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. 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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. A decreased titre of antibody concentration was observed when the protein was fixed at pH 4.0 and 7.2 and there was only an increase in serum antibody in the protein fixed at pH 5.5. However the best response was seen in the immunizations with the wild type protein, especially in the group three where the protein was combined with the HIV inhibitory drug DS003. 5References 1. UNAIDS. (2010). Worldwide HIV and AIDS statistics. Available: http://www.avert.org/worldstats.htm. Last accessed 16 April 2011. 2. Fanales-Belsio.E, Raimondo.M, Suligoi.B and Butto.S. (2010). HIV virology and pathogenetic mechanisms of infection: a brief overview.HIV virus and pathogenicity,46(1): 5-14 3.Pillay.S, Shephard.E, Meyers.A, Williamson.A, Rybicki.E. (2010). HIV-1 sub-type C chimaeric VLPs boost cellular immune response in mice .Journal of Immune based therapies and vaccines, 8 (7). 4.Hook, E. W., III, R. O. Cannon, A.J. Nahmias, F.F. Lee, C. H. Campbell, Jr., D. Glasser, and T.C. Quinn. (1992). 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J, and Akkina. R. (2007). Complete knockdown of CCR5 by lentiviral vector-expressed siRNAs and protection of transgenic macrophages against HIV-1 infection. Gene therapy: 14, 1287-1297 26. Mines.M.A, Goodwin.J.S, Limbird.L.E, Cui.F, and Fan.G-H. (2009). Deubiqitination of CXCR4 by USP14 Is critical for both CXCL12-induced CXCR4 degradation and chemotaxis but not ERK activation. J. Biol. Chem: 284 (9), 5742-5752 27. Moriuchi. M, Moriuchi. H, Turner. W, and Fauci. A.S. (1997). Cloning and analysis of the promoter region of CXCR4, a coreceptor for HIV-1 entry. J.Immunol: 159 (9), 4322-4329 28. Curran.R, Donnelly.L, Morrow.R, Fraser.C, Andrews.G, Cranage.M, Malcolm.K, Shattock.R, Woolfson.D. (2009). Vaginal delivery of the recombinant HIV-1 clade-C trimeric gp140 envelope protein CN54gp140 within novel rheologically structured vehicles elicits specific immune responsese. Vaccine: 27 (48), 6791-6798. 29. Conant.M, Hardy. D, Sernattinger. J, Spicer. D, and Levy. J. (1986). condoms prevent transmission of the AIDS-associated retrovirus by oral-genital contact. JAMA: 225, 1706 30. Ngugi. E, Plummer. F, Simonsen. J, Cameron. D, Bosire. M, Waiyaki. P, Ronald. A, and Ndinya-achola. J. (1988). prevention of transmission of human immunodeficiency virus in Africa: effectiveness of condom promotion and health education amongst prostitutes. Lancet: ii, 887-890 31. van der Straten. A, Kang. M, Posner. S, Kamba. K, Chipato. T, and Padian. N. (2005). predictors of diaphragm use as a potential sexually transmitted disease/HIV prevention method in Zimbabwe.sex.transm.dis:32, 64-71 32. Nag. P, Kim. J, Sapiega. V, Landay. A, Bremer. J, Mestecky. J, Reichelderfer. P, Kovacs. A, Cohn. J, Weiser. B, and Baum. L. (2004). women with cervicovaginal antibody-dependent cell-mediated cytotoxicity have lower genital HIV-1 RNA loads.j.infect.dis: 190, 1970-1978 33. Cameron. D, Simonsen. J. N, D’Costa. L. J, Ronald. A. R, Maitha. G. M, Gakinya. M. N, Cheang. M, Ndinya-Achola. J. O, Piot. P, Brunham. R. C, and Plummer. F. A. (1989). Female to male transmission of human immunodeficiency virus type 1: risk factors for seroconversion in men. Lancet. ii: 403-407 34. Levy. J. A. (2004). prospects for an AIDS vaccine: encourage innate immunity. AIDS: 18,2085-2086 35. Levy.J. A, Scott. I, and Mackewicz. C. (2003). protection from HIV/AIDS: the importance of innate immunity. clin.immunol: 108, 167-174 36. Pashine. A, Valiant. N, and Ulmer. J. (2005). Targeting the innate immune response with improved vaccine adjuvants. nat.med: 11, s63-s68. 37. Miller. C. J, Li. Q, Abel. K, Kim. E. Y, Ma. Z. M, Wietgrefe. S, Franco-Scheuch. L. La, Compton. L, Duan. L, Shore. M. D, Zupancic. M, Busch. M, Carlis. J, Wolinsky. S, and Haase. A. T. (2005). Propagation and dissemination of infection after vaginal transmission of simian immunodeficiency virus. J. Virol: 79, 9217-9227 38. Karim. Q, Karim. S, Frohlich. J, Grobler. A, Baxter. C, Mansoor.L, Kharsany. A, Sibeko. S, Mlisana. K, Omar. Z, Gengiah. T, Maarschalk. S, Arulappan. N, Mlotshwa. M, Mprris. L, and Taylor. D. 2010. Effectiveness and safety of tenofovir Gel, an antiretroviral Microbicide, for the prevention of HIV infection in women. Science: 329 (5996), 1168-1174 39. Pitisuttithum. P, Gilbert. P, Gurwith. M, Heyward. W, Martin. M, van Griensven. F, Hu. D, Tappero. J, and Bangkok Vaccine evaluation group. 2001. Randomized, double-blind, placebo- controlled efficacy trial of a bivalent recombinant glycoprotein 120 HIV-1 vaccine among injection users in Bangkok, Thailand. J. Infec. Dis: 194(12), 661-1671 40. Nitayaphan. S, Pitisuttithum. P, Karnasuta. C, Eamsila. C, de Souza. M, Morgan. P, Polonis. V, Benenson. M, VanCott. T, Ratto-Kim. S, Kim. J, Thapinta. D, Garner. R, Bussatid. V, Singharaj. P, el Habib. R, Gurunathan. S, Heyward. W, Birx. D, McNeil.J, and Brown. A. (2004). Safety and Immunogenicity of an HIV subtype B and E prime-boost Vaccine combination in HIV-negative Thai adults. J. Infec. Dis: 190, 702-706 41. Jones. N, DeCamp. A, Gilbert. P, Peterson. M, Gurwith. M, and Cao. H. 2008. AIDSVAX immunization induces HIV-specific CD8+ T-cell responses in high- risk HIV-negative volunteers who subsequently acquire HIV infection. Vac. 27: 1136-1140 42. Pillay.S, Shephard.E, Meyers.A, Williamson.A, Rybicki.E. (2010). HIV-1 sub-type C chimaeric VLPs boost cellular immune response in mice.Journal of Immune based therapies and vaccines: 8 (7) 43. Graham.B. S. (2009). What does the report of the USMHRP phase III study in Thailand mean for HIV and for vaccine developers. The journal of Transitional Immunoligy: 158, 257-259. 44. Montefiori. D and Mascola. J. (2009). Neutralizing antibodies against HIV-1: can we elicit them with vaccines and how much do we need. Cuur Opin HIV AIDS: 4 (5), 347-351. 45. Gnanakaran.S, Daniels.M, Bhattacharya.T, Lapedes.A, Sethi.A, Li.M, Tang.H, Gao.H, Haynes.B, Cohen.M, Shaw.G, Seaman.M, Kumar.A, Gao.F, Montefoiri.D, Korber.B. (2010). Genetic signatures in the Envelope Glycoprotein of HIV-1 that associate with broadly neutralizing antibodies. PLoS COMPUTATIONAL BIOLOGY, 6 (10). 46. Garg. A, Nuttall. J, and Romano. J. (2008). The future of HIV microbicides: challenges and opportunities. Antiv. Chem Chemo: 19, 143-150 47. Nuttall. J, Romano. J, Douville. K, Galbreath. C, Nel. A, Heyward. W, Mitchnick. M, Walker. S, Rosenberg. Z. (2007). The future of HIV prevention: Prospects for an effective anti-HIV microbicide. Infect. Dis. Clin. N. Am: 21, 219-239 How to cite Human Immunodeficiency virus 1 (HIV-1), Essay examples

Miss Julie Commentary Essay Example For Students

Miss Julie Commentary Essay In the play, Miss Julie, the element that grabbed my interest was the natural behaviors of the characters, and how they interacted with each other. Especially the conversations between Miss Julie and Jean, they were two different ranked individuals, but still the upper ranked Miss Julie wanted to have an affair, or have a date with the lower ranked Jean. Jean and Kristin were both workers and helpers for Miss Julie, and from experience and common sense, it is quite surprising that the owner falls in love with the workers. When Miss Julie finds out that she doesnt have a dance partner, she decides to bring Jean for her partner. However, Jean is already taken by his fianci , Kristin. Miss Julie, of course, doesnt care whether Jean is taken from; she just cares about her own needs and desires. She says, {softly} Dont take it as a command. This evening we are all just enjoying ourselves together, and any rank is laid aside. So give me your arm Dont worry Kristin! I shant run off youre your fianci! This shows how much the author, August Strindberg, writes all of the characters behaviors through dialogue, but not words. In the past literature works we have read, it was quite common for the author to characterize the individuals in the plot, and did not characterize much in the dialogue. However, in this work, Miss Julie, the Strindberg did not use any explanation in the plot, nor did he write a small caption how each individual behaves. From this one quote, {softly} Dont take it as a command. This evening we are all just enjoying ourselves together, and any rank is laid aside. So give me your arm Dont worry Kristin! I shant run off youre your fianci! we can already see who Miss Julie is. The reader, including myself, was very into the play mostly because it made us think who the characters are, and made us find it out ourselves. Another element that caught my interest and attention was the behaviors and the actions each character took. Jean, Kristins fianci , takes good care of Miss Julie. Even though Miss Julie told him and Kristin that all ranks are put aside for the night. It is expectable from the readers point of view, that Jean and Kristin will spend time together, and not be any helper for anyone, but a normal engaged couple. However, Jean took the other side of the choice, and stuck with Miss Julie. It was seen the Jean had to dance with Miss Julie, but Jean was with her and helped even after the dance. Also, Miss Julie, the fact that she fell in love with Jean was true, and this shows how she lacks in nervousness. This will catch the readers attention because there is a twist into the play.

Case Study of Qantas Airways-Free-Samples-Myassignmenthelp.com

Question: Discuss about the Case Study of Qantas Airways. Answer: Introduction Human resource frame of a corporation focuses on recruiting employees as well as deciding their role, responsibilities, duties, values, interests, and interactions; in other words, it manages the human capital of an organisation. The human resource frame is administered by the HR department of a company that performs various actions relating to human capital such as recruitment, training, development, retention, compensation, and others. An effective human resource frame efficiently manages employees requirements and increases their productivity through motivation, training, incentives, maintaining a positive organisational environment and removing the conflict between workers and company. In modern business environment, corporations focus on maintaining an effective human resource frame since it provides them a competitive advantage over their competitors; the insightful understanding of human resource frames strengths and weaknesses allow top-level management to formulate policies that improve the productivity of employees and sustain future development of the company. This report will focus on analysing the strengths and weaknesses of human resource frame maintained by Qantas Airways. Further, the report will provide suggestions to reduce the drawbacks of human resource management and to improve the effectiveness of human capital in Qantas Airways. Qantas Airways The Qantas Airways was founded on 16th November 1920, and it is the third oldest airline company is the world; currently, it is the flag carrier and largest airline of Australia based on international destinations offers, overseas flights, and fleet size. The Qantas is an acronym made by the corporation for Queensland and Northern Territory Aerial Services. The company started its international passenger flights services in May 1935; now the company has a total of 118 fleets, and it provides services in more than 85 countries (Qantas, 2017). The head office of Qantas is situated in Mascot, Sydney, Australia and its secondary hubs are in Dubai International Airport, Adelaide airport, and Perth airport. In 2015, the corporation accumulated revenue of AU$16.2 billion and an operating profit of AU$1.03 billion (Ong and Letts, 2016). Qantas Airways has more than 33,826 employees. The subsidiaries of the company include Impulse airlines, Qantas link, Australia Asia airlines, Jetconnect, Jetstar Airways and Network aviation. Qantas Airways is one of the top ten largest airlines in the world, and it is the number one domestic airline in Australia (Rosen, 2017). The company comprises multiple activities through its front and backstage operations. Qantas Airways human resource department focuses on recruiting professionals and providing them extensive training to ensure that they provide excellent services. The productivity and performance of the workers are refined by exceptional training programs that are arranged by the organisation. Qantas management hierarchy includes three levels which are top level, secondary level, and ground level. The top-level management consists of eight members that are headed by a president; the apex level is divided into various departments that manage and formulates policies for various functions such as nomination, audit, recruitment, remunerations, environment, health, security and many others (Day, 2009). The secondary level comprises various executives that look after the policies established by the top level management. This level is divided into 12 central departments which include IT, legal, domestic, finance, loyalties, resources, and others. These departments include managers and air professionals that provide necessary training to workers from ground level in order to develop their skills and abilities. The ground staffs consist of employees that work at ground level such as runways, airports, planes and other places. The diligence and hard work of ground staff allow organisation to provide a high level of service to its customers and sustain their future growth (Sarina and Wri ght, 2015). Strengths of Human Resource Frame Qantas Airways focus on attracting the best talent and developing their skills to ensure they perform at their maximum capacity and provide high-quality services to its customers; the human resource department of the company is highly efficient while selecting professionals and providing them appropriate training (Latemore, 2013). Following are some strengths of Qantas Airways human resource frame. Effective recruitment process Qantas Airways has a robust recruitment process that focuses on hiring professional and qualified candidate for different posts. The HR department thinks outside the box while selecting candidates for the job; they look for extra value in employees rather than just focusing on their credentials (Weller, 2007). To assess unique qualities of candidates, HR department conduct various exercises that provide necessary information which can be utilised while assigning candidates in different positions. Below is a diagram of Qantas Airways recruitment process: Recruitment Process of Qantas Airways Submission of Online Application Interview Psychometric Testing: numerical reasoning, personality tests, verbal reasoning, etc. Assessment Centre In-Tray Exercises Reference Background Checks: Citizenship, Visa, Residency and Medical Check Selection Diversity in Qantas According to Hewett (2016), the CEO of Qantas Airways Alan Joyce provided that maintaining diversity in top-level management assisted them in remaining strong during difficult times. The HR department is committed to maintaining a diverse environment which provides equal respect, value, and compensation to people from different background and gender. The HR department also establishes policies to promote and support recruitment of Aboriginal and Torres Strait Islanders peoples in Australia. As per the diversity report of Qantas Airways, following are the diversity policies of the company (Qantas, 2016): Training employees to treat each other with dignity and respect. Developing inclusive leaders that promote and value diversity in team and environment. Developing diversity programs to ensure employees work at their highest potential. Communicating organisational diversity policies and its growth with workers. Identifying and creating new partnerships that promote and develop diversity in the organisational Building an including culture that is flexible and provides services to all employees. Women Workers Qantas Airways has a long history of promoting and nurturing their women workers and providing various services for their development. Following facilities are provided by HR department to women employees (Qantas, 2016): Equal opportunity, harassment, and anti-discrimination policies Talent management in order to put women in leadership positions Increasing female engineers Female pilots in advertisement and promotion for recruitment Women @ Qantas development program Flexible work, safety, and other benefits Employees Talent Programs Qantas Airways human resource department has implemented various talent and education programs that are focused on improving knowledge and skills of its employees. The company provides scholarships and loans to employees who decided to study further in order to enhance their knowledge (Bamber, 2015). These programs attract a large number of qualified candidates in the recruitment procedure of Qantas Airways. Following are few programs provided by the corporation to its workers: Harvard leadership program Graduate program Women @ Qantas Development Program Emerging Leader program Indigenous careers @ Qantas team University cadetships and School-based traineeships Training and Development Qantas Airways provide world-class training to its employees and professionals to improve their productivity. The company focus on self-learning policies and foster it by establishing a flexible environment. The self-assessment assignments, tasks, and evaluation techniques help the employees to dwell deep and enhance their capabilities and skills (Gittell and Bamber, 2010). The training programs are divided into four main sections to fulfill the requirements of each trainee; this segregation allows employees to learn comfortably, and it reduces the feeling of discrimination. The trainers provide training based on the readiness of employees and motivate them to improve their performance. Weaknesses of Human Resource Frame Along with several strengths, there are few weaknesses in HR frame of Qantas Airways due to various challenges faced by them. Lack of New Talent More than 95 percent of the Qantas Airways operations are situated in Australia, in order to reduce cost; the company hires workers from local cities (Haynold and Rosander, 2006). The use of local recruitment policy narrows down the scope of the organisation to select talented and skilled employees. The number of airlines professionals is also low, which increases their bargaining demand at the time of recruitment. Qantas Airways provide training to improve the performance of employees, but workers without any knowledge or degree in airline find it difficult to learn new topics. New and talented employees bring new ideas with them that are beneficial for a corporations growth. Therefore, lack of talented employees is significant weaknesses of HR frame of Qantas Airways. High Employee Turnover Qantas Airways is known for the benefits provided by them to their employees, but in return, they constantly put high pressure on them to perform better. Especially in peak season, the employees face a high level of workload that can be dangerous for their physical and mental health. The company also provide flexible hours to its employees, but still, many workers do long shifts during both day and night (Pauka, 2016). These factors increase the employee turnover ratio, and HR department find it difficult to retain workers in the organisation. Employee Engagement In Qantas Airways, most of the employees find it difficult to engage with top-level management due to lack of effective HR policies; it is difficult for low-level workers to communicate their problems directly to the senior management of the company (Oxenbridge, Wallace, White, Tiernan and Lansbury, 2010). The HR staff also failed to effectively assess the requirements and needs of their workers which made employees disconnect from the organisation and it reduced their productivity as well. Lack of Ability to Change The human resource frame of Qantas Airways lacks the ability to change their policies as per the marketing requirements. The HR frame has rigid traditional policies that are not enough to fulfill the requirements of modern employees (Healey, 2016). The company has implemented various modern policies regarding diversity and discrimination, but there is still space for more. The HR department did not incorporate modern technology into their functions which reduces their ability to assess qualities of candidates using digital methods, it increases the time of recruitment and did not analyse the full potential of candidates (Allen, 2006). Recommendations Overall Qantas Airways has an efficient Human resource frame, but it includes few weaknesses as well. Following suggestions can assist the company in reducing their weaknesses and improve the effectiveness of their processes. The company should start international recruitment processes that allow them to attract and hire talented employees from all over the world. Using this strategy will widen the choices for company, and they will be able to hire employees as per their specific technical requirements. The company should recruit part-time employees during their peak season to reduce the pressure from their employees. They should also establish a positive environment in the workplace by assessing the requirement of their workers and fulfilling them. The senior-level HR management should enhance their engagement with low-level staff; the employees must be able to tell their problems and feel like a part of the family. A quarterly meet up program should be established which allow workers to give their feedback to top-level management, and in return, management motivates the employees. The HR department should implement modern technology into its operations to enhance their efficiency. For example, using digital assessment methods to measure capabilities and knowledge of the candidates will reduce the recruitment time and assist in selected of suitable candidates. Conclusion From the above observations, it can be concluded that human resource frame deals with human capital of an organisation. Effective management of human capital is necessary to sustain future development of a corporation. The HR frame of Qantas Airways has several strengths along with few weaknesses. The strengths include diversification in the workplace, equal opportunities to women, effective recruitment process that focus on selecting the best talents, talent programs for workers and high-quality training programs. The weaknesses include high turnover ratio, lack of implementation of modern technology, shortage of new talent, and low employees engagement. The company can adopt various policies to reduce its weaknesses such as international recruitments, hiring part-time workers, quarterly employees engagement program and using modern technologies in recruitment procedure. These strategies will benefit organisation by increasing the effectiveness of HR frame and sustain their future growth. References Allen, J., 2006. How to adjust to a new corporate culture after a merger.Keeping Good Companies,58(1), p.51. Bamber, G.J., 2015. Low-cost airlines product and labor market strategic choices: Australian perspectives.Members-only Library. Day, Q.I., 2009. Alan Joycee, Ceo Qantas.Retrieved July,5, p.2010. Gittell, J.H. and Bamber, G.J., 2010. High-and low-road strategies for competing on costs and their implications for employment relations: international studies in the airline industry.The International Journal of Human Resource Management,21(2), pp.165-179. Healey, B., 2016. How did Qantas improve its human resources management?. 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