Monday, September 30, 2019

Honda (a)

Q1. What’s the strategy that allowed Honda to be so successful in the US market? It is a fact that Honda had experienced a remarkable success during the period from the late 50’s to the end of the 70’s. This success is a result of the strategy the company developed at that stage of its lifecycle. This strategy in principle is a technologically based strategy; where Honda established Honda Technical Research Institute (HTRI) in 1946. In the meantime, Honda developed manufacturing expertise to become fully integrated producer of motorbikes components.In order to minimize the associated risk, Honda offered a multiproduct line, taking leadership in product innovation through the HTRI and exploiting opportunities for economies of mass production by gearing design to production objectives. How is possible that Honda’s many expensive investments lead to lower costs? The company achieved a significant product advantage through a heavy commitment to R&D and advance d manufacturing techniques.Honda used its productivity-based cost advantage and R&D capability to introduce new models to the market very quickly and at prices below those of competitive machines. Over time, Honda had smoothly descending real prices. This price reduction is experience-based which means that as long as more experience is acquired, Honda is capable to develop new methodologies to produce motorcycles at lower cost. This was mainly due to high production volume and improved productivity of the Japanese labor. Q2. How did Honda come to have this strategy?Honda’s strategy was a result of Honda’s underlying philosophy which is a marketing philosophy; i. e. grapping more market share and increasing the sales volume. Selling and Distribution system was a tool to implement this philosophy. What evidence do we have of forethought and planning in the company approach to production? Honda built a manufacturing plant with a capacity 10 times in excess of demand at t he time of construction in 1958. Honda developed manufacturing expertise to become fully integrated producer of motorbikes components.Honda minimized risk through: offering a multiproduct line, taking leadership in product innovation through the HTRI and exploiting opportunities for economies of mass production by gearing design to production objectives. Marketing? In the 1958 Honda’s market research identified a large untapped market segment seeking a small, unintimidating motorcycle that could be used by small motorcycle business for local deliveries. Honda advertizing concerted to change the image of motorcyclist through heavy campaigning towards middle class consumers. Distribution?Honda followed a policy of developing the market region by region beginning with the west coast and moving eastward. Design? Honda designed the lightweight motors to match the market research results. In general, Products are updated or redesigned whenever a market threat or opportunity is perc eived, prices are set at levels to achieve market share targets and will be cut if necessary, effective marketing systems are set up in all markets where serious competition is intended – regardless – of short term cost and finally plans and objectives look to long term payoff.Q3. To what degree was the US entry strategy thought out in advance? Honda was already the world’s largest motorcycle producer. Honda established a US subsidiary and began its push in the market by offering very small lightweight motorcycles. Honda followed a policy of developing the market region by region beginning with the west coast and moving eastward until it established the largest dealership network in the US. Honda (a) Q1. What’s the strategy that allowed Honda to be so successful in the US market? It is a fact that Honda had experienced a remarkable success during the period from the late 50’s to the end of the 70’s. This success is a result of the strategy the company developed at that stage of its lifecycle. This strategy in principle is a technologically based strategy; where Honda established Honda Technical Research Institute (HTRI) in 1946. In the meantime, Honda developed manufacturing expertise to become fully integrated producer of motorbikes components.In order to minimize the associated risk, Honda offered a multiproduct line, taking leadership in product innovation through the HTRI and exploiting opportunities for economies of mass production by gearing design to production objectives. How is possible that Honda’s many expensive investments lead to lower costs? The company achieved a significant product advantage through a heavy commitment to R&D and advance d manufacturing techniques.Honda used its productivity-based cost advantage and R&D capability to introduce new models to the market very quickly and at prices below those of competitive machines. Over time, Honda had smoothly descending real prices. This price reduction is experience-based which means that as long as more experience is acquired, Honda is capable to develop new methodologies to produce motorcycles at lower cost. This was mainly due to high production volume and improved productivity of the Japanese labor. Q2. How did Honda come to have this strategy?Honda’s strategy was a result of Honda’s underlying philosophy which is a marketing philosophy; i. e. grapping more market share and increasing the sales volume. Selling and Distribution system was a tool to implement this philosophy. What evidence do we have of forethought and planning in the company approach to production? Honda built a manufacturing plant with a capacity 10 times in excess of demand at t he time of construction in 1958. Honda developed manufacturing expertise to become fully integrated producer of motorbikes components.Honda minimized risk through: offering a multiproduct line, taking leadership in product innovation through the HTRI and exploiting opportunities for economies of mass production by gearing design to production objectives. Marketing? In the 1958 Honda’s market research identified a large untapped market segment seeking a small, unintimidating motorcycle that could be used by small motorcycle business for local deliveries. Honda advertizing concerted to change the image of motorcyclist through heavy campaigning towards middle class consumers. Distribution?Honda followed a policy of developing the market region by region beginning with the west coast and moving eastward. Design? Honda designed the lightweight motors to match the market research results. In general, Products are updated or redesigned whenever a market threat or opportunity is perc eived, prices are set at levels to achieve market share targets and will be cut if necessary, effective marketing systems are set up in all markets where serious competition is intended – regardless – of short term cost and finally plans and objectives look to long term payoff.Q3. To what degree was the US entry strategy thought out in advance? Honda was already the world’s largest motorcycle producer. Honda established a US subsidiary and began its push in the market by offering very small lightweight motorcycles. Honda followed a policy of developing the market region by region beginning with the west coast and moving eastward until it established the largest dealership network in the US.

Sunday, September 29, 2019

Business Communications Report Essay

The purpose of this report is to evaluate the importance of oral communications within the workplace, as well as to identify the aspects and skills that are valued within the workplace. This report will also give recommendations to the problems it highlights. 1. 2. 3 Questions * Is oral communication really a problem among graduates in the workplace? * Which aspects of oral communication are valued more within the workplace? * Which oral communication skills are lacking in graduates within the workplace? * What solutions could we recommend to the NUS management? 1. 3 Scope The ability to express ideas convincingly should therefore be an aspect to be focused on. 3. 4 Need for Clarity of Content The findings have made obvious that clear content is of high priority within the workplace. This oral communication skill is needed because the content is the crux of the message being sent to others and it needs to be clear and concise to avoid confusion. As such this oral communication skill should be focused on. 3. 5 Need for Confidence in Oral Communications The importance of this skill has been noted in the findings. This communication skill complements the other conclusions, as confidence is something that is required for all aspects of oral communications. Clearly, confidence is a skill that should be focused on. 4. Recommendations Based on the conclusions made above, this report recommends changing the NUS curriculum to involve more opportunities to practice oral communications skills, as well as to introduce a public forum, providing chances for all students to speak publicly more. 4. 1 Change in NUS Curriculum This proposal involves a tweaking of all modules in NUS to accommodate even more usage of oral communications skills than at current levels. For example, module marks could be adjusted to allow for more emphasis on presentations or interaction with the lecturer and the class. This would not only allow students to practice their oral communications as a whole, but also improve on specific aspects and skills, like their confidence and clarity. It is noted that this proposal would be difficult to implement and would also take time to adjust to for the entire university. However, this strategy would help all NUS students to improve their oral communications skills greatly over their time in NUS as they are encouraged to work on their oral communications. Thus this would be a long term solution for the problems stated above. 4. 2 Creating Student Forums The Management should also consider utilizing a key aspect of education in NUS: student life. Through providing a forum for students to speak up, especially about issues that the students have passion for, they will get more practice at oral communications. They would also debate and discuss among themselves about such issues, further boosting their proficiency in key skills like being convincing in oral communications. This suggestion does have certain drawbacks, like difficulty in implementing instantly as students are not used to it. However, over time, and with proper promotion and support from student leaders, it could be a good avenue for students to air their views and practice their oral communications skills at the same time.

Saturday, September 28, 2019

On the Morality of Non-Legal Political Tactics

Morality arises from virtue. It makes the determination of good or bad weighed before proper conduct, logic and experience. Yet what is logical and proper? Can we equate moral with legal? If not, how do we create an intersection of the two such that obedience to the law is also a moral act? The overlap between morality and law may be an ideal. A code of conduct becomes the framework of morality which is rooted in cultural norm and tradition, religion, ideology, philosophy and individual conscience. Laws as embodiment of political beliefs and values are ideally made when morals become a major basis. Yet, in the same way that no one is perfect, society is also an imperfect system and most of the time morals and laws do not meet in the same direction. The policy-makers are mostly humans with vested interests neglecting majority needs and demands. The shaping of laws ergo is a difficult task for the citizens in order to create a law-abiding and morally-upright population. If not, moralists who pursue their perceived moral code of conduct might afford to disobey societal rules to satisfy morality or the law-abiders might dissatisfy the latter. However, the sources of morality at some point would contend each other vis a vis proper judgment. When this happens, every issue must represent a relative field of concern and must base the measure of morality on which aspect is dominant. In this paper I would like to present how activities of activists and political interest groups have transgressed legalities however may find justification on moral grounds. The example chosen are the environmentalists’ illegal pursuits against companies that degrade the ecology. In a political sense; sabotage, rallies, arson and eco-terrorism which are done for the achievement of their political interests are considered political tactics. These are the measures adopted by organizations to create an impression of sustained efforts and operations to make the government act according to their wishes. These political tactics are made outside of state means and are labeled anti-government. Using the framework of the existence of social movements, these strategies are expressed only when sympathizers lost trust in their lobbying capacities and find better attention in the non-state parameters. Yet advocates are confident on the righteousness of their position and actions. Here is an illustration of where the conviction is coming from. Let us concentrate on Eco-terrorism. This simply has been defined as terrorism that is conducted for the sake of environmental causes. Violence is employed but is directed against property of the target offenders. The damage that eco-terrorism has caused amounted to $42.8 million for decades which in 2003 increased to 200 million dollars in property damage. Environmentalists have argued that the government have misinterpreted the acts of the so-called eco-terrorists and must refer to the acts of persons, companies and governments that are involved in ecologically irresponsible activities like deforestation, construction of infrastructures in agricultural suburbs, destroying habitat of animals and various life-forms.[1] This has served the justifying principle for eco-terrorism; the sustainability of environment is more important than profit of companies and no amount of consumerism could be utilized as reason for the business expansions and material development. The relative avoidance of killings and deaths resulting from activism is attributed to the value that any culture, religion and ideology places over life and the right to life. The environmentalists have been serious about relating environmental degradation to an indirect act of taking away the lives of people due to anticipated outcomes of mishandling of the ecology and imbalance in the ecosystem. Nature has its way of getting back at modernization and industrialization; global warming, flash floods, extinction, etc. Philosophically speaking, the morality of eco-terrorism also begs ethical consideration. Rights are given importance where morality is present. Human rights have included the right to life, a healthy habitat, and a safe and clean society. In a political sense, â€Å"power† is an element that is not confined to the government and may be given to civil society and interest groups. Authority hence may be questioned and rendered illegitimate where the general public sees selfish interests dominate the society’s politics. The other sources of proper conduct such as norm or tradition, religion and individual conscience may be subjective yet there are some arguments that favor the activists; again following the cost-benefit analysis of eco-terrorism. The costs may be high now, but it is highest where the environmental destruction is not hindered. Following Aristotle’s idea of the dependence of morality on law as it appeared on the Nicomachean Ethics- law is essential should we want to nurture our moral virtues. This is so because the law can yield to the enhancement of the intellectual virtues and improve our lives in general. However the context with which Aristotle speaks of consists of a society where citizens have relegated authority to political leaders who are most capable and just. Justice is achieved where the benefits of the majority is promoted. It is an unlucky occasion for the present time to be marred with inequalities implicating morality to a downfall. In which case, morality can not base itself on laws as the latter have contained contradictions to the former. Put it in another way, laws have given intrinsic worth to values other than those that are held by philosophy, ideology, norm or religion; instead to individualistic benefits. What transpires in laws are political culture, market-oriented beliefs and modernized attitudes and behavior; none of which is ready to face the non-profit, organized and strong sentiments of environmental activists. [1] Cited from http://en.wikipedia.org/wiki/Eco-terrorism   

Friday, September 27, 2019

The Legitimacy of Global Warming Essay Example | Topics and Well Written Essays - 250 words

The Legitimacy of Global Warming - Essay Example The Sept. 6, 2000 ozone hole was the largest on record at 11.5 million square miles (29.9 million square kilometers). (Climate Change) This is one evidence that the hole is getting smaller and smaller and this is extremely bad for the human race, it will fail to protect us from the ozone layers and terrible diseases like skin cancer would spread at the drop of a hat. Global warming is believed to be changing the current climate and this will have an adverse effect on a lot of things, the climate will be highly unstable and it will inevitably affect the human race. The imbalance will result in excessive rainfall, drought in some areas, tsunamis, hurricanes, cyclones and so on, this will cause damage beyond repair. To conclude it is very fair to say that global warming is not a hoax, it is happening and it will inevitably have an adverse effect on the human beings, it is preventable but more measures need to be taken in order to prevent it.

Thursday, September 26, 2019

Critically Analysis Of TOYOTA Motor Europe's Environmental Management Essay

Critically Analysis Of TOYOTA Motor Europe's Environmental Management System and Life Cycle Assessment - Essay Example 176). This ambitious plan was unveiled in order to ensure the success of their business. Unlike other global business, Toyota has effectively cultivated its social relationship through its EMS program. This has contributed to its success and sustainability. The success of its EMS program has been enhanced by its guiding principles, which provide a philosophical management that is geared towards sustainability of their business and giving back to the society. The guiding principles are also in line with the principles of the Rio earth summit, which is concerned, with conservation of forests and water resource. The choice of Toyota for this project was realized through a comparison of EMS programs from other equivalent companies. Toyota Motor Europe: Company Outline Currently Toyota motor corporation (TMC) is the third largest automobile marketer and manufacture in the globe. TMC has well established European manufacturing and marketing units through the Lexus and Toyota brand names. I n addition, TMC Europe has a well-designed structure that facilitates manufacturing and distribution of automobiles and spare parts. Toyota design and development centre (EDD). This organization is tasked with designing of all automobiles under the Toyota and Lexus brands. The organization is also tasked with the responsibility of designing environmental friendly motor vehicles. This is done through its comprehensive research scheme and consultation with various stakeholders. Current researches undertaken by the centre are aimed at establishing fuel-efficient cars and vehicles that can use alternative sources of energy. The centre is responsible for the development of the modern hybrid cars that run on a combination of electric and fuel energy. The unit is concerned with both the body and engine designs. This is done in relation to the European terrain and weather. The Toyota Prius is the latest model from the unit. This automobile has an engine capacity of 1.5 litters and petrol co nsumption on of 5.1 L/ 100 km. In addition, the vehicle has an emission capacity 120 g/km and 71 dB (A)2 carbon dioxide and noise emissions respectively. This has been achieved through the effective body and engine design of the car. Toyota Motor Europe Marketing & Engineering (TMME), this organization is concerned with marketing and conducting market research across Europe. In addition, the organization has the responsibility of collecting information from various stakeholders with the prospects of creating sustainable business. The TMME handles marketing of both the Lexus and Toyota brands across Europe on both the wholesale and retail basis. Toyota Logistic Service France (TLSFR), this organization is a subsidiary arm of the TMME, which undertake marketing of automobiles band spare parts across France and other French speaking nations. The organization also handles logistics TMC logistics across Europe. This organization is also responsible for collecting data aimed at improving the sustainability of the company from different stakeholders. TLSFR is also representing TMC in the on going anti global warming campaigns. Toyota Motor Europe Manufacturing (TMEM) is the other organization under Toyota. This organization oversees manufacturing of Toyota vehicles across Europe. The organization also coordinates all manufacturing operations in Europe. To

The Englishtenment Period and the Value of Women's Rights Article

The Englishtenment Period and the Value of Women's Rights - Article Example The fact that she is from China is telling and relevant. Chinese culture has not been known for gender equality, and in fact, women from the country have always been compelled to follow certain rules and strictures on how to behave. Foot binding, or the process of binding feet of girl-children so that they get deformed and resemble lotuses, is one of China’s anachronistic traditions. But the article is telling us that those days are drawing to an end and Chinese women are claiming their place and breaking new barriers. This recognition of women is one of the contributions of the Enlightenment period. To quote Carla Hesse, writing on women writers during the Enlightenment period, â€Å"the data on women writers suggests that the economic and commercial vision of the Enlightenment and Revolution opened up possibilities for female participation in an absolutely central arena of modern public life that was at odds with the dominant male conception of appropriate relations between the sexes.† How do we relate these Enlightenment values with the values of our times? In the modern days, the legitimacy of the struggle for women’s rights is largely accepted. Women are believed to be equal to men in most respects – they can work, vote, run for public office, and aspire towards their goals. Feminists all over the world have fought for these rights and have fought for the mainstreaming of gender values. Admittedly, there is still a lot that needs to be done and so much more than needs to be accomplished. In the developing world, women’s needs are still considered secondary to men, and in conflict and poverty situations, women tend to be at higher risk of hunger and malnutrition.  

Wednesday, September 25, 2019

The Effects Of Social Networks On E-Commerce Dissertation

The Effects Of Social Networks On E-Commerce - Dissertation Example Over the past years, the internet was used by majority of people as a medium for interaction and making fun by engaging in activities such as online gaming. With numerous improvements made on the internet, there has been an ever rising increase in trust in social links among the users. For this reason, online marketplaces have been augmented by many online business people as a way of developing and building honest and trust in their transactions with their partners as well as maximizing customer satisfactions. The outcome is the growth in the size of sales over the internet leading to a shift in demand. Through social links, customers can chart through forums with their clients, exchange useful business information concerning a particular company or product. Therefore, customers have a better bargaining power. This paper gives a deep exploration into how social media instruments affects the frequent purchasing behaviors and business transaction activities, notwithstanding the implica tions on the demand for products in the online-fashion industry. Like the trends in fashions, the tools of social media are innovative yet unpredictable because they are getting utilized and improved each and every time. The fashion industry is getting transformed by facets that are more related as a result of movement of more professionals of fashion in the online media, for this case the social media. The accessibility into the industry by has been made easy by these tools thus involving the general public and the entire customers of the fashion industry updated on the current fashion trends. Specifically this study explains the effects of social media on the behaviors of demand and customer inflow into the market. 1.0 INTRODUCTION In the recent years, the way people communicate with each other and share information has been revolutionized by social media. Initially the social networks were being used in large numbers by the teenagers for making new friends as well as keeping in t ouch with old ones. However, the situation has changed with now the focus being directed to online commerce for the purpose of generating solid and reliable revenues1. The e-mail which was earlier seen as the only method of communication electronically has been out-fashioned by the social media which is currently leaving its mark in the e-commerce, and the online fashion industry is not an exception. The fashion industry traces its roots to the traditional and old business practices hence making it lag behind in adopting new technologies. Almost everything done in the industry is more dependent on human labor rather than machines, beginning with designs sketches of designs to the process of purchasing. In most situations, the customers were the determiners of what kind of stock was to be put in stock because of their different tastes and preferences. However, with the existence of fast-acting technologies, fashion brands no longer seat back and wait for their customers to meet their needs. Social media is a to-way form of media under which users develop personal identities through dialogue and self-presentation.2 This has led to the social media receiving immense attention from consumers who go shopping online, specifically the fashion industry consumers. The global platforms such Tweeter, LinkedIn and Facebook creates room for designers to blog and keep their profiles updated with the aim of molding their brands and personalities on real-time basis. The interaction between

Tuesday, September 24, 2019

Rhetorical essay Example | Topics and Well Written Essays - 1250 words

Rhetorical - Essay Example The ad that caught everybody’s attention read â€Å"Wanted: Somebody to go back in time with me. This is not a joke. P.O. Box 91 Ocean View, WA 99393. Youll get paid after we get back. Must bring your own weapons. Safety not guaranteed. I have only done this once before† (Trevorrow, 2012). Curiously, a young intern name Jeff Schewensen (Jake Johnson) who works at a magazine based in Seattle embarked on an investigative mission to find the author of the strange posting. He assembled the oddest team with her friends Kenneth (Mark Duplass), Arnau, and Darius. Darius found out that the source of the ad came from Kenneth Calloway and so she made contact with Kenneth and from there, the story took unusual turn that made it interesting. It has to be noted that The idea of time travel in the film ‘Safety Not guaranteed’ is obviously not feasible and the film’s narrative is not meant to elucidate with using some technological discovery or marvelous science but to make the audience smile with how the odd characters in the film reacted with such technological possibility with each with their own agenda that reveals human nature. Thus, the story is told not from the logos or logical point of view but rather from pathos or emotional appeal to make the film entertaining (albeit not necessarily believable). Synthesizing the film through the lens of rhetorical devices made one realize that the film is not trying to convince or persuade the audience of any argument or some position of the author. Looking at it from the point of logos, the film does not make sense because time travel is not possible (at least in real world application but only in theory) and its technological possibility is not even explained. Even the sequencing of events such as placement of an ad that invites time travel with no safety guarantee to Darius and Kenneth’s trysts are just improbable in

Monday, September 23, 2019

Cloning Essay Example | Topics and Well Written Essays - 1000 words

Cloning - Essay Example By discussing these to the full extent, we will be able to come to a more conclusive and knowledgeable objective on this subject of interest. The aim of this paper is to discuss of this, as well as the other key factors in regards to this issue. This is what will be dissertated in the following. The term cloning is literally referred to as "using specialized DNA technology to produce multiple, exact copies of a single gene or other segment of DNA to obtain enough material for further study." ("CDC", 2006). The process itself is actually referred to as cloning DNA, and the resulting cloned (or copied) collections of DNA molecules are called clone libraries. In more simple terms, cloning is the process of creating an identical copy of something original. In the cloning of a gene, there must be extraction from one gene from an organism and insert that into another organism, where it can then be used and studied. There is also a related technique to this which is called subcloning, which refers to the transferring of a gene from one plasmid into another, again for further study. Cloning is the process of making a genetically identical organism through nonsexual means. Although the idea of cloning may seem rather revolutionary to some, it has in fact been around for some while. It has been used for many years to produce plants (in fact even growing a plant from a cutting is a type of cloning). The terms 'recombinant DNA technology', 'DNA cloning', 'molecular cloning', or 'gene cloning' all refer to the same process: the transfer of a DNA fragment on interest from one organism to a self-replicating genetic element such as a bacterial plasmid. "This technology has been around since the 1970s, and it has become a common practice in molecular biology labs today." (Cloning Fact Sheet, 2006). When the media report on cloning in the news, they are usually only talking about one type of cloning, which is called reproductive cloning. However, there are many different forms of cloning, including human cloning, animal cloning, and cellular cloning. As for reproductive cloning, it is a technology which is used to generate an animal that has the same DNA as another currently or previously existing animal. The majority of reproductive cloning has so far been tried and tested on animals, with the modern cloning techniques involving nuclear transfers being successfully performed on several species. These land mark experiments in chronological order are: Tadpole (1952) Carp (1963) Sheep (1986) Rhesus monkey: Tetra (female, January 2000) Cattle: Alpha and Beta (males, 2001) and (2005) Brazil Cat: Copycat "CC" (female, late 2001), Little Nicky (2004) was the first cat cloned for commercial reasons Mule: Idaho Gem (2003) Horse: Prometea (2003) Human (2005) Dog: Snuppy (2005) The recent success in cloning animals has sparked fierce debates among scientists, politicians and the general public about the use and morality of cloning plants, animals and possibly humans. Although some argue that cloning can be used for many positive reasons, such as to preserve and increase the number of various endangered species, others are outraged at the 'misuse and mistreatment of science'. Currently the most successful cloning technique is that of the somatic cell nuclear transfer. This is the same cloning technique which allowed Dolly the sheep to be cloned, is also the same technique used by ACT - the first company to

Sunday, September 22, 2019

Cognitive Effects of Early Bilingualism Essay Example for Free

Cognitive Effects of Early Bilingualism Essay The American educational system has fallen behind other leading nations in the world in many respects, one of which is in bilingual instruction. This has traditionally been overlooked in the United States until the high school level. Children in today’s society should be made more prepared for the growing globalism and technological advances throughout the world instead of losing educational opportunities due to economic downfall and lack of resources. This includes a second language acquisition introduced earlier in the program. On top of political reasons, the positive effects to the cognitive development of the brain when introduced to a second language are many. The age of acquisition is crucial due to the plasticity of the brain which, according to the critical period hypothesis, begins to plateau after five years of age. The current policy in early education limits greatly the amount of extracurricular lessons provided in accordance with government policies such as No Child Left Behind, which restricts school funding based on standardized testing only in certain subject areas. School programs, realistically beginning in elementary education, should include foreign language study due to the strong evidence that bilingualism in children can develop higher cognitive abilities which can be enhanced with proficiency and positively influence skills in other areas. Old arguments suggest that, â€Å"children who are instructed bilingually from an early age will suffer cognitive or intellectual retardation in comparison with their monolingually instructed counterparts† (Diaz 24). Much of the research from the past supporting this argument focused on older bilinguals, mostly adults who may have shown competent abilities in a second language but who had much later ages of acquisition and who usually acquired the second language outside of the home. Many early studies in this field worked with children of immigrants who showed lower abilities in cognitive tasks most likely because of the lack of proficiency in the second language (L2) and lack of proper schooli ng in relation to this deficiency (Kovà ¡cs 307). In correlation with poorly chosen test subjects, the studies were typically done with orthographic representations of words that would have been more difficult for younger test subjects to work with. For example, a study done by Ton Dijkstra, Professor of Psycholinguistics and Multilingualism at the Donders Institute, which focused only on adult English/Dutch bilingualsthe youngest being fifteen years old, all of whom studied their L2 in a middle or high school level. This study included only written examples of words and had the subjects determine if the word was English or Dutch. The results were able to somewhat prove Dijkstra’s theory of Bilingual interactive activation (BIA) which underlines the effects orthography has on L1 and L2 word retrieval that is â€Å"assuming, of course, that the same orthography is used in the input† (Dijkstra 217). If this study were done on younger children, it is sure they would not have performed as well since children are typically less familiar with the written language than with the spoken. Older language learners would make more use of the written approach to learning, such as a textbook, while younger learners typically lear n more from a speech-based approach, like conversationally in the home. The textbook approach is a symbolic processing which differs from the more embedded cognitive retrieval of the speech-based learning approach utilized by younger children to understand the two languages. There have been many studies over the past few years that have proven the opposite of these older arguments. Many of the studies have tested the cognitive abilities of young children, usually aged six and under in accordance with the critical period hypothesis, with both monolingual and bilingual proficiency. These experiments are concerned with cognitive tasks including false-belief tasks and grammar testing to determine the ability to hold abstract thought in the L2 as well as phonemic testing in order to find if there is an ability to distinguish between the phonemes of the different languages. The majority of these studies have tested subjects using visual representations and vocal experiments with proctors who have experience working with children and are trained in both languag es being tested. The more useful subjects are usually taught implicitly, or passively in the home. Although some make use of explicitly taught subjects, meaning they learned actively in a class setting. It has been proven that an infant of four months has the incredible linguistic discrimination abilities to distinguish languages with different prosody and phonemes (Kovà ¡cs 303). An infant is then better equipped to attain more native-like proficiency later in life when exposed this early to the sounds and rhythm of the L2. Doctor in Communication Sciences, Karsten Steinhauer explains, â€Å"that late L2 learners stabilize at some point short of native-like attainment [which] most recently has been discussed in terms of phonological/prosodic interference from L1† (Steinhauer 15). When a young child is introduced to two separate languages, the mechanisms of attention, selection, and inhibition become more fine-tuned due to the experience of attending to one language and ignoring the other (Kovà ¡cs 303, 308). The training in encoding and the association of two correspondi ng words with a common concept underlines the superior representational abilities a bilingual retains especially when the L2 is entrenched in the brain the way early acquisition allows. Linguist à gnes Melinda Kovà ¡cs presents research proving that monolinguals typically attain these abilities at the age of four years while young bilinguals gain these skills much earlier (Kovà ¡cs 316). The brain’s plasticity allows the young child to hold and use the two languages without interference and with continued usage the child will be more likely to attain full native-like proficiency in both languages. Kovà ¡cs also explains that since the brain remains active during demanding tasks, the brain may take on the extra load of two languages as a constructive challenge. The young, malleable brain may possibly â€Å"greatly adapt to [the challenge], for example, by changing its morphology† (Kovà ¡cs 308). A type of adaptation has been proven in studies done by neuroscientist Andrea Mechelli, which were concerned with the grey matter surrounding the left inferior parietal cortex, the general area associated with language use containing the Broca’s are a. These studies confirmed that the grey matter in this area is denser in early-acquired bilinguals. The density decreases in correlation with proficiency in the L2 with monolinguals having the least dense matter (Mechelli 757). This may be the case because a later acquired L2 is held at a more surface level of the brain and requires the use of the declarative memory instead of the procedural memory. Many tests have been done to determine the amount of brain activity associated with language in the left inferior parietal cortex through the use of event-related brain potential, or ERPs. Dr. Steinhauer describes ERPs as â€Å"reflecting the real-time electrophysiological brain dynamics of cognitive processes with an excellent time resolution in the range of milliseconds,† and that ERPs â€Å"have been hypothesized to be linked to rule-based automatic parsing† (Steinhauer 16). Measurements of ERPs are taken while subjects perform syntactically poignant tasks. Since it is thought that syntactic processes are generally automatic or a part of â€Å"implicit grammar processi ng† (Steinhauer 17), the ERP components would be more difficult to elicit in later acquired bilinguals. Steinhauer et al. performed several studies in this area, working with many real and one artificial language labeled BROCANTO 2. In each case, the subjects were given grammaticality judgment tasks in the given language, such as subject-verb agreement violations and lexical anomalies. For each group, the early acquired or implicitly taught subjects educed the same type of ERP responses as native speakers. Late-acquired or explicitly instructed subjects showed more shallow responses, if any at all in this area. These findings show that â€Å"syntactic processes appear to be sensitive to delays in L2 acquisition† (Steinhauer 19). One of the most prominent issues in L2 proficiency is attaining the phonemic boundary between the two languages. Monolinguals are usually unable to distinguish the sounds of a language other than their own. The more proficient a bilingual is in their L2, the more able they are to perceive the two types of phonemes and to determine which is correct in a given phonological circumstance. The phonemic boundary is the least likely area to be fossilized in a late-acquired bilingual. There have been several studies done which have proven this, including a 2008 study done by Adrian Garcia-Sierra, professor of Communications at the University of Texas. In this study, the voice onset time, or VOT, of thirty college students was tested. Half of the students were English monolinguals while the other half were English/Spanish bilinguals who described themselves as fluent speakers of both languages and who learned their L2 at home. This study was done in Austin, Texas where some Spanish is integrated into the daily culture. The results showed that the more fluent bilinguals were more apt to â€Å"a perceptual shift†¦associated with high level of confidence in English and Spanish†¦[and] that highly confident L2 bilinguals are more likely to possess a double phonemic boundary† (Garcia-Sierra 378). This shows that more proficient bilinguals will have a stronger ability to determine different phonemes, which also underlines the effects bilingualism has on a dvanced discrimination and attention skills. Another recent study performed on early bilinguals was done by a group of psychologists headed by Michael Siegal. The experiments tested the pragmatic skills of 41 children in northeastern Italy. All were between the ages of three and six years old, with 19 Italian monolinguals and 22 Italian/Slovenian bilinguals who attended the same preschool taught only in Italian. The children were tested on the Gricean maxims of conversational understanding. These are four basic rules which provide a foundation for pragmatic competence including quality, quantity, relevance, and politeness. The groups of children were shown cartoons with characters having conversations that contained one response created in order to break one of the maxims. The children were then asked which of the characters said something strange or rude and to provide a more appropriate response when the statement was positively identified. The main thesis in this study was that bilingualism requires â€Å"the capacity for flexibility in the representation of language and objects [which] suggests that early bilingualism should be accomp anied by advanced meta-pragmatic skills† (Siegal 115). This theory was upheld by the results of these tests in which the bilingual children outperformed the monolinguals by much more than a chance margin, especially in the maxims of politeness and quality even though many bilinguals had a delayed vocabulary in their L2. The psychologists behind this study suggest that bilingualism can be â€Å"accompanied by an enhanced ability to appreciate effective communicative responses† (Siegal 115). The results of this research seem to highlight the idea that the acquisition of a second language allows a child to remove themselves from the comfortable context of their native language and to realize that it is more necessary to provide useful information and use polite tones for more a successful exchange in both languages. Recently, studies have been performed concerning the effects and importance of early-acquired bilingualism in patients with neuropsychiatric disorders such as Parkinson’s and Alzheimer’s diseases. Research in this area shows that it is less likely for a bilingual individual to be affected by these types of diseases. The majority of the hypotheses behind this statistic pertain to the activity in the brain that is needed to think and speak bilingually. This constant activity exercises the brain in a way that is counterintuitive to the deterioration involved with these disorders (Paradis 216). The research behind Parkinson’s disease explains that the procedural memory is affected greatly sometimes causing a loss of the L1. This is partnered with a tendency to â€Å"produce a smaller portion of grammatical sentences†¦and exhibit deficits in comprehension of complex syntactic forms† (Paradis 217). This is likely linked to the deterioration of the left inferior parietal cortex, the same area in the brain discussed earlier, which is associated with syntactic processes and holding the L1. On the other hand, bilingual patients with Alzheimer’s show a loss in t heir L2 as well as in semantic abilities and a gradual loss of pragmatic, phonological, and syntactic structures. More common in this type of dementia is a puzzlingly inappropriate mixture of the two languages (Paradis 222). This is due to the break down of the declarative memory caused by the dementia. The declarative memory is involved with metacognition, which is why it affects such things as the less familiar language, pragmatic skills, and the selective attention abilities of bilinguals. The major finding in these studies is that â€Å"the differences observed in psychotic conditions as well as in dementias are caused by the increased reliance on declarative-memory-based (and hence consciously controlled) explicit metalinguistic knowledge† (Paradis 222). The advances made in early bilingual research have been great over the past few decades. Through these studies and so many more, it has been made clear that bilinguals with early ages of acquisition not only achieve more native-like proficiency but also tend to have more advanced cognitive abilities than their monolingual peers. These include but are not exclusive to increased analytical, representational, selective, and control abilities. Bilingualism also implies more developed metalingustic awareness and mental flexibility. Early bilinguals have also shown greater abilities in pragmatics and phonemic discrimination. In opposition to old arguments, Kovà ¡cs writes, â€Å"The bilingual condition could be stimulating for the highly plastic developing mind of the child, and induces specific changes in the brain and cognitive systems† (Kovà ¡cs 317). The higher development has been seen in ERP testing and in the density of grey matter involved in the linguistically apt area of the brain. Educators and policy makers should consider this information when planning early education programs. Those enriched with the benefits of a bilingual education are not only better off cognitively, but in the modern world, would be more prepared for the global society and workplace. Works Cited Diaz, R â€Å"Thought and Two Languages: The Impact of Bilingualism on Cognitive Development.† Review of Research in Education 10 (1983): 23-54 Dijkstra, Ton. â€Å"Task and Context Effects in Bilingual Lexical Processing.† Cognitive Aspects of Bilingualism (2007): 213-235. Garcia-Sierra, Adrian, Randy L. Diehl, and Craig Champlin. â€Å"Testing the double phonemic boundary in bilinguals.† Speech Communication 51 (2009): 369-378. Kovacs, Agnes Melinda. â€Å"Beyond Language: Childhood Bilingualism Enhances High- level Cognitive Functions.† Cognitive Aspects of Bilingualism (2007): 301-323. Mechelli, A., Crinion, J. T., Noppeney, U., O’Doherty, J., Ashburner, J., Frackowiak, R. S., and Price, C.J. 2004. Structural plasticity in the bilingual brain. Nature. 431: 754. Siegal, Michael, Laura Iozzi, and Luca Surian. â€Å"Bilingualism and conversational understanding in young children.† Cognition 110 (2009): 115-122.

Saturday, September 21, 2019

Streptozotocin (STZ) Induced Diabetes Experiment

Streptozotocin (STZ) Induced Diabetes Experiment MATERIALS AND METHODS 3. MATERIALS AND METHODS 3.1 Animal Selection and Care STZ induced diabetes are commonly performed in Male Wistar Kyoto rats. In these models, male Wistar rats at 8 weeks of age (200–300 g) are selected from animal supply facility of SGPGIMS Lucknow, and house in a 12-h light, 12-h dark cycle environment, temperature 25 °C, with standard rat diet and water in metabolic cages for one week prior to STZ treatments. The study had clearance from Institute animal ethics committee. 3.2 Study Design Wistar Rat (Preparation and Validation of animal model of DN) 50 mg/kg STZ induced Diabetic Rat (n=3) Normal Rat (n=3) After 8th week rat are sacrificed for validation of Diabetic nephropathy 3.3 Induction and Validation of Diabetes by Streptozotocin There is a lack of appropriate animal model that could spontaneously develop DN. It has been a significant limitation to find out specific factors that is underlying this disease and also the development of new therapeutic methods or strategies to prevent progressive renal disease in diabetes (Morcelo A. Nobrega et. al. 2004). Most Sprague-Dawley (SD), Wistar-Kyoto (WKY) rats are used as model for performing STZ-induced diabetic nephropathy. Here, in these models, 8 weeks old male rats (200-250gm) are starved or kept on fasting for 18hrs and after that STZ was injected intraperitoneal (WKY-50mg/kg, SD-55mg/kg and SHR-45mg/kg) with sodium citrate buffer (1ml/kg) (Ma G et. al. 2004 and Cooper ME et. al. 1988). STZ given here intraperitoneally to the rats, however, it is a very less common procedure as intravenous injections are comparatively easy to perform in rats and give more reliable results. In addition to the STZ dosage required for inducing diabetes via an intraperitoneal route is relatively higher as compared to other route. After giving STZ, the rats should be given ample amount of drinking water with sucrose for 48 hrs (15g/L), to avoid early mortality of rats as stored insulin is released from damaged pancreatic Islets of Langerhans. After completion of 1 week of STZ induction, the rats must be assessed for hyperglycaemia and also those with fasting blood glucose of over 280 mg/dl (15 mmol/L), which is usually around 90%, and it should be included in the studies of diabetic nephropathy. In order to prevent subsequent development of ketone uria, subsequent injections of long-acting insulin (approx. 2-4 U/rat) should be given daily to maintain the blood glucose levels in a desirable range(300-600 mg/dl, 16-33 mmol/l) (Davis BJ et. al. 2003). Studies exploring the effects of treatment on the development of DN should not be underway until at least 3 weeks after STZ when the kidneys have improved from the acute mild nephrotoxic effects of STZ (Kraynak AR et . al. 1995) This drug i.e., STZ has been shown to induce a diabetic state in 72 hrs (3 days) as documented by examining tail blood samples using a Glucometer (Optimum Exceed) Diabetic animals and non-diabetic control group were kept in metabolic cages individually and separately and under feeding and metabolism control. Glucose in the blood of diabetic rats exceeded that of the non-diabetic control ones. Food consumption was measured in terms of (gm), water consumption was measured in terms of (ml) and urine volume was measured in terms of (ml) on a daily basis and glucose in blood serum were also measured, so that chemical diabetes was verified in rats injected with Streptozotocin. 3.4 Estimation of Creatinine Modified Jaffe’s method was used for colorimetric estimation of creatinine concentration in urine samples. Principle: Creatinine + picric acid Creatinine picrate (Yellow) (Orange) The resultant orange color is measured colorimetrically. However, the intensity of the resultant orange color is directly proportional to the concentration of creatinine in the sample. Method: Draw the Blood from a vein and then transferred into the vial. Centrifuge the blood for 10 minutes and Serum is obtained. Separate out the serum in different eppendrofs. The concentration of creatinine is calculated in the serum sample using the Jaffe’s method, as follows: In a clean dry test tube add 0.5 ml distilled water (blank) or serum (test), add 0.5 ml NaOH and then 0.5 ml picric acid. Mix all the contents of each tube. Left to stand for 15 minutes. The absorbance is measured at ÃŽ »max 500 nm. If a standard creatinine solution (0.55 mg/dl) has an absorbance value of 0.30, then the concentration of creatinine in the provided serum sample is calculated by using the following equation: Ctest = Cstd x 3.5 Estimation of Urinary Albumin Enzyme linked immunosorbent assay (ELISA) for detection of rat albumin in serum, plasma or urine. Other biological fluids that contain Rat Albumin, such as faeces or saliva, may be suitable samples. 3.5.1 Principle: The antigen present in urine sample are allowed to stick to a poly vinyl and then plate is washed to separate antigen and antibodies from remaining sample components. To this plate a corresponding second antibody is added to get fixed to the already adhered first antigen in the plate. To this added second antibody, an enzyme is also tagged is that, when a suitable substrate is added, the enzyme reacts with it to produce a colour. This colour produced is measurable as a function or quantity of antigen present in the urine sample and there by identified. 3.5.2 Chemical and material required: 96-well plate ELISA Coating Buffer ELISA Wash Solution ELISA Blocking Buffer Sample/Conjugate Diluent (ELISA Blocking Buffer + Tween 20) 10% Tween 20 Enzyme Substrate, TMB ELISA Stop Solution Additional Materials Required: Ultrapure water Precision pipettors, with disposable plastic tips Polypropylene, polyethylene or glass tubes to prepare standard and samples Containers to prepare buffers An aspiration device or an automated 96-well plate washer Disposable reagent reservoirs A standard microtiter plate reader for measuring absorbance at 450 nm 3.5.3 Procedure: Added 100  µl of diluted coating antibody to each well. Samples were run in duplicate. Incubated at room temperature (20-25 C) for 1 hour. Washed plate FIVE times. Added 200  µl of Blocking Solution to each well. Incubated at room temperature for 30 minutes. Washed plate FIVE times. Added 100  µl of standard or sample to well. Incubated at room temperature for 1 hour. Washed plate FIVE times. Added 100  µl of diluted HRP detection antibody to each well. Incubated at room temperature for 1 hour. Washed plate FIVE times. Added 100  µl of TMB Substrate Solution to each well. Developed the plate in the dark at room temperature for 15 minutes. Reaction was stopped by adding 100  µl of Stop Solution to each well. Absorbance was measured on a plate reader at 450 nm. 3.5.4 Precautions: Store all reagents at 2-8 ºC. Do not freeze reagents. All reagents must be at room temperature (20-25  ºC) before use. Vigorous plate washing is essential. Use new disposable pipette tips for each transfer to avoid cross-contamination. Minimize lag time between wash steps to ensure the plate does not become Completely dry during the assay. Avoid microbial contamination of reagents and equipment. Automated plate Washers can easily become contaminated thereby causing assay variability. Take care not to contaminate the TMB Solution. Do not expose TMB Substrate solution to glass, foils, or metal. If the solution is blue before use, do not use it 3.6 Oral Glucose Tolerance Test Rats are fasted overnight (12-16 hours) before the test, sedated rats, will be given 50 % dextrose (3ml/kg body weight) intraperitoneally. Whole blood will be collected from the tail vein at 0, 15, 30, 60, 120, and 180 min after the administered of glucose for the measurement of glucose with a glucometer. 3.6.1 Material Required: Glucometer and glucose strips Dextrose Gauge needles Timer and Pen Table of mice for record keeping of glucose values 3.6.2 Preparations before the test: Weigh the animals before the test. The animals are fasted overnight (approximately 16 hours: 5 pm to 9 am). Place each of the animals in a separate fresh cage with no food, but make sure they have water bottles. Make sure that there is no activity in the animal room before and while you are performing the test. Prepare the glucose solution the night before the test. 3.6.3 Procedure: After the overnight fast, blood glucose was determined (time 0) in a drop of blood as following: Rats were placed on top of the cage (let it hold onto the grid). The tail tip was pricked with a needle, wiped off the tail tip with gauze, and the next drop of blood was used for the determination of glucose with a glucometer. Glucose solution was given orally. Blood glucose was determined at 10, 20, 30, 60, 90 and 120 min after the administration of glucose. For the determination of glucose at each of these time points collected a drop of blood as following: wiped the cut end of the tail to break any blood clot that had formed and collected the next drop of blood. If the blood does not appear spontaneously â€Å"milk† the tail by holding the tail between your thumb and index finger and move along the tail from the base of the tail to the tip of the tail while applying gentle pressure. 3.7 Assessment of Renal Histopathological Injury 3.7.1 Tissue preparation for histology: After 8 weeks the rats were weighed, sacrificed out in accordance to the Institutional animal ethics committee by using suitable anaesthetic agent (Ketamine) and their kidneys were taken out. Left kidneys were perfusion fixed for Histopathological and IHC studies and right kidneys were freezed immediately for western blotting and RT-PCR. Preparation of paraffin blocks of kidney: Kidneys were preserved in Para- formaldehyde is taken out in a glass slab for sectioning. A two cross section of upper half of kidney was done with sterile blade for paraffin embedding. The whole process for preparation of paraffin blocks took two days. Day first: The Formalin fixed kidneys were picked up and place in plastic cassettes was sequentially dipped in alcohol for dehydration. The schedule is as followed: 50% Alcohol →1.5hrs 70%Alcohol →1 hrs. 80% Alcohol →1 hrs. 90% Alcohol →1hrs 100% Alcohol →1hrs (twice) The cassette containing the tissue was left overnight in 100% absolute alcohol. Day Second: On 2nd day we perform the following treatment to tissue containing cassette Cassette was removed from 100% alcohol and dipped for CHCl3 treatments Chloroform (A) 1.5 hours. Chloroform (B) 1.5 hours. Chloroform (C) 1.5 hours. The cassette was then kept in melted paraffin wax (at 58 °C 65 °C in water bath) following two changes of paraffin wax for proper blocking. The steel chocks are kept at the 65 °C electronic heater and the paraffin treated kidney in plastic cassettes are opened and place in pre-heated steel chocks together with melted paraffin wax and closed with cassette. Block was kept at room temperature to solidify the melted wax. The paraffin block containing tissue was sectioned with microtome. The block was fit properly in the Microtome machine 5 µ sections were cut. 3.8 Periodic Schiff’s Acid (PAS) Staining 3.8.1 Principle: PAS (Periodic Acid Schiff) is a method of staining used for the detection of polysaccharides i.e., glycogen and mucosubstances that is glycoprotein’s and glycolipids. PAS stain is a histochemical reaction. In the reaction, periodic acid oxidises vicinal diols in these sugar. It oxidized the aldehyde formed by carbon-carbon bonding that react with fuchsin-sulphurous acid and forms the magenta colour. This periodic acid exposes the glycogen to give a colouring product. The Schiff’s reagent is a very specific agent that only reacts with the carboxylic group compounds. Material required: Glass slides and cover slips Poly Lysine (Sigma Aldrich, USA) Xylene Graduated Alcohol (30%, 50%, 70%, 90% and 100%) Periodic acid Schiff Reagent Haematoxylin Acid alcohol (1% HCl in 70% alcohol) DPX mountant 3.8.3 Protocol for PAS Staining: Keep the Poly-Lysine coated slides on hot plate for 15-20 min. Dip in warm Xylene for 10 min twice. Pass the slide through graded alcohol 100% alcohol- (10 minutes) twice 90% alcohol (5 minutes) 70% alcohol (5 minutes) 50% alcohol (5 minutes) 30% alcohol (5 minutes) Dip in distilled water for 5 min. Place the slide containing section into 0.5 % Periodic acid for 5 minutes. Rinse with distilled water. Schiff Reagent was added for 10 min until deep magenta colour appear. Wash in running tap water for 5 min. Counter stain in haematoxylin Rinse with distilled water. If high stain come, then wash with acid alcohol (1% HCl+70% alcohol) Wash the slide in tap water. Dehydrate in alcohol, and dry the slides. After drying mount the slides by DPX mountant. Masson’s Trichrome Staining 3.9.1 Principle: MT Staining method is used to demonstrate the increase of collagen in diseases. This method of staining uses three dyes of contrasting colours for the selective staining of basic tissue components i.e., muscle, collagen fibers, fibrin, and erythrocytes. The general phenomenon of trichome staining is that smallest dye molecule colours or stains the less porous tissues. However, the penetration of dye of larger molecular size is depends on the expense of small molecules. Some others suggests that the acid dye firstly stained the tissue then the Biebrich Scarlet will binds with the acidophilic components of the tissue, after which when treated with the phosphor acids, the components that are less permeable will retain the red colour, because of the collagen this red colour is pulled out and at the same point of time causing a link with the collagen to bind with the aniline blue. . At the outset it must be made clear that the methods control how ionised acid dyes react with ionised basic tissues. 3.9.2 Material required: Bouin’s solution Xylene Graduated Alcohol (30%, 50%, 70%, 90% and 100%) Weigert’s iron hematoxylin solution Biebrich scarlet acid fusin phosphomolybedic-phosphotungustic acid solution Aniline blue solution 3.9.3 Protocol for MT Staining: Keep the Poly-Lysine coated slides on hot plate for 15-20 min. Dip in warm Xylene for 10 min twice. Pass the slide through graded alcohol 100% alcohol- (10 minutes) twice 90% alcohol (5 minutes) 70% alcohol (5 minutes) 50% alcohol (5 minutes) 30% alcohol (5 minutes) Dip in distilled water for 5 min. For formalin fixed tissue, re-fix in Bouin’s Solution for 30 minutes to 1 hr. at 56 ºC to improve the staining quality although this step is not necessary. Rinse in running tap water for 5-10 minutes to remove the yellow color. Stain in Weigert’s iron hematoxylin working solution for 10 minutes. Rinse in running warm tap water for 10 minutes. Wash in D/W Stain with Biebrich Scarlet Acid Fusin for 5 minutes. Differentiate in phosphophosphomolybedic-phosphotungustic acid solution for 10-15 minutes. Transfer the section directly (without rinse) to aniline blue solution and stain for 5-10 minutes. Rinse directly in D/W and differentiate in 1% Acetic acid solution for 2 minutes. Rinse slides, dehydrate through Alcohol Clean slides in xylene Mount with DPX mountant. NoteNuclei-Black, Muscle Fibre-Red, Collagen-Blue, Cytoplasm-Red Modified Immunohistochemistry 3.10.1 Principle: Immunohistochemistry (IHC) is the process whereby antibodies are used to detect proteins (antigens) in cells within a tissue section (for instance liver, pancreas or the heart). Immunohistochemistry exploits the principle that in biological tissues antibody binds to the specific antigens. This tool is used to localize specific antigens in tissue sections with labelled antibodies based on antigen-antibody interactions. This antigen-antibody interaction can be visualized in number of ways i.e., the immune reactive products can be visualized by a marker including fluorescent dyes, enzymes in general; radioactive elements or colloidal gold. This IHC technique is widely used by the researcher in basic research for understanding the differentially expressed proteins and for the localization and distribution of biomarkers in different- different parts of biological tissues. 3.10.2 Material required: Poly-Lysine coated slide. Xylene 1X- PBS buffer.(Sigma Aldrich Inc., USA) Graduated Alcohol (30%, 50%, 70%, 90% and 100%) DPX mountant for microscopy Acid alcohol (1% HCl in 70% alcohol) citrate buffer Sodium Borohydrate Hydrogen Peroxide Blocking solution Primary antibody Secondary antibody Streptovidin HRP 3.10.3 Protocol for Modified Immunohistochemistry: Cut the section of 3 to 5  µm. Warm the slide on hot plate (55 °c) for 30’. Dip the slide in warm Xylene twice for 10 minutes. Wash the slides three times in TBS or PBS for 5 minutes each. Dehydration using 100% Alcohol twice for 10 minutes each and 90, 80, 40, 50 and 30 percent for 5’ each in coupling jar. Wash thrice in TBS or PBS for 5 minutes each. Antigen retrieval -10 minutes for 98 °c in citrate buffer pH-6 (Note: 0.294 gm in 100 ml MQ with pH=6, 1 10mm citrate buffer) Here we are using pressure cooker for Antigen retrieval Cool the citrate Buffer slides. Give the Sodium Borohydrate treatment in coupling jar (To remove Background). (Dark Condition) (Note:- 1% NaBH4) Wash slides thrice in TBS or PBS for 5 minute each. Hydrogen Peroxide Blocking (3% H2O2 in Methanol or water –Dip the slides in it and keep it for 30’). Wash thrice in TBS or PBS for 5 minutes each. Blocking solution[0.3% Triton X and 5% sheep serum in 1xPBS or 1xTBS] For 2ml (6 µl Triton, 100  µl sheep serum, 19 of 94  µl (1xPBS or 1xTBS)) 14. Blocking for 30minutes at 37 °c in lab (some time 1hour also) Wash the slides thrice in TBS or PBS for 5’ each. Primary antibody in TBS or PBS solution, left overnight at 4 °c (Note: 1:200 dilution for fibronectin 1:500 dilution of Collagen IV). Wash the slides thrice in 1xTBS or 1xPBS Secondary antibody added on slide and keep it for 30’ to 1hour. But parafilm on it. (Note: 1:200 dilution) Wash the slide thrice in 1xTBSor 1XPBS for 5 minutes. Streptovidin HRP (1:200) in 1xTBS or 1xPBS for 20 minutes at Room temperature. Three wash in 1xTBS 5 minutes each. DAB (Till color in Brown) (15 minutes) 23.1xPBS (5minutes) wash 24. Counterstain for 30 seconds with Hematoxylene (Directly put on slide) 25. 1xTBS or PBS immediate wash 26. Dehydrate and dry and mount

Friday, September 20, 2019

Truman’s Horrible Mistake to Use the Atomic Bomb Essay -- Argumentativ

The war was coming to a victorious conclusion for the Allies. Germany had fallen, and it was only a matter of time until Japan would fall as well. Secretary of War Henry L. Stimson was at the forefront of the American war effort, and saw atomic weaponry as a way out of the most monumental war ever. As discussed in Cabell Phillips’ book, The Truman Presidency: The History of a Triumphant Succession, Stimson was once quoted as saying that the atomic bomb has â€Å"more effect on human affairs than the theory of Copernicus and the Law of Gravity† (55). Stimson, a defendant of dropping the bomb on Japan, felt that the world would never be the same. If the world would change after using atomic weapons, could it possibly have changed for the better? One would think not. However, that person might be weary of the biased opinion of White House personnel. He or she should care more for the in depth analytical studies done by experts who know best as to why America should or should not have dropped the atomic bomb. As more and more evidence has been presented to researchers, expert opinion on whether or not the United States should have dropped the two atomic bombs on Hiroshima and Nagasaki has also changed. More and more researchers seem to feel that the atomic bomb should never have been used (Alperovitz 16). Despite several officials’ claims to enormous death estimations, an invasion of Japan would have cost fewer total lives. In addition, post atomic bomb repercussions that occurred, such as the Arms Race, were far too great a price to pay for the two atomic drops. However, possibly the most compelling argument is that Japan would have surrendered with or without the United States using the atomic bomb. In defiance of top... ... Conference.† Reader’s Companion to American History. Houghton Mifflin Company, 1991. Online. Internet. Available at HTTP: http://www.historychannel.com/. 23 Sept 2001. Franck, James, et al. The Franck Report. June 11, 1945. Online. Internet. Available at HTTP: http://www.dannen.com/decision/franck.html. 1 Oct. 2001. Miles, Rufus E. Jr. â€Å"Hiroshima: The Strange Myth of Half a Million American Lives Saved.† International Security (1985): 121-140. Newman, Robert P. â€Å"Hiroshima and the Trashing of Henry Stimson.† New England Quarterly (1998): 5-32. Phillips, Cabell. The Truman Presidency: The History of a Triumphant Succession. New York: MacMillan, 1966. â€Å"Truman, Harry S.† The Concise Columbia Electronic Encyclopedia. Columbia: Columbia, 1994. Online. Internet. Available at HTTP: http://www.historychannel.com/. 24 Sept. 2001.

Thursday, September 19, 2019

Seven Years War Essay -- essays research papers fc

  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   The Seven Years’ War The first true World War. †¦Cause and effects!   Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚     Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   What would the state of the free world be today if the alliance of the war of the Austrian Succession had not reversed in the Seven Years’ War? Would we speak French, still be â€Å"New England†, or perhaps New Spain? The fact is that while we may not know for certain that today’s world would be different, you can rest assured that the Seven Years’ War set the tone in Europe, and more importantly in North America for the next half century. The history of the 18th century in Europe was always uncertain. In fact, the history of Europe will show that the fate of the continent, perhaps even the world, was always on the brink. Nations constantly were maneuvering for the upper hand looking to the highest bidder to choose sides with. The war of the Spanish Succession and the war of the Austrian Succession will show us that this new â€Å"world war† would be no different. The degree of uncertainty on the continent in 1755 is unparalleled. Russia, Bohemia, and even France and England could have swung in either direction. In fact France and England did change â€Å"loyalties† if you will between the Treaty of Aix-la-chapelle and Frederick’s invasion of Bohemia in 1756. Maria Theresa, although agreed to the aforementioned treaty to end the war of her accession, would always seek revenge on Frederick for the humiliation he had inflicted on her. If these loyalties or interests I should say hadn’t changed, what would the effect on the world be today? Would you or I be speaking some other language? French perhaps? The Enlightened Despots, Frederick? Was he? Maria Theresa? Hardly, Catherine had absolutely no impact whatsoever, and William Pitt, while he was an effective military strategist, was no despot, and surely not enlightened. Louis the XV, who was led around by the nose by Mme de Pompadour, was as ineffective as all the Kings of France would be after his grandfather. Britain obtained Prussia as her ally, but you might ask, why? Surely you can’t fuel Frederick’s massive army any more? Pitt the Elder argued though that while true... ... have none of that. The war would lead Prussia to the forefront on the European continent, however Britain clearly gained much more than any other did. France would not recover, and Louis knew all too well what lay in store when he said â€Å" After me, the deluge.†   Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Works Cited 1.  Ã‚  Ã‚  Ã‚  Ã‚  Anderson, Fred â€Å"Crucible of War†, The Seven Years’ War and the fate of Empire in British North America. Random House: New York, NY 2000 2. Durant, Will and Ariel â€Å"Rousseau and Revolution† The Story of Civilization. Simon and Schuster: New York, NY 1967 3.  Ã‚  Ã‚  Ã‚  Ã‚  Kennedy, Paul â€Å"The Rise and Fall of the Great Powers† Random House: New York, NY 1987 4.  Ã‚  Ã‚  Ã‚  Ã‚  Leckie, Robert â€Å"A Few Acres of Snow†, The Saga of the French and Indian Wars. John Wiley & Sons: New York, NY 1999 5.  Ã‚  Ã‚  Ã‚  Ã‚  Margiotta, Franklin D., Ed. â€Å"Brassey’s Encyclopedia of Military History and Biography†, Washington: Brassey’s, Inc. 1994

Wednesday, September 18, 2019

Pro Legalization of Euthanasia Essay -- Argument for Euthanasia

I am poured out like water, and all my bones are out of joint, my heart is like wax, it is melted within my breast, my mouth is dried up like a potsherd, and my tongue sticks to my jaws, you lay me in the dust of death (Psalm 22) Some might say euthanasia is wrong, it is murder, it is an inhumane act. But is it not inhumane to see another person’s unbearable suffering and not release them when they long to be released? Is it not cruel to watch a person in a vegetative state, with no brainwaves showing, fed by a machine, â€Å"breathing† with the help of a machine and not let them be liberated from their pain? Prolonging one’s suffering when an easier, more painless way out is possible is not â€Å"the right thing to do†. Giving the person the choice of release is. Euthanasia is. What is euthanasia? The basic definition of euthanasia is the practice of ending a life so as to release an individual from a painful, incurable disease or intolerable suffering. There are said to be two types of euthanasia: passive and active. Passive euthanasia is â€Å"stopping (or not starting) some treatment which allows a person to die, the persons condition causes his or her death† (Mackinnon, 12). An example would be â€Å"withdrawing a respirator or feeding tube† (Landau, 80). â€Å"Passive euthanasia is a brutal death. Allowing someone to starve to death and die of thirst, the way we do now, is barbaric†. â€Å"That’s what the Nazis did in the concentration camps† (quoted in Manipulating Life, 33). Active euthanasia on the other hand is an act of â€Å"merciful killing†. It is defined as â€Å"The act of administering a lethal drug, or using other means that cause a persons death† in case of incurable terminal illness (MacKinnon, 126). Euthanasia is... ...hical Choices in an American Hospital, New York: Oxford University Press, 1991. Landau, Elaine, The Right to Die, Franklin Watts, USA, 1988 MacKinnon, Barbara. Euthanasia, Ethics Theory and Contemporary Issues, second edition. Wadworth Publishing Co 1998. McCuen, Gary E., Manipulating life: debating the genetic Revolution Gary E. McCuen Publication, Inc, 1985 Report of the Board of Trustees of the American Medical Association. (Transcript), v10, Issues in Law & Medicine, summer, 1994, p 81-90 Thomas A. Shannon, Bioethics: basic writing on the key ethical questions that surround the major, modern biological possibilities and problems, Third Edition, New Jersey, 1987 Varga, Andrew C. The Main Issues in Bioethics. Paulist Press, 1980 Hollard, Ninnet B. Controversy: Shall It Be Legal? www.freeesays.com/topic/PAS_47A/Middles_pon_1/controversy5/ Pro Legalization of Euthanasia Essay -- Argument for Euthanasia I am poured out like water, and all my bones are out of joint, my heart is like wax, it is melted within my breast, my mouth is dried up like a potsherd, and my tongue sticks to my jaws, you lay me in the dust of death (Psalm 22) Some might say euthanasia is wrong, it is murder, it is an inhumane act. But is it not inhumane to see another person’s unbearable suffering and not release them when they long to be released? Is it not cruel to watch a person in a vegetative state, with no brainwaves showing, fed by a machine, â€Å"breathing† with the help of a machine and not let them be liberated from their pain? Prolonging one’s suffering when an easier, more painless way out is possible is not â€Å"the right thing to do†. Giving the person the choice of release is. Euthanasia is. What is euthanasia? The basic definition of euthanasia is the practice of ending a life so as to release an individual from a painful, incurable disease or intolerable suffering. There are said to be two types of euthanasia: passive and active. Passive euthanasia is â€Å"stopping (or not starting) some treatment which allows a person to die, the persons condition causes his or her death† (Mackinnon, 12). An example would be â€Å"withdrawing a respirator or feeding tube† (Landau, 80). â€Å"Passive euthanasia is a brutal death. Allowing someone to starve to death and die of thirst, the way we do now, is barbaric†. â€Å"That’s what the Nazis did in the concentration camps† (quoted in Manipulating Life, 33). Active euthanasia on the other hand is an act of â€Å"merciful killing†. It is defined as â€Å"The act of administering a lethal drug, or using other means that cause a persons death† in case of incurable terminal illness (MacKinnon, 126). Euthanasia is... ...hical Choices in an American Hospital, New York: Oxford University Press, 1991. Landau, Elaine, The Right to Die, Franklin Watts, USA, 1988 MacKinnon, Barbara. Euthanasia, Ethics Theory and Contemporary Issues, second edition. Wadworth Publishing Co 1998. McCuen, Gary E., Manipulating life: debating the genetic Revolution Gary E. McCuen Publication, Inc, 1985 Report of the Board of Trustees of the American Medical Association. (Transcript), v10, Issues in Law & Medicine, summer, 1994, p 81-90 Thomas A. Shannon, Bioethics: basic writing on the key ethical questions that surround the major, modern biological possibilities and problems, Third Edition, New Jersey, 1987 Varga, Andrew C. The Main Issues in Bioethics. Paulist Press, 1980 Hollard, Ninnet B. Controversy: Shall It Be Legal? www.freeesays.com/topic/PAS_47A/Middles_pon_1/controversy5/

Tuesday, September 17, 2019

Don Benito Cereno: No Future without the Past

A. P. English September 22, 2012 No Future Without the Past By: Bernice Mojica â€Å"But the past is past; why moralize upon it? Forget it. See, yon bright sun has forgotten it all, and the blue sea, and the blue sky; these have turned over new leaves. † â€Å"Because they have no memory,† he dejectedly replied; â€Å"because they are not human. † â€Å"But these mild trades that now fan your cheek, Don Benito, do they not come with a human-like healing to you? Warm friends, steadfast friends are the trades. â€Å"With their steadfastness they but waft me to my tomb, Senor,† was the foreboding response. â€Å"You are saved, Don Benito,† cried Captain Delano, more and more astonished and pained; â€Å"you are saved; what has cast such a shadow upon you? † â€Å"The Negro. † (pg. 75) -Melville, Herman. Benito Cereno. New York: Dover Publications, Inc. , 1990 Don Benito Cereno’s epiphany over the mistake of underestimating the Neg roes keeps him moralized upon the past, rather than accepting his wrong doings and modifying them into optimistic beneficial accomplishments.To prove that his way of thinking is keeping him from moving on, Captain Delano advices him that, â€Å"the past is past; why moralize upon it? † Delano is letting him know that he can’t hold himself back from a great future based on a rotten past. Although selfishness is looked upon as a sinful thought, pleasing one’s self is still considered a good intention. Delano comforts Cereno by indirectly assuring him that he should never regret what has been done, because during the moment of that action, there was always a reasonable purpose to be followed.Those experiences taught him how to become a better human being; hence, if he didn’t go through any of that, he would not have the current regretful feelings he has at the end of the novel. Delano alludes to Benito that if the world is able to metamorphose, then so can h e; â€Å"See, yon bright sun has forgotten it all, and the blue sea, and the blue sky; these have turned over new leaves. † These are all forms of our dynamic nature which juxtapose Benito’s static character. DonBenito comes to realize that the population he had once tortured is the same as those who have morally and mentally saved him. This is translated when Captain Delano asks him, â€Å"you are saved; what has cast such a shadow upon you? † and Cereno replies that â€Å"the Negro† is what has cast the shadow upon him. The Negroes aboard the San Dominick represent â€Å"blackness†. Therefore, Cereno is implying that blackness is the evil within human nature, which the black ethnic group metaphorically represents.Blackness is a connotation of an actual color, and in this case, it is also the live image of what us humans believe stands for worthlessness and diabolic. This comparison is an oxymoron to what Cereno is referring to. If you recall, Cer eno had stated that he was saved because of this blackness; which I had just mentioned was unholy. This contradiction establishes how there is no future without the past; how you cannot grow without a base to keep yourself standing. There is no need for change if there is nothing destructive keeping you from reaching your goals.Cereno believes he himself was the destructiveness keeping the Negroes from fulfilling their wishes of being able to see their families again. This brings about the empathy that the reader feels towards him in the end. With this passage being at the end of the novel, it clearly exhibits a stable theme for the novel as a whole. Race seems to be the common denominator throughout the story. This is supported by the fact that it was written during the 1850’s; when slavery was most popular.Melville seems to realize that this sort of power over other humans was morally wrong and he did not agree with what was socially acceptable. Our generations have been ta ught that we should never judge a book by its cover, therefore, we should not judge a man by his color. You should never underestimate a man based on what he doesn’t have, but rather estimate his power based on what he does have. Now that Don Benito Cereno comes to terms with the equality of all men, he took the first step into admitting his mistakes and chooses to follow a leader rather than being one.

Monday, September 16, 2019

Asn report

Materials and products must be inspected to assure that the quality characteristics conform to requirements. Inspection may occur as the product is being produced, at final Inspection on completed product at the producer, or at receiving Inspection at the consumer. There are three ways that a lot of N quantity may be Inspected: complete Inspection of the lot, no Inspection of the lot, or a partial inspection of the lot. Complete inspection of the lot, referred to as 100% inspection, can be extremely time consuming, and as such expensive. 0% inspection should be reserved for those situations where even a single defect is associated with unacceptable risk, as in medical or aerospace applications. 100% inspection may also be necessary if there is reason to believe that the lot is of particularly low quality, or if no information is available to estimate the lot quality. No inspection of the lot, or 0% inspection, is the ideal inspection level from a financial view, as there is no cost a dded. However, 0% inspection Is risky, as even one bad lot of material can have a significant monetary impact, easily erasing any savings realized from the lack of Inspection.Regardless, certain situations do lend themselves to Inspection. The material may be so Inexpensive, Like a screw or nut, that there Is no Justification for Inspection. Or there may be sufficient statistical and/or historical evidence that the lot will meet the required quality level that inspection is unwarranted. A partial inspection of the lot, called sample inspection, provides an alternative to the extremes of 100% or 0% inspection, and is the most common method of lot inspection. Sample inspection Lana typically use statistically derived tables from a know standard, such as the ubiquitous IEEE military standard.These sampling plans allow for the selection of an Acceptable Quality Level (SQL) with a corresponding sample size (n) based on lot size (N). The inspector then uses the specified acceptance number (c) and rejection number (d or r) to decide if the lot should be accepted or rejected, called sentencing the lot. The lot Is accepted and considered of adequate quality when the number of Identified defects In the sample is less than or equal to the acceptance number; otherwise the lot Is rejected. A refinement to single sampling plans, where a single sample is used for lot sentencing, is a double sampling plan.Instead of a single sample (ml), a second sample size (no) is also defined by the selected plan, as well as a second set of acceptance and rejection numbers (ca and do). If the number of defects identified in the first sample is less than CLC, the lot is accepted; if the defects are greater than ca, the lot is rejected. If the number of defects is greater than CLC, but less than or equal to ca, a second sample is drawn. If the sum of the defects identified in both samples is less than or equal to ca, the lot is accepted, if the sum is greater than ca, the lot Is rejected.Dou ble sampling plans can be psychological appealing, with a perceived â€Å"second chance† for accepting a lot. The reality Is that no such advantage exists, as both single and double sampling plans are designed to provide similar probable's for accepting or rejecting lots of Identical quality. The actual advantage of double sampling plans over single sampling plans Is found In the sampling plan will always have a lower ASSN then a single sampling plan; the inspection took less time and so cost less.

Sunday, September 15, 2019

A Game of Thrones Chapter Twenty-six

Jon Jon was showing Dareon how best to deliver a sidestroke when the new recruit entered the practice yard. â€Å"Your feet should be farther apart,† he urged. â€Å"You don't want to lose your balance. That's good. Now pivot as you deliver the stroke, get all your weight behind the blade.† Dareon broke off and lifted his visor. â€Å"Seven gods,† he murmured. â€Å"Would you look at this, Jon.† Jon turned. Through the eye slit of his helm, he beheld the fattest boy he had ever seen standing in the door of the armory. By the look of him, he must have weighed twenty stone. The fur collar of his embroidered surcoat was lost beneath his chins. Pale eyes moved nervously in a great round moon of a face, and plump sweaty fingers wiped themselves on the velvet of his doublet. â€Å"They . . . they told me I was to come here for . . . for training,† he said to no one in particular. â€Å"A lordling,† Pyp observed to Jon. â€Å"Southron, most like near Highgarden.† Pyp had traveled the Seven Kingdoms with a mummers' troupe, and bragged that he could tell what you were and where you'd been born just from the sound of your voice. A striding huntsman had been worked in scarlet thread upon the breast of the fat boy's fur-trimmed surcoat. Jon did not recognize the sigil. Ser Alliser Thorne looked over his new charge and said, â€Å"It would seem they have run short of poachers and thieves down south. Now they send us pigs to man the Wall. Is fur and velvet your notion of armor, my Lord of Ham?† It was soon revealed that the new recruit had brought his own armor with him; padded doublet, boiled leather, mail and plate and helm, even a great wood-and-leather shield blazoned with the same striding huntsman he wore on his surcoat. As none of it was black, however, Ser Alliser insisted that he reequip himself from the armory. That took half the morning. His girth required Donal Noye to take apart a mail hauberk and refit it with leather panels at the sides. To get a helm over his head the armorer had to detach the visor. His leathers bound so tightly around his legs and under his arms that he could scarcely move. Dressed for battle, the new boy looked like an overcooked sausage about to burst its skin. â€Å"Let us hope you are not as inept as you look,† Ser Alliser said. â€Å"Halder, see what Ser Piggy can do.† Jon Snow winced. Halder had been born in a quarry and apprenticed as a stonemason. He was sixteen, tall and muscular, and his blows were as hard as any Jon had ever felt. â€Å"This will be uglier than a whore's ass,† Pyp muttered, and it was. The fight lasted less than a minute before the fat boy was on the ground, his whole body shaking as blood leaked through his shattered helm and between his pudgy fingers. â€Å"I yield,† he shrilled. â€Å"No more, I yield, don't hit me.† Rast and some of the other boys were laughing. Even then, Ser Alliser would not call an end. â€Å"On your feet, Ser Piggy,† he called. â€Å"Pick up your sword.† When the boy continued to cling to the ground, Thorne gestured to Halder. â€Å"Hit him with the flat of your blade until he finds his feet.† Halder delivered a tentative smack to his foe's upraised cheeks. â€Å"You can hit harder than that,† Thorne taunted. Halder took hold of his longsword with both hands and brought it down so hard the blow split leather, even on the flat. The new boy screeched in pain. Jon Snow took a step forward. Pyp laid a mailed hand on his arm. â€Å"Jon, no,† the small boy whispered with an anxious glance at Ser Alliser Thorne. â€Å"On your feet,† Thorne repeated. The fat boy struggled to rise, slipped, and fell heavily again. â€Å"Ser Piggy is starting to grasp the notion,† Ser Alliser observed. â€Å"Again.† Halder lifted the sword for another blow. â€Å"Cut us off a ham!† Rast urged, laughing. Jon shook off Pyp's hand. â€Å"Halder, enough.† Halder looked to Ser Alliser. â€Å"The Bastard speaks and the peasants tremble,† the master-at-arms said in that sharp, cold voice of his. â€Å"I remind you that I am the master-at-arms here, Lord Snow.† â€Å"Look at him, Halder,† Jon urged, ignoring Thorne as best he could. â€Å"There's no honor in beating a fallen foe. He yielded.† He knelt beside the fat boy. Halder lowered his sword. â€Å"He yielded,† he echoed. Ser Alliser's onyx eyes were fixed on Jon Snow. â€Å"It would seem our Bastard is in love,† he said as Jon helped the fat boy to his feet. â€Å"Show me your steel, Lord Snow.† Jon drew his longsword. He dared defy Ser Alliser only to a point, and he feared he was well beyond it now. Thorne smiled. â€Å"The Bastard wishes to defend his lady love, so we shall make an exercise of it. Rat, Pimple, help our Stone Head here.† Rast and Albett moved to join Halder. â€Å"Three of you ought to be sufficient to make Lady Piggy squeal. All you need do is get past the Bastard.† â€Å"Stay behind me,† Jon said to the fat boy. Ser Alliser had often sent two foes against him, but never three. He knew he would likely go to sleep bruised and bloody tonight. He braced himself for the assault. Suddenly Pyp was beside him. â€Å"Three to two will make for better sport,† the small boy said cheerfully. He dropped his visor and slid out his sword. Before Jon could even think to protest, Grenn had stepped up to make a third. The yard had grown deathly quiet. Jon could feel Ser Alliser's eyes. â€Å"Why are you waiting?† he asked Rast and the others in a voice gone deceptively soft, but it was Jon who moved first. Halder barely got his sword up in time. Jon drove him backward, attacking with every blow, keeping the older boy on the heels. Know your foe, Ser Rodrik had taught him once; Jon knew Halder, brutally strong but short of patience, with no taste for defense. Frustrate him, and he would leave himself open, as certain as sunset. The clang of steel echoed through the yard as the others joined battle around him. Jon blocked a savage cut at his head, the shock of impact running up his arm as the swords crashed together. He slammed a sidestroke into Halder's ribs, and was rewarded with a muffled grunt of pain. The counterstroke caught Jon on the shoulder. Chainmail crunched, and pain flared up his neck, but for an instant Halder was unbalanced. Jon cut his left leg from under him, and he fell with a curse and a crash. Grenn was standing his ground as Jon had taught him, giving Albett more than he cared for, but Pyp was hard-pressed. Rast had two years and forty pounds on him. Jon stepped up behind him and rang the raper's helm like a bell. As Rast went reeling, Pyp slid in under his guard, knocked him down, and leveled a blade at his throat. By then Jon had moved on. Facing two swords, Albett backed away. â€Å"I yield,† he shouted. Ser Alliser Thorne surveyed the scene with disgust. â€Å"The mummer's farce has gone on long enough for today.† He walked away. The session was at an end. Dareon helped Halder to his feet. The quarryman's son wrenched off his helm and threw it across the yard. â€Å"For an instant, I thought I finally had you, Snow.† â€Å"For an instant, you did,† Jon replied. Under his mail and leather, his shoulder was throbbing. He sheathed his sword and tried to remove his helm, but when he raised his arm, the pain made him grit his teeth. â€Å"Let me,† a voice said. Thick-fingered hands unfastened helm from gorget and lifted it off gently. â€Å"Did he hurt you?† â€Å"I've been bruised before.† He touched his shoulder and winced. The yard was emptying around them. Blood matted the fat boy's hair where Halder had split his helm asunder. â€Å"My name is Samwell Tarly, of Horn . . . † He stopped and licked his lips. â€Å"I mean, I was of Horn Hill, until I . . . left. I've come to take the black. My father is Lord Randyll, a bannerman to the Tyrells of Highgarden. I used to be his heir, only . . . † His voice trailed off. â€Å"I'm Jon Snow, Ned Stark's bastard, of Winterfell.† Samwell Tarly nodded. â€Å"I . . . if you want, you can call me Sam. My mother calls me Sam.† â€Å"You can call him Lord Snow,† Pyp said as he came up to join them. â€Å"You don't want to know what his mother calls him.† â€Å"These two are Grenn and Pypar,† Jon said. â€Å"Grenn's the ugly one,† Pyp said. Grenn scowled. â€Å"You're uglier than me. At least I don't have ears like a bat.† â€Å"My thanks to all of you,† the fat boy said gravely. â€Å"Why didn't you get up and fight?† Grenn demanded. â€Å"I wanted to, truly. I just . . . I couldn't. I didn't want him to hit me anymore.† He looked at the ground. â€Å"I . . . I fear I'm a coward. My lord father always said so.† Grenn looked thunderstruck. Even Pyp had no words to say to that, and Pyp had words for everything. What sort of man would proclaim himself a coward? Samwell Tarly must have read their thoughts on their faces. His eyes met Jon's and darted away, quick as frightened animals. â€Å"I . . . I'm sorry,† he said. â€Å"I don't mean to . . . to be like I am.† He walked heavily toward the armory. Jon called after him. â€Å"You were hurt,† he said. â€Å"Tomorrow you'll do better.† Sam looked mournfully back over one shoulder. â€Å"No I won't,† he said, blinking back tears. â€Å"I never do better.† When he was gone, Grenn frowned. â€Å"Nobody likes cravens,† he said uncomfortably. â€Å"I wish we hadn't helped him. What if they think we're craven too?† â€Å"You're too stupid to be craven,† Pyp told him. â€Å"I am not,† Grenn said. â€Å"Yes you are. If a bear attacked you in the woods, you'd be too stupid to run away.† â€Å"I would not,† Grenn insisted. â€Å"I'd run away faster than you.† He stopped suddenly, scowling when he saw Pyp's grin and realized what he'd just said. His thick neck flushed a dark red. Jon left them there arguing as he returned to the armory, hung up his sword, and stripped off his battered armor. Life at Castle Black followed certain patterns; the mornings were for swordplay, the afternoons for work. The black brothers set new recruits to many different tasks, to learn where their skills lay. Jon cherished the rare afternoons when he was sent out with Ghost ranging at his side to bring back game for the Lord Commander's table, but for every day spent hunting, he gave a dozen to Donal Noye in the armory, spinning the whetstone while the one-armed smith sharpened axes grown dull from use, or pumping the bellows as Noye hammered out a new sword. Other times he ran messages, stood at guard, mucked out stables, fletched arrows, assisted Maester Aemon with his birds or Bowen Marsh with his counts and inventories. That afternoon, the watch commander sent him to the winch cage with four barrels of fresh-crushed stone, to scatter gravel over the icy footpaths atop the Wall. It was lonely and boring work, even with Ghost along for company, but Jon found he did not mind. On a clear day you could see half the world from the top of the Wall, and the air was always cold and bracing. He could think here, and he found himself thinking of Samwell Tarly . . . and, oddly, of Tyrion Lannister. He wondered what Tyrion would have made of the fat boy. Most men would rather deny a hard truth than face it, the dwarf had told him, grinning. The world was full of cravens who pretended to be heroes; it took a queer sort of courage to admit to cowardice as Samwell Tarly had. His sore shoulder made the work go slowly. It was late afternoon before Jon finished graveling the paths. He lingered on high to watch the sun go down, turning the western sky the color of blood. Finally, as dusk was settling over the north, Jon rolled the empty barrels back into the cage and signaled the winch men to lower him. The evening meal was almost done by the time he and Ghost reached the common hall. A group of the black brothers were dicing over mulled wine near the fire. His friends were at the bench nearest the west wall, laughing. Pyp was in the middle of a story. The mummer's boy with the big ears was a born liar with a hundred different voices, and he did not tell his tales so much as live them, playing all the parts as needed, a king one moment and a swineherd the next. When he turned into an alehouse girl or a virgin princess, he used a high falsetto voice that reduced them all to tears of helpless laughter, and his eunuchs were always eerily accurate caricatures of Ser Alliser. Jon took as much pleasure from Pyp's antics as anyone . . . yet that night he turned away and went instead to the end of the bench, where Samwell Tarly sat alone, as far from the others as he could get. He was finishing the last of the pork pie the cooks had served up for supper when Jon sat down across from him. The fat boy's eyes widened at the sight of Ghost. â€Å"Is that a wolf?† â€Å"A direwolf,† Jon said. â€Å"His name is Ghost. The direwolf is the sigil of my father's House.† â€Å"Ours is a striding huntsman,† Samwell Tarly said. â€Å"Do you like to hunt?† The fat boy shuddered. â€Å"I hate it.† He looked as though he was going to cry again. â€Å"What's wrong now?† Jon asked him. â€Å"Why are you always so frightened?† Sam stared at the last of his pork pie and gave a feeble shake of his head, too scared even to talk. A burst of laughter filled the hall. Jon heard Pyp squeaking in a high voice. He stood. â€Å"Let's go outside.† The round fat face looked up at him, suspicious. â€Å"Why? What will we do outside?† â€Å"Talk,† Jon said. â€Å"Have you seen the Wall?† â€Å"I'm fat, not blind,† Samwell Tarly said. â€Å"Of course I saw it, it's seven hundred feet high.† Yet he stood up all the same, wrapped a fur-lined cloak over his shoulders, and followed Jon from the common hall, still wary, as if he suspected some cruel trick was waiting for him in the night. Ghost padded along beside them. â€Å"I never thought it would be like this,† Sam said as they walked, his words steaming in the cold air. Already he was huffing and puffing as he tried to keep up. â€Å"All the buildings are falling down, and it's so . . . so . . . â€Å" â€Å"Cold?† A hard frost was settling over the castle, and Jon could hear the soft crunch of grey weeds beneath his boots. Sam nodded miserably. â€Å"I hate the cold,† he said. â€Å"Last night I woke up in the dark and the fire had gone out and I was certain I was going to freeze to death by morning.† â€Å"It must have been warmer where you come from.† â€Å"I never saw snow until last month. We were crossing the barrowlands, me and the men my father sent to see me north, and this white stuff began to fall, like a soft rain. At first I thought it was so beautiful, like feathers drifting from the sky, but it kept on and on, until I was frozen to the bone. The men had crusts of snow in their beards and more on their shoulders, and still it kept coming. I was afraid it would never end.† Jon smiled. The Wall loomed before them, glimmering palely in the light of the half moon. In the sky above, the stars burned clear and sharp. â€Å"Are they going to make me go up there?† Sam asked. His face curdled like old milk as he looked at the great wooden stairs. â€Å"I'll die if I have to climb that.† â€Å"There's a winch,† Jon said, pointing. â€Å"They can draw you up in a cage.† Samwell Tarly sniffled. â€Å"I don't like high places.† It was too much. Jon frowned, incredulous. â€Å"Are you afraid of everything?† he asked. â€Å"I don't understand. If you are truly so craven, why are you here? Why would a coward want to join the Night's Watch?† Samwell Tarly looked at him for a long moment, and his round face seemed to cave in on itself. He sat down on the frost-covered ground and began to cry, huge choking sobs that made his whole body shake. Jon Snow could only stand and watch. Like the snowfall on the barrowlands, it seemed the tears would never end. It was Ghost who knew what to do. Silent as shadow, the pale direwolf moved closer and began to lick the warm tears off Samwell Tarly's face. The fat boy cried out, startled . . . and somehow, in a heartbeat, his sobs turned to laughter. Jon Snow laughed with him. Afterward they sat on the frozen ground, huddled in their cloaks with Ghost between them. Jon told the story of how he and Robb had found the pups newborn in the late summer snows. It seemed a thousand years ago now. Before long he found himself talking of Winterfell. â€Å"Sometimes I dream about it,† he said. â€Å"I'm walking down this long empty hall. My voice echoes all around, but no one answers, so I walk faster, opening doors, shouting names. I don't even know who I'm looking for. Most nights it's my father, but sometimes it's Robb instead, or my little sister Arya, or my uncle.† The thought of Benjen Stark saddened him; his uncle was still missing. The Old Bear had sent out rangers in search of him. Ser Jaremy Rykker had led two sweeps, and Quorin Halfhand had gone forth from the Shadow Tower, but they'd found nothing aside from a few blazes in the trees that his uncle had left to mark his way. In the stony highlands to the northwest, the marks stopped abruptly and all trace of Ben Stark vanished. â€Å"Do you ever find anyone in your dream?† Sam asked. Jon shook his head. â€Å"No one. The castle is always empty.† He had never told anyone of the dream, and he did not understand why he was telling Sam now, yet somehow it felt good to talk of it. â€Å"Even the ravens are gone from the rookery, and the stables are full of bones. That always scares me. I start to run then, throwing open doors, climbing the tower three steps at a time, screaming for someone, for anyone. And then I find myself in front of the door to the crypts. It's black inside, and I can see the steps spiraling down. Somehow I know I have to go down there, but I don't want to. I'm afraid of what might be waiting for me. The old Kings of Winter are down there, sitting on their thrones with stone wolves at their feet and iron swords across their laps, but it's not them I'm afraid of. I scream that I'm not a Stark, that this isn't my place, but it's no good, I have to go anyway, so I start down, feeling the walls as I descend, with no torch to light the way. It gets darker and darker, until I want to scream.† He stopped, frowning, embarrassed. â€Å"That's when I always wake.† His skin cold and clammy, shivering in the darkness of his cell. Ghost would leap up beside him, his warmth as comforting as daybreak. He would go back to sleep with his face pressed into the direwolf s shaggy white fur. â€Å"Do you dream of Horn Hill?† Jon asked. â€Å"No.† Sam's mouth grew tight and hard. â€Å"I hated it there.† He scratched Ghost behind the ear, brooding, and Jon let the silence breathe. After a long while Samwell Tarly began to talk, and Jon Snow listened quietly, and learned how it was that a self-confessed coward found himself on the Wall. The Tarlys were a family old in honor, bannermen to Mace Tyrell, Lord of Highgarden and Warden of the South. The eldest son of Lord Randyll Tarly, Samwell was born heir to rich lands, a strong keep, and a storied two-handed greatsword named Heartsbane, forged of Valyrian steel and passed down from father to son near five hundred years. Whatever pride his lord father might have felt at Samwell's birth vanished as the boy grew up plump, soft, and awkward. Sam loved to listen to music and make his own songs, to wear soft velvets, to play in the castle kitchen beside the cooks, drinking in the rich smells as he snitched lemon cakes and blueberry tarts. His passions were books and kittens and dancing, clumsy as he was. But he grew ill at the sight of blood, and wept to see even a chicken slaughtered. A dozen masters-at-arms came and went at Horn Hill, trying to turn Samwell into the knight his father wanted. The boy was cursed and caned, slapped and starved. One man had him sleep in his chainmail to make him more martial. Another dressed him in his mother's clothing and paraded him through the bailey to shame him into valor. He only grew fatter and more frightened, until Lord Randyll's disappointment turned to anger and then to loathing. â€Å"One time,† Sam confided, his voice dropping from a whisper, â€Å"two men came to the castle, warlocks from Qarth with white skin and blue lips. They slaughtered a bull aurochs and made me bathe in the hot blood, but it didn't make me brave as they'd promised. I got sick and retched. Father had them scourged.† Finally, after three girls in as many years, Lady Tarly gave her lord husband a second son. From that day, Lord Randyll ignored Sam, devoting all his time to the younger boy, a fierce, robust child more to his liking. Samwell had known several years of sweet peace with his music and his books. Until the dawn of his fifteenth name day, when he had been awakened to find his horse saddled and ready. Three men-at-arms had escorted him into a wood near Horn Hill, where his father was skinning a deer. â€Å"You are almost a man grown now, and my heir,† Lord Randyll Tarly had told his eldest son, his long knife laying bare the carcass as he spoke. â€Å"You have given me no cause to disown you, but neither will I allow you to inherit the land and title that should be Dickon's. Heartsbane must go to a man strong enough to wield her, and you are not worthy to touch her hilt. So I have decided that you shall this day announce that you wish to take the black. You will forsake all claim to your brother's inheritance and start north before evenfall. â€Å"If you do not, then on the morrow we shall have a hunt, and somewhere in these woods your horse will stumble, and you will be thrown from the saddle to die . . . or so I will tell your mother. She has a woman's heart and finds it in her to cherish even you, and I have no wish to cause her pain. Please do not imagine that it will truly be that easy, should you think to defy me. Nothing would please me more than to hunt you down like the pig you are.† His arms were red to the elbow as he laid the skinning knife aside. â€Å"So. There is your choice. The Night's Watch†Ã¢â‚¬â€he reached inside the deer, ripped out its heart, and held it in his fist, red and dripping—†or this.† Sam told the tale in a calm, dead voice, as if it were something that had happened to someone else, not to him. And strangely, Jon thought, he did not weep, not even once. When he was done, they sat together and listened to the wind for a time. There was no other sound in all the world. Finally Jon said, â€Å"We should go back to the common hall.† â€Å"Why?† Sam asked. Jon shrugged. â€Å"There's hot cider to drink, or mulled wine if you prefer. Some nights Dareon sings for us, if the mood is on him. He was a singer, before . . . well, not truly, but almost, an apprentice singer.† â€Å"How did he come here?† Sam asked. â€Å"Lord Rowan of Goldengrove found him in bed with his daughter. The girl was two years older, and Dareon swears she helped him through her window, but under her father's eye she named it rape, so here he is. When Maester Aemon heard him sing, he said his voice was honey poured over thunder.† Jon smiled. â€Å"Toad sometimes sings too, if you call it singing. Drinking songs he learned in his father's winesink. Pyp says his voice is piss poured over a fart.† They laughed at that together. â€Å"I should like to hear them both,† Sam admitted, â€Å"but they would not want me there.† His face was troubled. â€Å"He's going to make me fight again on the morrow, isn't he?† â€Å"He is,† Jon was forced to say. Sam got awkwardly to his feet. â€Å"I had better try to sleep.† He huddled down in his cloak and plodded off. The others were still in the common room when Jon returned, alone but for Ghost. â€Å"Where have you been?† Pyp asked. â€Å"Talking with Sam,† he said. â€Å"He truly is craven,† said Grenn. â€Å"At supper, there were still places on the bench when he got his pie, but he was too scared to come sit with us.† â€Å"The Lord of Ham thinks he's too good to eat with the likes of us,† suggested Jeren. â€Å"I saw him eat a pork pie,† Toad said, smirking. â€Å"Do you think it was a brother?† He began to make oinking noises. â€Å"Stop it!† Jon snapped angrily. The other boys fell silent, taken aback by his sudden fury. â€Å"Listen to me,† Jon said into the quiet, and he told them how it was going to be. Pyp backed him, as he'd known he would, but when Halder spoke up, it was a pleasant surprise. Grenn was anxious at the first, but Jon knew the words to move him. One by one the rest fell in line. Jon persuaded some, cajoled some, shamed the others, made threats where threats were required. At the end they had all agreed . . . all but Rast. â€Å"You girls do as you please,† Rast said, â€Å"but if Thorne sends me against Lady Piggy, I'm going to slice me off a rasher of bacon.† He laughed in Jon's face and left them there. Hours later, as the castle slept, three of them paid a call on his cell. Grenn held his arms while Pyp sat on his legs. Jon could hear Rast's rapid breathing as Ghost leapt onto his chest. The direwolf's eyes burned red as embers as his teeth nipped lightly at the soft skin of the boy's throat, just enough to draw blood. â€Å"Remember, we know where you sleep,† Jon said softly. The next morning Jon heard Rast tell Albett and Toad how his razor had slipped while he shaved. From that day forth, neither Rast nor any of the others would hurt Samwell Tarly. When Ser Alliser matched them against him, they would stand their ground and swat aside his slow, clumsy strokes. If the master-at-arms screamed for an attack, they would dance in and tap Sam lightly on breastplate or helm or leg. Ser Alliser raged and threatened and called them all cravens and women and worse, yet Sam remained unhurt. A few nights later, at Jon's urging, he joined them for the evening meal, taking a place on the bench beside Halder. It was another fortnight before he found the nerve to join their talk, but in time he was laughing at Pyp's faces and teasing Grenn with the best of them. Fat and awkward and frightened he might be, but Samwell Tarly was no fool. One night he visited Jon in his cell. â€Å"I don't know what you did,† he said, â€Å"but I know you did it.† He looked away shyly. â€Å"I've never had a friend before.† â€Å"We're not friends,† Jon said. He put a hand on Sam's broad shoulder. â€Å"We're brothers.† And so they were, he thought to himself after Sam had taken his leave. Robb and Bran and Rickon were his father's sons, and he loved them still, yet Jon knew that he had never truly been one of them. Catelyn Stark had seen to that. The grey walls of Winterfell might still haunt his dreams, but Castle Black was his life now, and his brothers were Sam and Grenn and Halder and Pyp and the other cast-outs who wore the black of the Night's Watch. â€Å"My uncle spoke truly,† he whispered to Ghost. He wondered if he would ever see Benjen Stark again, to tell him.