Cellular Molecular Mechanisms Of Hutchinson Gilford Progeria Syndrome Biology Essay

Cellular Molecular Mechanisms Of Hutchinson Gilford Progeria Syndrome Biology Essay Hutchinson-Gilford Progeria Syndrome (HGPS or progeria) is a very rare autosomal dominant disorder which results in premature aging and eventually death. Patients are often very underweight at birth and will display conclusive symptoms of premature aging within 18-24 months, such as reduced body fat, hair loss and aged skin, alongside tissue and organ degeneration. Average lifespan for sufferers of HGPS is thirteen years and cause of death is invariably due to heart failure. Due to the nature of these symptoms, HGPS is referred to as an accelerated aging disorder. Currently, only 100 cases of HGPS have been formally documented, around 50 of which are children who are alive today; however it is estimated that a further 150 children worldwide currently suffer from HGPS, but have not yet been formally diagnosed. The identification of the HGPS mutation has only recently been identified and located on codon 608 of the LMNA gene, which codes for four types of lamin proteins: lamin A, lamin C, lamin Aà ¢Ã‹â€ Ã¢â‚¬  10 and lamin Cà ¢Ã‹â€ Ã¢â‚¬  2. These, along with the B-type lamin are responsible for keeping the structure of the nucleus together by forming a scaffold which lines and interacts with the nuclear membrane. The LMNA mutation leads to the expression of progerin, rather than lamin A, altering the structure of the nuclear membrane and leading to loss of nuclear integrity, DNA damage and a compromised DNA double-strand-break repair mechanism. This causes a variety of problems relating to normal cellular function as well as resulting in increased apoptosis and significantly decreased proliferation rates. Until recently, medical assistance for children with HGPS only involved treating the symptoms which accompany the disease, rather than the effects of the HGPS mutation; however in August 2009, clinical trials began testing the effects of farnesyltransferase inhibitors (FTIs) on HGPS patients. This was following experimental evidence from Toth et al (2005) who showed that human fibroblast cells expressing progerin had reversed nuclear alterations when treated with farnesyltransferase inhibitors in vitro. Introduction Aging is a normal and natural physiological process which all living things must experience, unless death should occur prematurely. Aging impairs verve and mobility, causes hair loss, weakens bones and inevitably will ultimately lead to death. As humans, the price that must be paid for our unique self-awareness is the knowledge that we will eventually age and die, forcing us to come to terms with our own mortality and compelling us to value youth. Patients suffering from HGPS are not given the opportunity to experience their youth in the same way as most. By 18-24 months their bodies are already showing signs of physiological aging and by 5 years old they will show more signs of age-related disease than many adults in their 70s. In young, healthy humans, cells are able to cope with the assault of DNA damage which all livings organisms will unavoidably encounter over time. Over time, the ability to deal with this damage lessens and the physiological process of aging will gradually occ ur. HGPS cells are not capable of dealing with this DNA damage to the same extent and so aging happens quickly and prematurely. Despite this, intelligence and cognitive function in children with HGPS are often above average compared to other children within their age bracket (Progeria Research Foundation, 2006). To understand the cellular and molecular mechanisms of HGPS, it is first important to understand the process of normal aging in healthy humans. Normal Physiological aging Aging on an evolutionary level may be described as a progressive decline in fitness (the ability to survive and reproduce) due to a decline in tissue functionality with increasing age (Partridge Gems, 2002). On a cellular level it may be described as a progressive functional decline and increase in cell mortality (Lombard et al., 2005). Although many theories have been proposed, no conclusive theory has been agreed upon to explain how or why we age. Aging is thought to be caused by various genetic and environmental factors. At the cellular level this progressive malfunction of tissue is thought to be due to accumulation of damage by various biomolecules which leads to cell loss or damage (Vijg, 2000). Usually the body has the ability to regenerate these damaged or lost cells through pools of stem cells, however the body does not have the ability to regenerate infinitely and over time this causes a decline in tissue functions typical of aging. This is consistent with the disposable soma theory of aging (Kirkwood Holliday, 1979), which states that cell repair and maintenance (including DNA repair, defence against oxidants, etc) are costly activities. To work to extend life indefinitely would make little sense, as in the wild many organisms have an extremely high mortality rate. Therefore, animals have evolved in such a way that energy invested in maintaining the soma is limited, so that the animal is kept alive long enough for it to reproduce, but not to keep it alive indefinitely. Past this point, the bodys regeneration mechanisms fail and physiological aging becomes apparent. From an evolutionary point of view, this theory makes more sense than others which are based around aging being a genetically inbuilt process, as this would provide no benefit whatsoever to the individual animal or the species as a whole. Aging may involve damage to a variety of cellular components; however damage to various DNA and RNA molecules is likely to be a major contributing factor. Despite the cells inbuilt mechanisms designed to repair damage, damaged DNA which has not been restored perfectly can lead to mutations with detrimental consequences. Balaban et al. (2005) outlines the potential role for damaged mitochondria DNA in the process of aging, however nuclear DNA is a more likely culprit. Mitochrondial DNA has thousands of copies present within the cell which can be replaced if damaged, whereas there are only two copies of nuclear DNA. Several studies have shown that as age increases, so does the risk of mutation (Martin et al., 1996) and there is substantial evidence to suggest a causal link between damage to nuclear DNA and physiological aging. Sedelnikova et al. (2008) showed that the level of double strand breaks (DSBs) that take place increase with age and older cells are less able to repair these DS Bs. Finally, there is the observation that symptoms of progeria are caused by defined mutations in DNA maintenance proteins, which leads to impaired DNA repair mechanisms (Musich Zou, 2009). HGPS belongs to a group of disorders known as segmental progeroid syndromes, characterised by early manifestation of features usually associated with normal physiological aging. They are consequently referred to as accelerated aging disorders. HGPS Background HGPS was first described over 120 years ago by Hutchinson (1886) and again later by Gilford (1904). Since its original classification in 1886, just over 100 cases of the disease have been documented. It affects around 1 in 4-8 million newborns all over the world with no preference for gender. Figure 1 Locations of children around the world who have been diagnosed with HGPG. There are currently 50 living children diagnosed with HGPS, most of whom reside in affluent Western countries. It is estimated that a further 150 children worldwide suffer from progeria but have not yet been identified due to insufficient means to diagnose in less prosperous countries as well as the rarity and complexity of the disease and the fact that the HGPS gene has only recently been identified. (Adapted from the Progeria Research Foundation, 2006) Children with progeria are often born appearing healthy, however shortly after birth it becomes apparent that weight and height gain are below that of which is expected of healthy children (Merideth et al., 2008). By 18-24 months of age they begin to display signs of premature aging, which can be seen in figure 2 below. Figure 2: A 3 and a half year old boy with HGPS showing typical progeroid symptoms. These include stunted growth, loss of hair and body fat, aged skin and unusually prominent eyes. Non-visible symptoms include stiff joints, atherosclerosis., osteoporosis and fatigue. Impaired growth does not seem to be due to any hormone inbalance/resistance or malnutrition. Cardiovascular problems are generally thought to be caused by loss of smooth muscle cells, disruption of the extracellular matrix and other irregularities in vascular structure. Life span ranges from 8 21 years with the average being around 13 years old. Death is almost invariably caused by atherosclerosis (Progeria Research Foundation, 2006). HGPS has recently been acknowledged as part of a family of diseases known as laminopathies, as it is caused by a dominant mutation on the LMNA gene. Before the identification of the gene responsible for progeria, diagnosis was carried out solely on the symptoms that have been mentioned. Thanks to the recent discovery of the mutated gene (Eriksson et al., 2003), diagnosis can be carried out based on the appearance of this mutation. HGSP is caused by a de novo mutation on the LMNA gene, although other progeroid syndromes may be hereditary. The lamins The LMNA gene codes for the nuclear A-type lamin proteins: lamin A, lamin C, lamin Aà ¢Ã‹â€ Ã¢â‚¬  10 and lamin Cà ¢Ã‹â€ Ã¢â‚¬  2 (Fisher et al., 1986). These, along with the B-type lamins are responsible for keeping the structure of the nucleus together by forming a scaffold which lines and interacts with the nuclear membrane. The structure of the nuclear lamina within the nuclear envelope is shown in figure 3. Figure 3: Structure and function of the nuclear lamina The lamina exists on the inner nuclear membrane (INM), providing structural support and helping with chromatin organisation as well as binding nuclear pore complexes (NPCs), nuclear proteins (purple) and transcription factors (pink). Barrier to auto integration factor (BAF) is a chromatin-associated protein which binds to the lamina and various nuclear envelope proteins (Coutinho et al. 2009) Fawcett (1966) deduced that the lamins are components of the nuclear lamina, a layer found between the chromatin and the nuclear envelope initially thought to provide structural support for the nuclear membrane and a location for the chromatin to attach. Recently, further roles of nuclear lamins have been proposed, including DNA synthesis, transcription, apoptosis and assembly of the nuclear envelope. It has been determined that several isoforms of lamins are encoded by three different genes: The LMNA gene encodes lamin A, lamin C, lamin Aà ¢Ã‹â€ Ã¢â‚¬  10 and lamin Cà ¢Ã‹â€ Ã¢â‚¬  2 and the LMNB1 gene encodes lamin B1 and LMNB2 encodes B2 and B3 lamins (Stuurman et al., 1998). Whilst every vertebrate cell expresses at least one type of B lamin, lamins encoded by the LMNA gene are only expressed in differentiated tissue. This observations suggest that lamin encoded by LMNA may have specific roles within certain cells. It is possible that the role of these lamins is in fact to induce or maintain differentiation within these cells, although no conclusive evidence has been presented to support this. It is likely, however, that A type lamins have roles related to and dependant on correct chromatin organisation and nuclear structure. This includes reformation of the nuclear envelope post-mitosis, transcription, DNA replication and nuclear positioning (Holaska et al., 2003; Spann., 20 02; Moir et al., 2000; Haque et al., 2006). Although studies have shown that LMNA deficient mice develop normally, shortly after birth growth problems occur (Sullivan et al., 1999). Shortly before cell division in the late prophase stage of the cell cycle, the phosphorylation of lamin subunits takes place, causing the nuclear envelope to break down. A-type lamins are the first to be disassembled, occurring during early prophase, whilst B-type lamins are disassembled during prometaphase (Georgatos et al., 1997). This course of action is thought to be a necessary requirement for the reassembly of the nuclear envelope after cell division have taken place (Burke and Gerace, 1986). However, studies such as those by Newport et al. (1990) have provided evidence against these theories, stating that when these disassembled lamin subunits are imported, they are done so into a formed nuclear envelope with fully functional pores. Perhaps is it possible that a finite number of lamin monomers are used in the reassembly of the nuclear envelope and the rest are transported in at a later stage. The role of lamins in DNA replication is unclear, however several studies have suggested that Xenopus interphase extracts were depleted of Lamin B3 which resulted in a lack of DNA replication (Newport et al., 1990), however it remained unclear whether this was entirely due to the absence of lamins or if other factors, such as a smaller and more fragile nuclear envelope, contributed. A later study by Moir et al. (2000) provided evidence to suggest that normal nuclear lamin organisation is required for DNA synthesis and that this dependency is completely unrelated to the insufficient formation of the nuclear envelope. It appears that when nuclear lamin organisation is incorrect, the elongation phase of replication fails to take place, probably due to an alteration in distribution of elongation factors, Replication Factor Complex and Proliferating Cell Nuclear Antigen. The potential role of nuclear lamins in transcription has been put forward by Spann et al. (2002), who disrupted the normal nuclear organisation of nuclear lamins using a dominant negative mutant lamin lacking the NH2- domain. This resulted in the inhibition of RNA polymerase II activity in both mammalian and embryonic Xenopus cells. Notably, RNA polymerases I and III were not affected. The role of lamins in apoptosis is directly related to the state of the nuclear envelope. Apoptosis is a very precise physiological mechanism for effective destruction of unwanted cells without causing inflammation or distress to other cells, as would occur in necrosis. Lamin degradation is one of the processes that occur during apoptosis, however several studies have suggested nuclear lamins play a role in the induction of apoptosis (Rao et al, 1996; Di Matola et al., 2001). Post mitosis, protein phosphatise 1 (PP1) is dispatched to the nuclear envelope to initiate lamin B reformation. If this process is abolished, lamin B is degraded and apoptotic signals take place. As previously mentioned, expression of lamins A/C is limited to differentiated cells. Due to their role in DNA replication and transcription, several researchers have hypothesised that they play a role in gene expression as well. Gupta and Saumyaa (2008) propose that specialised A/C lamin expression regulates gene expr ession in such a way which may prevent cell division and cause the cell to undergo terminal differentiation a form of programmed cell death. Lamins are also used for chromatin organisation and positioning within the nucleus, so cells with LMNA mutations exhibit a range of problems involving abnormal chromosome organisation. Glass et al (1993) showed that A lamins interact with chromatin by binding histones as well as indirectly through lamin-binding proteins such as LAP2ÃŽÂ ± and barrier-ro-autointegration (BAF) (Holaska et al., 2003). Cellular and molecular mechanisms of HGPS The most commonly reported mutation responsible for causing HGPS is LMNA  codon 608 in exon 11 (c. 1824 C>T). Although the LMNA gene encodes both A and C type lamins, only A lamins are affected as exon 11 is not present in lamin Cs. Whilst this point substitution does not result in an amino acid change (G608G ) it partially activates a cryptic splice site, resulting in the deletion of ~50 amino acids near the carboxyl terminal in lamin A (LAà ¢Ã‹â€ Ã¢â‚¬  50) but maintaining the CAAX site. Amongst the deleted amino acids is the ZMPSTE24 cleavage site, which is necessary for the maturation of lamin A. This results in farnesylation and carboxymethylation of lamin A, resulting in progerin (Capell et al., 2005). This cryptic splice site is only partially activated and it is estimated that only 10-50% of splices mRNA in transcribed. Since the second LMNA allele is normal, there is still some presence of wild-type lamin A although it is present in much lower levels. Whilst most HGPA pat ients are heterozygous for LMNA p.G608G, mutations have been reported on other location on the LMNA gene, such as one patient with a p. E145K mutation and another with 471C and R527C mutations (Goldman et al., 2002). These mutations have lead to various laminopathies very similar to HGPS (often referred to as atypical HGPS) however the pathophysiological manifestation of these diseases is probably different as they do not result in the production of progerin. HGPS cells are significantly larger than normal cells and usually have large cytoplasmic vacuoles, an abnormally shaped and sized nucleus, often distorted with chromatin extrusion (De Sandreà ¢Ã¢â€š ¬Ã‚ Giovannoli et al., 2003). Goldman et al. (2004) also report evidence of misshapen nuclear envelopes and abnormally thick lamina, both of which are associated with other mutations on the LMNA gene. Not surprisingly, these structural abnormalities cause a variety of cellular dysfunctions including loss of structural nuclear integrity and certain mitotic problems such as irregular chromosome separation, delays in cytokinesis and nuclear assembly and binucleation (Dechat et al., 2007). The loss of nuclear integrity can have a detrimental effect of the function of the cell, however it does not remove all structural ability of the nucleus and cell, it only reduces it. This means that the tissues most likely to be affected by a comprised nucleus are those which are consistently subjected t o mechanical stress, such as blood vessels. Indeed, vasculature in HGPS patients is often severely compromised and death is almost always caused by heart failure. HGPS cells have abnormal chromosome organization in interphase nuclei and can show a loss of peripheral heterochromatin, possibly due to various epigenetic changes. These include upregulation of genes such as H3K9me3 and H4K20me3, both of which are involved in the definition of constitutive heterochromatin (Columbaro, 2005). The H3K27me3, responsible for the definition of facultative heterochromatin, is downregulated, possible due to a reduction in the expression of the histone methyltransferase enhancer EZH2 used for H3K27 trimethylation (Schumaker et al., 2006). Evidence has suggested that these changes in heterochromatin due to altered gene expression may often result in further changes in gene expression. An interesting line of investigation to follow would be to monitor the expression of various other genes in HGPS cells. It is important to understand if HGPS enhances or inhibits the expression of various other genes and, if so, what these genes are and whether these genes play a part in the HGPS phenotype. Since lamin A only appears in differentiated cells, it is possible that one of the roles of lamin A is to maintain differentiation of the cells by securing tissue-specific gene expression. This could mean that the premature aging phenotype is not actually related to normal physiological ageing, but occurs due to certain tissues being unable to perform their designated function due to incorrect gene expression, leading to cell death, tissue degeneration and organ failure. Several studies have indicated that HGPS cells show increased damage to DNA. This was demonstrated by Bridger and Kill (2004) whose experiments showed that HGPS cells were unable to proliferate sufficiently when kept in culture and showed increased rates of apoptosis as well as demonstrating early signs of cellular aging. All of these afflictions are caused by damage to nuclear DNA. Liu et al (2006) also demonstrated that HGPS cells have increased activity in their DNA repair pathways, strongly implying that DNA damage has occurred. The most compelling evidence to suggest DNA damage as a likely culprit for progeroid symptoms is presented in a study by Liu et al. (2005), which showed that HGPS cells have a double strand break (DSB) repair defect. These cells show less concentrated levels of DSB repair factors such as Rad50 and Rad51 as well as damage signaling molecules such as 53BP1. With this evidence, the conclusion can be drawn that Lamin A in necessary for complete DNA repair to take place when necessary, and also that DSB repair is diminished in HGPS cells. DSBs are the most dangerous type of DNA damage that can potentially occur as a result of the constant attack from various agents, as they can potentially result in loss or confusion of genetic information or cell death. To some extent, DSBs occur naturally within the body during normal processes such as meiosis (Keeney Neale, 2006), or during replication when the separated stands encounter blocking lesions. The level of DSBs that occur during these processes can be increased by UV radiation, ionizing radiation, various chemical agents and the presence of free radicals. When a DSB occurs, the cells defense mechanisms commands that the cell cycle halts and DSB repair mechanisms activate. DNA DSB repair mechanisms are thought to occur as one of two processes: Non-homologous end joining (NHEJ) and homologous recombination (HR). Although HR is thought to be considerably more accurate than NHEJ, neither mech anism will produce perfect results. Evidence has been presented to imply that as we age we become increasingly dependent on HR to repair DSBs as activity of NHEJ and all other mechanisms gradually decrease (Johnson-Schlitz Engels, 2006). Many of the changes to the nuclear membrane are not a direct result of insufficient lamin A, but instead are caused by the presence of Progerin, an abridged version of lamin A, and its accumulation of the INM. This accumulation results in alterations in the structure of the nuclear lamina. Liu et al. (2005) suggests that the inefficient DNA repair mechanisms which occur in HGPS cells are in fact due to raised levels of Progerin at the INM. Fitting with this theory, experiments designed to reduce the levels of Progerin have successfully managed improve the cellular phenotype of HGPS cells. Scaffidi and Mistelli (2005) proposed inhibiting the production of Progerin whilst leaving lamin A unaffected by using specific antisense morpholinos which inhibit splicing at the deviant site. Another approach is to treat HGPS cells with farnesly transferase inhibitors (FTIs) which, as the name suggests, inhibit farnesyl transferase, so preventing the modification of Progerin to the farnseylated ki nd seen in HGPS cells. Liu et al., (2006) have shown that this approach does not only improve the cellular and nuclear abnormalities of HGPS cells, but can actually improve the health of HGPS mice. However, FTIs are not specific to Progerin as antisense morpolinos are, and lamin A is likely to be inhibited as well as the modification of Progerin. It is also possible that several other unrelated proteins would be affected by this approach; however these negative side-effects have not yet been documented. Several potential cellular and molecular mechanisms which may contribute to the HGPS phenotype are described below in figure 4. Figure 4 Various cellular and molecular mechanisms which may contribute to the HGPS phenotype. Progerin becomes trapped within the nuclear membrane as a result of permanent farnesylation. Alteration of the normal lamina structure causes vulnerability to mechanical stress and some nuclear blebbing. Other consequences involve disruption of protein interactions, disorganization/loss of heterochromatin and disrupted interactions with RNA polymerase II, RNA splicing factors and transcription factors, causing misregulation of gene expression. (Coutinho et al. 2009) The way these cellular abnormalities cause the general HGPS phenotype are not yet understood and currently can only be speculated on. As further research is carried out regarding HGPS, the cause of premature aging throughout the whole body may become more transparent. Treatment Due to the rarity and complexity of HGPS, there is currently no known cure; however, there are a variety of treatments aimed at alleviating the symptoms which are associated with this disease (Progeria Research Foundation, 2006). It is recommended that patients suffering from HGPS have a regular and nutritional diet as well as supplementary vitamin tablets (in normal doses). Dental problems which occur as a result of HGPS are treated with fluoride supplements, and aspirin should be administered on occasion in order to reduce the risk of heart attacks and strokes, which are invariably the main causes of death in HGPS children. In the case of heart-related problems such as angina, drugs like nitroglycerin may be used, although the dose should be measured carefully based on weight and height as use of anesthetics on children can be potentially dangerous. There is little that can be done to reduce the effect of reduced bone mass except for vitamin and calcium supplements, so children sho uld be accompanied at all times as they are at significantly greater risk of fracturing bones. The hip bones are particularly susceptible to dislocation due to coxa valga. Certain surgical procedures can take place to reduce the risk of this, as well as physical therapy in order to keep all joints as mobile as possible. The use of growth hormones has been suggested as a potential treatment for HGPS children; however the long term effects of this have not been shown to be significant. Currently there are several areas of research into possible clinical therapeutic treatments for progeria. Wang et al. (2008) and Liu et al (2006) demonstrated the use of farnesyltransferase inhibitors (FTIs) which appear to reduce the nuclear abnormalities present in HGPS cells, reducing the severity of symptoms on HGPS mice, improving their general health. Subjects showed improved cardiovascular health, a reduction in the number of bone fractures and improved survival and growth rates. This has been substantiated with evidence from Toth et al (2005) who showed that human fibroblast cells expressing progerin had reversed nuclear alterations when treated with FTIs in vitro. Clinical trials testing the effect of FTIs in children with HGPS began in August 2009 and ended the following December. Results from these trials are still being awaited. Summary Most of the breakthrough discoveries regarding HGPS have been made within the last few years. These include the location of the HGPS mutation, the nature of the disease and potential clinical therapies which are aimed at preventing the HGPS phenotype on a cellular and molecular level instead of simply treating the symptoms. There are several ways that the effects of the point mutation on the LMNA gene on chromosome 1 could lead to the pathogenesis of HGPS and it is likely that the combined affects of this mutation results in the HGPS phenotype. Compromised nuclear integrity may lead to reduced structural support for the cell, so those tissues under constant mechanical pressure such as the vasculature will suffer more greatly than other tissues. Another possible cause of the HGPS phenotype is the accumulation of DNA damage. This is a logical conclusion to draw, as the mechanism of normal human aging is thought to occur in this way. HGPS cells have insufficient DNA DSB repair mechanisms and so the phenotype of premature ageing in children with progeria is simply due to the magnification of one of the factors that causes aging in healthy humans. The pool of stem cells that healthy humans rely upon to counter increased apoptosis as a result of DNA damage would be under more pressure to proliferate in HGPS children, perhaps exhausting supplies and causing tissue degeneration. It is also possible that the stem cells themselves are affected by the HGPS mutation, causing a decline in proliferative ability. The possibility that the HGPS mutation may lead to up or down regulation of other genes is not one that should be ignored. If this is the case, the vast variety of symptoms which accompany HGPS may be explained by the altered lev els of expression of other genes. Until more is understood about Hutchinson-Gilford progeria syndrome, it is impossible to conclusively explain the extraordinary symptoms of this disease. Perhaps the results of the recent clinical trials will shed more light on how the alteration of two proteins due to a single point mutation can cause a child to show such drastic physiological aging. Experimental data analysis Programmed cell death (PCD or apoptosis) is a necessary part of complex life in all sorts of multicellular organisms. In humans, it is not only essential during embryonic development (preventing all manner of deformities) but also consistently through life. Efficient apoptosis prevents a vast number of diseases by ensuring that any unnecessary or potentially harmful cells are destroyed safely, without harmful effects on neighbouring cells, which happens in necrosis. Apoptosis only causes a diseased state when its rate of action exceeds or falls short of that which is necessary to keep an individual healthy. Insufficient apoptosis is well known culprit for the development of cancers, when tumorous cells which should have been erased are allowed to proliferate and develop into tumours. Another well known example of diseases due to lack of apoptosis is the vast number of immunodeficiency diseases caused by self-targeting T and B lymphocytes. These cells should have been removed through apoptotic signals, but when these signals fail these self-targeting lymphocytes are allowed to survive and will target the bodys own tissues. Increased rates of apoptosis throughout the body are commonly observed in many diseases, for example, HGPS. Abnormalities either within or external to the cell cause apoptotic signals to occur, resulting in PCD of cells which would otherwise not have been destroyed. PLAC8 (placenta-specific 8) is a gene which encodes a small, highly conserved protein known as onzin. Experimental evidence has been presented to demonstrate that under expression of endogenous onzin results in reduced cell proliferation, whilst over expression results in an increased cell count (Rogulski et al, 2005). This data suggests that onzin has a negative effect on the rate of apoptosis. Li et al. (2006) suggest that expression of onzin within a cell protects it from apoptotic signals and that when onzin levels are depleted the cell becomes sensitive to apoptotic assault. To test the effects of onzin on the rate of apoptosis, CEM-C7 T-leukemic cells were transfected with either an expression construct containing PLAC8 or pcDNA3, where the pcDNA3 vector acted as the control. Cultures of these cells where then exposed to a range of apoptosis-inducing agents: Fas, Dexamethasone (dex), cisplatin, butyric acid, okadaic acid and UV exposure. This was in order to determine whether expression of the PLAC8 gene effectively reduces the rate of induced apoptosis. Cell counts were taken after 24, 48 and 72 hours. The significance of the difference between cell counts in PLAC8 and pcDNA3 cultures when exposed to all apoptosis inducing agents was determined using a two-sample T-test. The samples used in each test were the apoptosis-inducing agent transfected with PLAC8 and the same apoptosis-inducing agent transfected with pcDNA3. The results from these two-sample T-tests are shown below in table I. Table I. Apoptosis inducer p-value (24 hours) p-value (48 hours) p-value (72 hours) Dex 0.023 0.069 Cisplatin 0.026 0.045 0.042 Butyric acid 0.003 Okadaic acid 0.03

Literature Review Celebrity Chef Essay

This review should bring all relevant facts about the subject and facilitate practitioners and highlight areas for further research. In short celebrity endorsement is about endorsing products with the help from a celebrity. Consumer association towards a celebrity endorsed produced increases their purchase intention as many see the celebrity as a role model. However, it is important that the consumer can identify with the celebrity and that the celebrity? s image fits with the produced he or she endorses, only then will celebrity backing be an effective advertising strategy. . 2. Background and Definition Nowadays, celebrities are used in advertising in almost every context. Athletes such as Michael Ballack (Adidas) or Tiger Woods (Rolex) or models such as Cindy Crawford (Omega) or Heidi Klum (Katjes) endorse several products. These celebrities act as a spokesperson in order to advertise and promote products (Kambitsis et al. , 2002). Celebrities can create more positive responses to wards advertising and greater purchase intentions than non-celebrity endorsers (Byrne et al. , 2003). Using celebrity as an endorser for a given product can either be positive or negative for a company/brand. A campaign that turned out successfully was the campaign with Jamie Oliver as an endorser for the supermarket chain J. Sainsbury. The successful format of the TV production â€Å"The naked chef† provided an ideal platform to use for the advertising campaign within a context relevant for J. Sainsbury? s desire (Byrne, 2003). An example of a campaign that did not turn out successful was when J. Sainsbury used the actor John Cleese in the â€Å"value to shout about† campaign in 1998. Employees and customers alike felt that Cleese was not the right personality to personify the supermarket’s quality image (Whitehead, 2003). In the literature there are two different definitions of celebrity endorsers used. The definitions used are: â€Å"A celebrity endorser is an individual who is known to the public (actor, sports figure, entertainer, etc. ) for his or her achievements in areas other than that of the product class endorsed†. (Friedman, 1979, p. 63) â€Å"Any individual who enjoys public recognition and who uses the recognition on behalf of a consumer good by appearing with it in an advertisement†. McCracken, 1989, p. 310) In other words these definitions says that an individual who is known to the public in different ways. The individual is famous and utilizes his or her publicity to advertise a product that does not have anything to do with the individual fame. I consider McCracken? s definition as the most informative one as it is short and concise. The definition gives a clear view of what a celebrity endorser is. Previous studies have been done on consumers? response to celebrity endorsement in advertising. Results of these studies show that celebrities make advertising believable and enhance message recall. Furthermore, when celebrities are recognized with brand names, it creates a positive attitude toward the brand and a distinct personality for the endorsed brand. In the following chapter the method used for this review will be introduced. The chapter will start with how a literature review is defined, followed by the literature search, method problems and quality standards. 2. 1. Definition of literature review Conducting a literature review is about understanding a topic that has already been addressed, how it has been researched by other authors, and what the key issues are (Hart, 1998). According to the author Chris Hart (1998) the definition of a literature review is the selection of available documents; both published and unpublished (in my review I will only study published academic documents), on the topic, which contains information, ideas, data and evidence written from a particular standpoint. 2. 2. Literature search, methodology used When searching for articles I used the databases Emerald, Ebsco and ProQuest. These databases were used because of the highest ranked journals in the field of marketing were listed there. I used the article Journal quality list (2008) issued by Harzading. com, research in international and cross-cultural management. High ranked journals increase the validity of the thesis, as validity is defined as â€Å"The ability of a scale or measuring instruments to measure what is intended to be measured†(Zinkmund, 2000). I also tried to find books relevant to the topic by using the library here at Les Roches International School of Hotel management as well as the library database google. scholar. . 3. Description of sub-topics Purchase intentions: Describes what impact the celebrity endorser has on the consumers purchase intentions. A consumer is more likely to purchase complex or expensive products which are endorsed by celebrities rather than by non-celebrities. Daneshvary and Schwer (2000) point purchase intention as an environment of associations between endo rsement and consumer, depending if the consumer can identify with that association and purpose. Consumers? association to celebrity endorsement/endorser: How consumers associate/have a connection towards the celebrity endorsement/endorser. If a company want a consumer to associate to an endorsed product it is important to choose an endorser who uses the product and where that use is a reflection of professional expertise (Daneshvary and Schwer, 2000). A formula 1 driver endorsing helmets is good, while a tennis player endorsing car polish is less good (ibid). Consumers? attitudes towards the endorser: The consumers? often have a positive attitude towards the product and the celebrity, despite the fact, that it is well known, that the endorser earned a lot when promoting the product (Cronley et al. 1999). Effectiveness of celebrity endorsement/endorsers: How effective is the usage of celebrity endorsement. It is, most likely, more effective to use celebrity endorsement constantly to increase the strength of the link between the celebrity and the endorsed brand. It is also more effective to use a celebrity who is not associated with another product (Till, 1998). Positive/negative effects of celebrity endorsement: the usa ge of celebrity endorsement can be both positive and /or negative, which can influence the company / brand in the end. For example, using celebrities can be very costly; also, celebrities might switch to a competitor, which would then increase the risk of a negative impact (Agrawal and Kamakura, 1995). However, in can be said that celebrities in advertising are widely spread and persistent and the marketing executives continue to utilize celebrity endorsement as an advertising strategy (ibid). Profitability of celebrity endorsement: The use of celebrity endorsement will hopefully lead to increased profitability. A study indicates that there is a positive impact of celebrity endorsement on the expected future profits, which recommends marketing managers to use celebrity endorsers in their advertising campaigns (Agrawal and Kamakura, 1995). Friedman/Friedman (1979)Does effectiveness of celebrity endorsement depend on the product? Interviews with 360 house wives Celebrity endorsements are not effective for all products Till (1998)What are the effects of celebrities endorsing more than 1 product? Case study with 99 students, who were shown different advertisings using the same celebrity endorserThe use of the same celebrity endorser to advertise for various products has got a negative impact on the efficiency of the ad, the product and the endorser. Atkin / Block (1983)Is celebrity endorsement effective in advertising and how does it influence customer purchase intentionsExperiment with 196 test persons, where each participant was shown 3 versions of an ad. Each version featuring a celebrity endorser and a non-celebrity. Advertising using celebrity endorsement is under special conditions more effective than using non-celebrities. Choi et al. (2005)How can celebrities be used successfully in advertising? How effective is the recall-value and the emotional response to celebrity advertising? Experiment with different groups of test persons. Advertising with celebrities is more effective than using non-celebrities under special conditions. Sanbonmatsu / Kardes (1988)How does the credibility of a celebrity affect the consumer purchasing intention? Interviews with 542 persons. Consumer purchasing intentions are more effected using celebrities than using non-celebrities. Tripp et al. (1994) How do consumers judge the celebrity endorsement, the ad and the brand, if the celebrity endorses various products? What are the effects on purchasing behaviour? First Study: interviews with 461 students. Second Study: Interview with 10 test persons. Simultaneous advertising trough the same celebrity has a negative effect on the ad, the product and the celebrity. Agrawal/Kamakura (1996)Which economical effects on advertising are there when using celebrities? Event-Study in regards to the effects on share prices trough the announcement of celebrity endorsementCelebrity endorsement can have a positive and a negative effect on share prices. Agrawal/Kamakura (1995)Can a single celebrity have a positive effect on the company? s value? Analysis of the share price of a company after announcing a celebrity endorserCelebrity endorsement can have a positive and a negative effect on share prices. Charbonneau / Garland (2005)How does a company find the right celebrity endorser for its products? Which criteria should be considered? Questionnaires: 414 marketing managers at 148 advertising companies.

Polyhydroxybutyrate Phb

Polyhydroxybutyrate PHB Polyhydroxybutyrate (PHB) is a polyhydroxyalkanoate (PHA), a polymer belonging to the polyesters class. Progress Analysis: – Bacteria first used to make PHB in 1925 at Pastuer Institute in Paris. – In 1970’s adopted by Imperial Chemical Industries (ICI) – Alcaligenes Eutrophus grown in fermentation vats with Molasses – The bacteria is then starved of nutrients (glucose and nitrogen) – PHB is produced as an energy store for the bacteria. In 1980’s Maddison University (Virgina) successfully cloned the 3 genes of A. Eutrophus that control PHB production and transferred them to Escherichia coli – an easier bacteria to work with allowing easier manipulation of the polymer depended on the need. – In 1990’s the 3 genes were cloned into sugar beets and turnips – much larger scale production of polymer. Work continuing (with Monsanto – company bought patent) into cloning genes to corn. Production: The manufacturing process of PHB begins with sunlight.Through photosynthesis carbon dioxide from the atmosphere is converted to carbohydrates via sugar beets or sugar cane. These carbohydrates (like Lactose, Fructose and Glucose) are the raw material for the manufacturing of PHB. Uses: †¢In medicine, PHB is compatible with the blood and tissues of mammals. The monomer of PHB is a normal metabolic in the human blood. As the body reabsorbs PHB it might be used as a surgical implant, in surgery, as seam threads for the healing of wounds and blood vessels. In pharmacology, PHB can be used as microcapsules in therapy or as materials for cell and tablet packaging. †¢In packaging for deep drawing articles in the food industry, for example, bottles, laminated foils, fishnets, potted flower, hygiene, fast food, one-way cups, agricultural foils and fibres in textile. Advantages of PHB: †¢Good mechanical properties, especially when low density and price are taken int o account. †¢Recyclable, renewable and non-abrasive to manufacturing equipment. †¢PHB is hydrophobic, therefore protects natural ? res from moisture sensitivity. †¢Mechanical properties are comparable to other polymers used (e. g. PP). †¢PHB has a fast degradation rate compared to other plastics. (months in sewage and years in sea water) yet ultra violet light can speed up this process. †¢The main advantage in the medical field is that PHB is a biodegradable plastic which can be inserted into the human body and does not have to be removed again. †¢Can be manipulated genically therefore can be used for many things Disadvantages of PHB: †¢It has a tendency to be brittle. Costs four times as much as polyethylene (it’s major competitor) †¢Cheaper to produce necessary materials from petrochemical industry. †¢Large scale production in early stages. †¢Uses sugarcane which can decrease biodiversity such as in Brazil. Evaluative Conclu sion: PHB has huge potetial as polymer for future,this is due to its main advantages such as being biodegradable and it has an ease of manipulation. This is good although work must be done to make it cheaper. Also work must be done to reduce the very large scale production in the early stages of production.There needs to be a way to make this plastic from cellulose in plant cell walls rather than from sugarcane alone. As the petrochemical industry supplies become scarce the price of PHB will go down, which is good for future use. Links Used: http://sundoc. bibliothek. uni-halle. de/diss-online/02/02H017/t2. pdf http://en. wikipedia. org/wiki/Poly-3-hydroxybutyrate http://en. wikipedia. org/wiki/Biopolymer http://www. gotessays. com/essays/543/index. php http://umpir. ump. edu. my/696/1/Aimi_Salma_Awalludin. pdf

Analysis of Nestles Position in India - Free Essay Example

Sample details Pages: 7 Words: 1954 Downloads: 7 Date added: 2017/06/26 Category Marketing Essay Type Case study Did you like this example? Nestle is one of the largest companies in the world. It produces different types of products and was based in different parts of the world. It was started in 1868 by Henri Nestle, who set up a sales office in London. The company went under the new ownership when Henri Nestle retired in 1875.Nestle opened its first factory in 1901 in UK and got merged with the Anglo-Swiss milk company which was founded by Charles and George page. It has acquired its own brand image over the years by innovating new products and attracting people all over the world with its quality products. Nestle became one of the leading companies in the world when it started producing chocolates. The chocolate production began at Hayes in 1913 and the famous white chocolate Milky Bar was released into the market in 1937. The other products of Nestle are coffee, milk, chocolates etc. The company has its branches in 86 countries like India, Pakistan, US, South Africa, brazil, Sri Lanka, France , Middle eas t countries etc all over the world and nearly employs 283,000 people. It has over 250,000 shareholders, while most of them are from Swiss. Nestle products are well known in the whole word for their wide range of products which include coffee , infant nutrition, baby products ,chocolates , milk products etc. Nestle products are cheaper when compared to other milk products and chocolates and the reputation in the market is good which makes the company to release wide range of products in the market across the whole world. Nestle is the world leader in the categories of coffee, infant nutrition, chocolates etc. The company respects the opinions of its customers and makes sure that the products are manufactured according to their interests. FIVE COMPONENTS OF ARMSTRONGS MODEL: BASIS: the environmental factors that are to be taken into account as the essential background for the organisations HR strategies. These factors can be analysed by using SWOT or PESTLE analysis. CONTENT: the details of the proposed HR strategies of an organisation must be included RATIONALE: the business case for the strategy. IMPLEMENTATION: finding the ways and plans to implement the proposed HR strategies. COST AND BENEFIT ANALYSIS: analysing the benefits and costs of implementing a strategy and acting according to the outcomes that may occur. NESTLE follows the strategies like commitment strategy, developing trust, culture management etc. By following and implementing these type of strategies, NESTLE has innovated and developed itself throughout the years. Let us evaluate the HR strategy of NESTLE organisation using Michael Armstrongs model for the strategys design. BASIS: the environmental factors of the NESTLE organisation are evaluated using the SWOT analysis. STRENGTHS: The company has a great support from its parent company which has a great influence all over the world. It has huge brand strength all over the world. The brands like NESCAFE, MAGGI a nd CERELAC which are generic to their product categories are very strong in the Indian market. Around 67% of the population use Nescafe, Maggi and Cerelac for their daily uses. Another main Strength of the company is, it continuously introduces new products in to the market with innovative thoughts and attracts people towards it. It makes sure that its products are manufactured with quality. There are nearly 3500 scientists in the companys RD department to innovate new thoughts and ideas. It is a truly global company which is operated in 77 countries. The competition from other organisations doesnt affect Nestle because of the low cost and high quality it maintains. It has a very strong work force unlike the other organisations. WEAKNESS: Because of the uneven nature of the Indian market, the things get more complicated. It is very much important to maintain good hygiene standards, talented personnel in the food industries like Nescafe. It is very difficult to maintain thes e standards in the big countries like India which has a complex supply chain management. OPPURTUNITIES: There are lots of opportunities to the company in the huge country like India. There are lots of towns in India where the products of Nescafe are not available. It can extend to those towns and villages and can increase its market. The company can extend its product folio by introducing new products into the market. As India is a huge country with lots of resources and manpower, it can be made as the export hub to achieve better results. Everyone is becoming a health freak now days, including the United States. So, health based products are having great opportunities in the global market. THREATS: The main threat for any organisation is its competitor. There is a huge competition among the organisations. Nestle faces huge competition from the organised and unorganised sectors. Moreover, the Indian government has decreased the import duty of food products, making the things worst. The organisation does not have any threats from the small scale industries but it will have threats from the organisations which are established with large investments and big brand value. The performance of FMCG sector is very less in the past 2-3 years, even though there is a decent pace of growth in the economy of the organisation. Some of the markets in which the company is about to enter, are already mature regarding these products. CONTENT: The main principle of HR strategies of the NESTLE organisation is its products growth through innovation and renovation. The other important strategies are Attracting dynamic employees and giving training to them Developing the management Respecting other cultures and traditions Providing training to the new comers Some other principles of proposed HR strategies are maintaining diversity in the manufacturing of products, despite of introducing several products in to the organisation. RATIONALE: L et us have a look on the business case of proposed HR strategies of NESTLE organisation. NESTLE has its Kit Kat manufacturing plant in York England. Ian Jobson is the manger of that plant. For manufacturing a ton of Kit Kat chocolate, it used to take like 38 man hours. It is a waste of time and man power and even it costs a lot of money to the organisation. To overcome this problem, they have implemented new strategies in the manufacturing of the chocolate. They have innovated and renovated their strategies and came up with a new strategy. They have decreased the human labour and introduced robotics to make the work easier. They used robotics, automated packaging, and production line improvements and they have reduced the manufacturing time to 23 hours. IMPLEMENTATION: NESTLE makes sure that the proposed strategies are implemented without any failure. It provides the necessary resources and necessary funds to the HR department to implement their strategies. It follows di fferent strategies and concepts to reach the better heights and to sustain in this competitive world. It has a research development department, who works on innovation of the new products and renovating the available products of the company. It opens new branches every year to attain a greater grip on the market. It commits to a strong work to achieve better results NESTLE makes sure that it follows all the local laws and legislations, as reputation of the company affects the sales of the organisation It gives rights to the employees to question their superiors about an unfair evaluation It motivates its employees, train them in maintaining teamwork, cooperate and integrate with the organisations strategies. COST AND BENEFIT ANALYSIS: NESTLE tries to decrease the extreme utilization of natural resources. By doing this, it benefits in increasing the human resources, expands contact to the financial resources. The managers and the individual employees are very benefited by the HR strategies of NESTLE. The company believes in the individual achievement of the employees plays a major role in the development of an organisation. To encourage its employees, the HR department has designed a pay structure to its employees. The company even introduced practices like job enlargement to encourage their employees and to break the limitations of their work. Let us evaluate the pay structure of the NESTLE organisation: NESTLE suggests reasonable remunerations to the employees. The level of remuneration is maintained above the average in the industry. The unpredictable part of salary is relatively big in rewarding an individual performance. The variable part of salary for higher management is based on the performances and achievements of the individuals and their team. BENEFITS: The employees can apply up to particular number leaves either personal or medical. They can have the assistance scheme for their childrens education. T he organisation offers provident fund to their employees. The other benefits for the employees include retirement gratuity scheme, conveyance repayments, insurance for accidents etc. The other concepts that can be included are providing recruitment services, talent management and international HR etc. The recruitment services of NESTLE are very much recognised all over the world for their methods of recruitment. They dont just try to fill the jobs but makes sure that the right person is selected for the right job. They select the people who are dynamic, hard working, flexible and honest. Those candidates are selected whose values are similar to the companys culture. The HR officials are the one who takes the final decision in selecting a person. The company even maintains a Rewards and Employee relations team, which provides guide lines to the employees in necessary situations. They provide guidance to the line managers, HR managers, research and development department etc. It provides a health environment to its employees. In a multi-national company like NESTLE, the employee turnover is less than 5%. EMPLOYEE RELATIONS: Nestle gives importance to the personal life of their employees. So, it makes sure that the work life and personal life of their employees is balanced. It maintains a separate policy in which work/life balance is given importance. REWARDS AND INCENTIVES: Nestle encourages its employees by giving rewards and incentives. By doing this, the employees who are eager to win awards will work hard for the development of the organisation. Competitiveness among the employees will result in the growth of the organisation. So, the organisation even offers a competitive reward package to its employees. The company awards NESTLE idea award every quarter to the employees who come up with innovative ideas. These are the different concepts and theories of NESTLE. CONCLUSION: Michael Armstrong gave a brief explanation on the co mponents that are to be included in the Human Resource strategies of an organisation. He covered all the important aspects which play a major role in the development of an organisation. By evaluating the HR strategy of NESTLE using Michael Armstrongs strategy model, we can understand that NESTLE is very keen and careful regarding the strategies it had implemented and it is going to implement in the future. NESTLE concentrated on its strengths, its weaknesses, the opportunities it has and the threats it has to face and planned strategies to face these problems. By the Michael Armstrong five components model the HR strategies are evaluated. In the first component basis for the organisation, HR strategies are estimated using SWOT analysis and the strengths and weaknesses are evaluated and future opportunities and threats are analysed. In the second component content the innovation and renovation of the Nestle organisation are discussed where the strategies state the need for develop ment in the management and opportunities for the skilled employees. In the third component, Rationale shows the reduced man power to increase productivity by using robotics as a strategy which gave good results by decreasing man power to 23 hours from 38 hours. In the fourth component implementation stated that the need for urgency for the development of organisation by increasing the branches of nestle every year. In the fifth component cost and benefit analysis states the remuneration of the employees it depends on the individual performance which reflects the NESTLE organisations growth. Don’t waste time! Our writers will create an original "Analysis of Nestles Position in India" essay for you Create order