’s recorded balance? If no, describe additional procedures you believe should be performed. 711/12/091/22/09YesNoYes 721/12/092/5/09NoNoYes 731/12/091/26/09NoNoYes 741/12/092/9/09N/AN/AFollow up why they didn’t follow procedure and mail directly back to auditing firm. 751/12/092/6/09NoNoYes 761/12/091/30/09NoNoNo. Investigate BBB stamp on paper. Date received day after that noted on stamp 771/12/091/19/09YesYesCheck cash receipts. 4. ) Confirmation is the process of obtaining and evaluating a direct communication from a third party in response to a request for information about a particular item affecting financial statement assertions. Negative confirmation is a written request by the external auditor sent to a party having a financial relationship with the client a nd asking for a reply only in the case of disagreement. The negative form requests the recipient to respond only if he or she disagrees with the information stated on the request. Negative confirmation requests may be used to reduce audit risk to an acceptable level when (a) the combined assessed level of inherent and control risk is low, (b) a large number of small balances is involved, and (c) the auditor has no reason to believe that the recipients of the requests are unlikely to give them consideration. The auditor should consider performing other substantive procedures to supplement the use of negative confirmations. Negative confirmation requests may generate responses indicating misstatements, and are more likely to do so if the auditor sends a large number of negative confirmation requests and such misstatements are widespread. The auditor should investigate relevant information provided on negative confirmations that have been returned to the auditor to determine the effect such information may have on the audit. If the auditors investigation of responses to negative confirmation requests indicates a pattern of misstatements, the auditor should reconsider his or her combined assessed level of inherent and control risk and consider the effect on planned audit procedures. Although returned negative confirmations may provide evidence about the financial statement assertions, unreturned negative confirmation requests rarely provide significant evidence concerning financial statement assertions other than certain aspects of the existence assertion. Positive confirmation is a written or oral request by the auditor of a party having financial dealings with the client about the accuracy of an item. A response is required whether the particular item is correct or incorrect. A positive confirmation can be sent to customers to verify account balances. Some positive forms request the respondent to indicate whether he or she agrees with the information stated on the request. Other positive forms, referred to as blank forms, do not state the amount (or other information) on the confirmation request, but request the recipient to fill in the balance or furnish other information. Positive forms provide audit evidence only when responses are received from the recipients; non responses do not provide audit evidence about the financial statement assertions being addressed. Since there is a risk that recipients of a positive form of confirmation request with the information to be confirmed contained on it may sign and return the confirmation without verifying that the information is correct, blank forms may be used as one way to mitigate this risk. Thus, the use of blank confirmation requests may provide a greater degree of assurance about the information confirmed. However, blank forms might result in lower response rates because additional effort may be required of the recipients; consequently, the auditor may have to perform mor e alternative procedures. 5. ) A real-life situation where an auditor apparently did not maintain sufficient control over the confirmation process, was an incident in 1994, where Sky Scientific, Inc. ramatically overstated assets and understated expenses in its financial statements. An SEC investigation into those financial statements led to charges being filed against both the company and its auditors. This case takes a detailed look into the independent audit of Sky’s financial statements for the purpose of understanding how the auditors failed to see the extent to which Sky’s financial reporting was misleading and to adequately challenge the assertions presented in the financial statements before Sky filed them with the SEC. This case provides an opportunity to examine numerous issues related to the audit engagement process, including audit planning, the evaluation of management representations, audit evidence and the confirmation process. The roles and responsib ilities of the concurring auditor and an outside specialist are also discussed. SEC Charges In late July 1994 a senior geologist on the Commission’s staff visited some of Sky’s California and Nevada mining operations. At three of those properties she found no evidence of current or recent mining activity, no equipment, and no personnel. At another mine she found equipment and personnel but no tailings that might indicate that recent operations had existed. As a result of these observations and the recent filing of Sky’s audited financial statements, the SEC opened its investigation of Sky on August 26, 1994. In 1999 the SEC charged several members of Sky management with fraudulent accounting practices and for filing false and misleading financial statements with the Commission. They also charged American Capital Network and Robert Schlien with violations of several federal securities laws. Barry Scutillo and Mark Jensen were charged with engaging in improp er professional conduct in that they intentionally, knowingly and recklessly violated the applicable professional standards when auditing a public company. Specifically, Scutillo and Jensen were charged with recklessly failing to see that most of Sky’s assets were fictitious or massively overvalued and that millions of dollars in expenses had not been recorded on Sky’s books. Don’t waste time! Our writers will create an original "Buds Big Blue Manufacturing Case" essay for you Create order

Environmental Planning in Phoenix - 888 Words

Much like the economic laws of supply and demand, there is a public demand for land-use and infrastructure, the ways in which people get from point A to point B affect general planning, for example, biking to school or work in the city of Tempe is common, so the high demand for bike lanes gets incorporated into Tempes general plan. A vital purpose of general planning is to designate specific land-use, for instance a particular area in Phoenix can be reserved for commercial use or private use only, these delineations are useful when trying to separate industrial areas that can potentially contain harmful or unpleasant waste from neighborhoods. Many of the elements in general planning interconnect, for example as just explained ineffective land-use can directly and adversely contribute to the environmental element of planning. If one was to try to summarize environmental planning with two words they would be sustainability and waste management; when people talk about sustainability the y are typically referring to sustainable energy; energy that does not hinder the lives of future generations. Developed countries are entering a progressive era which requires the use of renewable energy and sustainable growth, as CO ¬2 levels sour past the 400PPM range--meanwhile corporations are lobbying for the right to continue polluting our air and water supply, it has become evident that the demand for sustainable energy is higher than ever. 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The Fame of Coco Chanel - 629 Words

In the roaring 1920’s, women were in corsets, that restricted many women to not eat nor breathe. That’s when Coco Chanel change everything for women, that made her career in the fashion industry go through the roof. Before Coco became an icon in the fashion industry, she was put in an orphanage ever since her dad had left her sister and her. Coco was then raised by nuns, which is where she was taught to sew, a skill will help her to fame. Though her name wasn’t always Coco, her birth name was Gabrielle Chanel. Her nickname came from her first job as a singer at clubs around where she lived, that is France. It was up until her relationship with Etienne Balsan, that funded Coco’s first store. Her first store was located in Paris Cambon in 1910, where she sold hats for women. Coco later on left Etienne for one of his friends, Arthur Capel, that was wealthier than him. Arthur also help start up Coco’s fashion career. It wasn’t till Coco was seen wearing her husbands jersey that made her a fashion icon. Since then her career has been lucrative. Chanel introduced men clothing to women, she designed suits without collars and fitted shirts that made her an instant fashion icon. Coco Chanel’s clothing accentuated comfort to women in the 1920’s. After her death Coco Chanel has left popular fashion industry, she was important in the 1920’s that changed how women dressed. Coco Chanel was influential in the roaring 1920’s because she introduced men clothing into the women fashion. CocoShow MoreRelatedPersonal Statement : Empty Closet885 Words   |  4 Pageswill not solicit the mood for romance. If a touch of flirtatious personality is added, the person may end up with a replica of Coco Chanel’s â€Å"†¦little black dress, which seemed, at first glance, artless, rustic grab and impersonal. 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Persuasive Essay On Abortion - 954 Words

Mother Teresa once said, â€Å"For me, life is the most beautiful gift of God to mankind, therefore people and nations who destroy life by abortion and euthanasia are the poorest. I do not say legal or illegal, but I think that no human hand should be raised to kill life, since life is God’s life in us, even in an unborn child.† Do you know what abortion really is? Abortion is the ending of pregnancy by removing a fetus or embryo before it can survive outside the uterus. Abortion seems to be more common in today’s society. Numerous amounts of individuals support abortion, and do not realize how it affects themselves and others. Those who do not support this poor decision may choose to let it interfere with their relationship with others. The†¦show more content†¦Putting forth effort to conceive a child means that whether they are healthy or have some type of deformity or disease, you will love them unconditionally, and not worry about the difficulties th ey may have down the road. A child does not deserve to die from the crimes of his or her father’s. One can not kill a five-year-old because his or her father is a rapist nor should a five-month-old be killed for the same reason. No child deserves to die because his or her mother and/or father chooses irresponsibility. A child is completely faultless to their actions. They do not get to decide whether or not their parents conceive nor do they get to decide if they use any type of contraception. All innocent, unborn children should not die. Time does not erase murder or ease the reality of what it really means. Having an abortion results in a cruel tragedy. This decision should never be considered. The future of an unborn child includes an abundance of experiences, projects, and activities. Every individual born or unborn has an extremely valuable future full of many opportunities. When killing these unborn children, there is a valuable life being destroyed. These children will never get the chance to become part of today’s society nor will they be able to make an impact in future state. When an abundance of women abort their children a toll on population occurs around the world. Some would even sayShow MoreRelatedPersuasive Essay About Abortion993 Words   |  4 Pagesin two peoples perspectives. The story consisted of a daughter (Deb) and mother’s perspective after the death of her father. The purpose of this layout is to show how they are both grieving in individual ways. Writing for the persuasive was presented as an essay on why abortion should be accessible in every country, and to also outline the shame women receive. Purpose Purpose for writing publication- Purpose for writing a real-estate article was to inform a potential buyer on the house showed. AlsoRead MoreAbortion Persuasive Essay1119 Words   |  5 Pages To begin with, there has always been an extensive issue with the topic of abortion, why? Well, it’s a tough subject and can get extremely emotional and very defensive. Honestly, people just need to open up their eye’s and understand that its murder and not just a choice. It is murder because two can create a blessing and the choice of aborting it, is more like a sacrifice for the reason a mother can’t carry a baby for nine months. Whether a person is a minor, young adult, or an adult and is inRead MoreAbortion Persuasive Essay755 Words   |  4 Pagesinstead these babies are torn limb by limb, burned, and many other tortuous methods to end the precious life. Abortion is murder and is not only very cruel to the unborn baby but it also harms the mother. Pro-choice is an escape from the harsh reality that a baby is being murdered, and in my eyes a life should only be terminated if God himself is ready to call them home. One reason why abortion is wrong is because they are many other safe solutions that would give the child a chance at life. AdoptionRead MorePersuasive Essay On Abortion917 Words   |  4 PagesAbortions in Texas consist of ending a pregnancy of an unborn child before it can live out of the mother womb. However if the pregnancy ends not on purpose before the twenty four week mark then technically it would be considered a miscarriage. Though inducing the abortion on purpose has caused a lot of controversy over the past century. Deliberately putting people on one of two sides of this issues, of either being for it, which would be considered Pro Choice. Or in the other category of the ProRead MorePersuasive Essay On Abortion1635 Words   |  7 Pagessolve this issue the option of abortion was brought to the forefront by scientists and advocates such as Dan Savage who mentioned that abortion should be mandatory for the next 30 years in or to control the population. (Ertlet) For countri es such as the U.S. with a growth rate being 1.6%, the proposal of widespread abortion is highly recommended as mentioned by obstetricians and gynecologists. (MumfordKessel) Who now leaves the question as to if the option of abortion should be used as a means ofRead MorePersuasive Essay On Abortion1228 Words   |  5 Pagesmillion pregnancies each year in the United States, 1.6 million end up in abortion† (Hern). Because of the numerous traumatic psychological, physical, and overall irresponsible behavior or actions constantly encouraged due to abortion, it should be banned, and forever illegal. Killing one’s own fetus has been linked to psychological distress which has been channeled into many different cases of substance abuse. â€Å"Induced abortion has been linked to increased rates of substance abuse, especially amongRead MoreAbortion Persuasive Essay804 Words   |  4 Pagesfamily with someone. Often times accidents happen in which people didn’t plan for, and can lead to many financial problems or neglect of one’s child because the parents weren’t prepared to be parents. This is why there should be the option of abortion. Abortion needs to be an option for everyone because it helps from long lasting mental states, infection, economical problems, and or having conceived from a non consensual circumstance. Pregnancy is a hard and unforgiving to the woman’s body. In factRead MoreAbortion Persuasive Essay736 Words   |  3 Pagesfavor of abortion rights. The decision was 7-2, it stated that it showed a â€Å"right of privacy.†Abortions have been performed for thousands of years all around the world, even though many women died from it. Women nowadays have a choice of whether they want to get an abortion. Abortions do come with risk, one of them being death, but women should still have a right and say what they want to do with pregnancy and not be judged by others for doing so. Today the debate across the world is if abortion shouldRead MorePersuasive Essay On Abortion1321 Words   |  6 Pagessomeone who may be suicidal or who is suicidal himself/herself. As for taking away fetus’ lives, the damage that abortion makes on life can be proven with the statistics. According to Casey, â€Å"over the past 44 years, one-sixth (60,000,000) of the American population has been killed by elective abortion. Twenty-five percent of African-Americans are killed in the womb America,† (Should Abortion Be Legal n.p.). The figure 60,000,000 is a depressing number considering that these children could have been futureRead MorePersuasive Essay On Abortion723 Words   |  3 Pagescontraception I want to discuss is sterilization. For an idea that is so broadly accepted in private, contraception certainly generates a lot of opposition when discussed in public. Some people think when we talk about contraception that it’s code for abortion. It’s obviously not the same thing. Let’s be honest, people are just uncomfortable with the topic because it’s about sex. And sex talk in this country is kept on the hush even though we are sold sex via advertising left and right. Some people worry

Human Immunodeficiency virus 1 (HIV-1) Free Essays

string(53) " RNA plasma levels of less than 1,500 copies per ml\." 1. Introduction 1.1Human Immunodeficiency virus 1 (HIV-1) 1. We will write a custom essay sample on Human Immunodeficiency virus 1 (HIV-1) or any similar topic only for you Order Now 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 main t ransmission 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].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 barr ier 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 cells 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].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].In addition to its fusion, it is essential for virus recognition and entry into target cells. 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].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 inn er 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. You read "Human Immunodeficiency virus 1 (HIV-1)" in category "Essay examples" 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 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 higher 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 efficacious 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 preventing 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 could potentially fill an important HIV prevention g ap [38]. 1.2.1vaccine 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 hypothe sis 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 fre e 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 50Â µg of CN54gp140 in different formulations. A total volume of 1100Â µl was for each rabbit, 800Â µl was used per rabbit immunization. The formulations per rabbit and groups are as follows: Group 1: 742.9 Â µl Wild type CN54 + 945Â µl LASTS+ 4612.1 Â µl PBS 2.4Immunization groups A table showing the immunization groups that the rabbits belong to. 2.5Immunization 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. 2.6Reagents 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 (? 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 Reagent preparation 1. Coating buffer, sterile PBS pH 7.4 1.1Prepare coating buffer by adding 50ml 10X PBS to 450ml de-ionised water. 2. Washing buffer, 0.1% TWEEN-20 in 1X PBS (PBST) 2.1Prepare washing buffer by adding 100ml 10X PBS to 900ml of deionised water. Add 500Â µ of TWEEN-20 and mixing thoroughly 3. Assay buffer, 10% FBS in PBST 3.1Prepare assay buffer by adding 10ml FBS to 90ml PBST 3.2Filter sterilise 2.7ELISA 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 50Â µl/well of HIV-1 gp140 at 5Â µg/ml in Phosphate Buffered Saline (PBS) for an hour at 37Â °C. The wells were washed (wash procedure was 4 washes in PBST) and blocked for one hour at 37Â °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 37Â °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 50Â µl TMB (Sureblue TMB 1-component peroxidase substrate (KPL) for five minutes in the dark. The reaction was terminated by the addition of 50Â 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. 2.8Zeta Potential Measurements of gp140 constructs The Zeta-potential, (?) of the gp140 molecules used for immunization was determined under a range of pH and salinity conditions with a Malvern ZetaSizer Nano ZS. 188 Â 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. 2.9Statistics 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. Results 3.1Zeta 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 of t he 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) 3.2.4Grouped 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 that thi s 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. 4.2.2End 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 additi on 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 conf ormations that were able to elicit a good immune response. Gp120, as previously described, works in conjunction with gp41 to allow the virus to get close enough to the cell membrane and inject its genome into the target cell cytoplasm. The difference between the native gp140 and wild type infectious protein is that the gp140 is not near the target cell membrane. There is also a mutation on the cleavage site that would normally result in a gp41 and gp120 molecule being created instead of a single gp140-but the same external face and glycosylation patterns on both types of proteins will be the same. 4.5 Conclusion This study was successful in revealing whether vaccine response using the trimer CN54gp140 fixed in different conformations would elicit a modified antibody response. 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