Buy a Baseball America`s Favorite Game Essay Online

Purchase a Baseball America's Favorite Game Essay Online Article about Baseball As spring comes in, when the magnificence of the earth wakes up once again, the incomparable American game starts in North America and Canada. It is far beyond a ball game played between two groups of at nine players on a precious stone formed field †with bases, gloves, bats and balls, a hill, earth on in infield, grass in the outfield, and individuals in the stands. Baseball is a round of elegant physicality, knowledge, and class. Now and again, baseball resembles an expressive dance, an exhibition and a psyche game, a round of chess. It’s altogether different than †even better than â€that heartless game called American football, not to be mistaken for soccer. Baseball is unmistakably more accommodating in nature than football, which is a brutish round of brutality excessively like war. Baseball is a gentleman’s game, a reasoning keeps an eye on game of minds, quality, endurance, speed, and reflex. Additionally, baseball, which is said to have developed from the British game cricket, was made in New England around the hour of the American Civil War, played by Union troopers to involve their vacation, to most likely divert them and help in helping them keep their rational soundness when not battling. It requires focus and rivalry, so it was a useful instrument in uniting them to accomplish something fun and pleasant. Next came proficient baseball classes in different pieces of the nation, in urban areas in many states in America. After some time, in some structure, individuals all over were playing the incredible game †regardless of whether for entertainment only on pastures or on a level, soil field. At that point it turned into an onlooker sport throughout the late spring months. Late evenings implied for baseball throughout the day, and individuals started paying cash to see games. It is as yet along these lines today. They were paying cash to see the best nearby players go up against the best players in different areas, districts or towns. From that point forward, since the turn of the 21st century, baseball has been a staple of American life, culture, and society. This might be the explanation baseball is called, over and over, â€Å"America’s Favorite Pastime.† Today, Major League Baseball has become the expert on proficient baseball in the United States †and has become a multi-million-dollar organization (if not a multi-billion-dollar one). Itsâ games and other retail things pull in a great many Americans consistently, and the expert association fan base reaches out to South America, Canada, Europa,â and Asia. Itsâ players †Major League Baseball players †make a huge number of dollars a year, through agreements with each group, which is basically an enormous enterprise, and through arrangements with sports gear organizations, supporters, and other such things. That is a great deal of cash for somebody to play a game that was played for the sake of entertainment by fighters in the Civil War, a game played by youngsters on long summer days. Over the most recent a long time since the game was made, baseball †the genuine game itself †presumably hasn’t changed excessively. It despite everything includes similar thoughts and requires a similar language, similar fundamentals of the game, itsâ basic standards and rules and principles and prerequisites. In any case, the game has without a doubt changed. A significant issue in American expert baseball these past couple decades has been the utilization of execution improving medications, similar to steroids, among the game’s best and most well known players. For somebody who grew up revering these players and needing to copy them, it very well may be a genuine killjoy to think they are human and untrustworthy ones at that, too human to even consider being legends and to do gallant things. They are just individuals who could play a basic game to the point they could get paid to do it day in and day and for quite a long time at once. It has become an indus try and not only a game. That game has changed †and not really to improve things.

The Canterbury Tales Writing Assignment Essay Example | Topics and Well Written Essays - 1000 words

The Canterbury Tales Writing Assignment - Essay Example Later on, the Miller’s Tale tells a somewhat obscene and indecent story of a Carpenter and his significant other who tricks her better half with an agent. Generally speaking, the evil and profane character of the Miller outperforms the repugnance of different characters from The Canterbury Tales. The Miller’s Tale adequately shows the prurient character of the Miller who portrays a vulgar story of a woodworker named, John and his significant other, Alisoun. John, who fills in as a craftsman leases a room of his home to an understudy named Nicholas. Another agent named Absolon is likewise there around who later begins to look all starry eyed at Alisoun. The story shows a high level of double dealing where Alisoun gets associated with Nicholas just as Absolon simultaneously while her better half leaves town for a couple of days. At another event, Nicholas fools John of a downpour of a similar force as that of Noah’s time. John moves into a bushel to spare himself f rom the flood while Nicholas and Alisoun are getting to know one another in their bed. Simultaneously, Absolon shows up and marks Nicholas whereupon he cries â€Å"Water!† (Chaucer 3815). After hearing this, John slices the rope to his container and tumbles down. The townspeople show up at the scene and chuckle at John. The entire story of the carpenter’s spouse, Alisoun tricking her better half by engaging in extramarital relations with two more youthful men simultaneously speaks to an obscene and animalistic side to the character of the Miller. Along these lines, this model proposes the scurrilous inside character of the Miller separated from his effectively offensive physical viewpoint. The Miller is a character had by a detestable inside nature as well as an absurd physical character. The insight about the Miller’s exercises and interests expand on to his solid and physical character. In the preface, Chaucer acquaints its perusers with the Miller’s mo st ordinary act of wrestling where he generally wins the slam (548). His capacity to break entryways with his head (Chaucer 550-551) and the wrestling matches he has won exhibit his solid physical abilities including to his gigantic and sickening standpoint. Further subtleties of his appearance uncover his unappealing outward picture that makes him a monstrous person. The portrayals of monstrous highlights including his red whiskers, colossal physical make-up, mole with tufts of hair, and immense dim nostrils show a loathsome character that evokes horrendous pictures in the brain of the perusers. As an individual intently watches the clarification of the Miller’s character, it isn't difficult to picture a nauseating character with terrible highlights that is exploitative and disgusting naturally. During his discussion with the Host preceding the portrayal of his story, he reports that he is flushed and that he ought to be pardoned on the off chance that he says anything incor rectly. At the point when he proceeds to tell his story, his story brings up the improper side of spouses. The Miller’s immense character alongside his red facial hair and shaggy mole speak to a coarse side to the character of the Miller who despite the fact that has an enormous constitution however little insight. Aside from the Miller’s discourteous and vulgar tendencies, he is likewise an unscrupulous man in his business. The Miller isn't just a licentious and genuinely appalling character, yet in addition an unscrupulous man who deceives his clients by taking corn or getting them to pay more (Chaucer 562). With the enormous

Effect of Hurricane Katrina on Children

Effect of Hurricane Katrina on Children PTSD Causes Print The Effect of Hurricane Katrina on Children Hurricane Katrinas Impact on Children By Matthew Tull, PhD twitter Matthew Tull, PhD is a professor of psychology at the University of Toledo, specializing in post-traumatic stress disorder. Learn about our editorial policy Matthew Tull, PhD Medically reviewed by Medically reviewed by Steven Gans, MD on August 05, 2016 Steven Gans, MD is board-certified in psychiatry and is an active supervisor, teacher, and mentor at Massachusetts General Hospital. Learn about our Medical Review Board Steven Gans, MD Updated on June 24, 2019 Post-Traumatic Stress Disorder Overview Symptoms & Diagnosis Causes & Risk Factors Treatment Living With In Children Joe Raedle/Getty Images News/Getty Images The effect of Hurricane Katrina is significant. Since the storm hit the United States Gulf Coast in late August 2005, a number of people and communities have felt its impact, and the negative effect of Hurricane Katrina continues to be felt today. The Effect of Hurricane Katrina Several studies have been done in an attempt to describe the impact of Hurricane Katrina. Many people were separated from their children, friends, neighbors and relatives. In addition, their homes were destroyed or they were displaced from their homes for long periods of time. Additionally, people were also exposed to increased crime and violence as a result of the hurricane. Given these experiences, it is not surprising that many developed symptoms of posttraumatic stress disorder (PTSD)  and depression following Hurricane Katrina. Symptoms include upsetting memories and thoughts about the hurricane, feeling upset when being reminded of the hurricane, trying to avoid thoughts and feelings about the hurricane, having worries about future hurricanes, and feeling on edge and tense. However, less is known about the effect of Hurricane Katrina on children specifically. Depression and Posttraumatic Stress   Children may be particularly vulnerable to PTSD stress following exposure to a natural disaster  like Hurricane Katrina. One group of researchers surveyed 2,362 4th- to 12th-grade children in the 2005-2006 school year and 4,896 4th- to 12th-grade children in the 2006-2007 school year. All children were from schools in Louisiana parishes that were affected by Hurricane Katrina. They found that many children had experienced a great deal of stress as a result of the hurricane. Most had been displaced by the hurricane, had seen their neighborhood destroyed or damaged, and had lost personal belongings. In addition, around a third had been separated from a caregiver and/or a pet during the storm or evacuation. Children also reported, to a lesser extent, seeing family members or friends injured or killed. Given the stress that these children were exposed to, it makes sense that many experienced severe symptoms of depression and PTSD. In fact, this study found that about half of the children experienced high levels of depression and PTSD symptoms. An  increased risk  for these symptoms was correlated with: Currently being separated from a caregiverLiving in a trailerHaving to stay in a shelterYounger age, being femaleHaving previous loss or traumaHaving had family members or friends killed as a result of the hurricaneHaving personal belongings destroyed or damaged   Coping With the Effects of a Natural Disaster Natural disasters such as Hurricane Katrina can have a major impact on a persons psychological health. If you are coping with the effects of a natural disaster, help is available. The National Center for PTSD provides a number of fact sheets on the effects of natural disasters and how to cope with them. You can also find treatment providers in your area through UCompare HealthCare  as well as the Anxiety Disorder Association of America.

Criminal Profiling The Public Face of Forensic Psychology Free Essay Example, 1750 words

Literature review Conducting forensic evaluations commonly involves the utilisation of psychological concepts in seeking to enhance the understanding of the criminal. The content of many evaluations is normally narrowed to become fully focused on gaining pertinent information regarding the psycholegal elements being reviewed in the evaluation (Kalmbach & Lyons, 2006). The main reason that leads to the requirement of psychological evaluations is commonly centered on identification of aspects of human behaviours which fall outside what can be described as normal behaviour. Such behaviours commonly cause disturbances upon the involved individuals, and could have potential effect on the administration of justice. Consideration of various ethical issues surrounding the evaluations is essential in seeking to ensure fairness in the justice system. In conducting psychological evaluations, there are various special considerations that might be involved in seeking to ensure effectiveness of the evaluation. These considerations are commonly involved as an approach for mitigating the negative effects which might results from issues related to the administration of the evaluation (Hoge, 2012). Forensic psychology makes consideration of the individual’s mental state in seeking to ensure accuracy of the assessment since the information gained might be utilised in making judgements (Neal & Grisso, 2014). We will write a custom essay sample on Criminal Profiling: The Public Face of Forensic Psychology or any topic specifically for you Only $17.96 $11.86/page

Innate immunity - Free Essay Example

Sample details Pages: 31 Words: 9313 Downloads: 4 Date added: 2017/06/26 Category Genetics Essay Type Research paper Did you like this example? Introduction Innate immunity is the ancient defence system against infection that is conserved in most of organisms including prokaryotes and invertebrates throughout evolution. Among the cells that participate in the early innate immune response, natural killer (NK) cells occupies special positions, not only owing to its unique cytotoxic ability but its role between the innate and adaptive immunity put it to the high level of interest. The NK cells were first discovered in 1975 by two groups, R.Kiessling and R.Herberman when studying specific cytotoxic effect of lymphocytes against target tumour cells. Don’t waste time! Our writers will create an original "Innate immunity" essay for you Create order The observation of the NK cells brought many controversial issues because of its reactivity level was observed from not only limited to the animals showing the active immune response, but also to those non-immunised one. After several studies suggested the capacity to kill the tumour cell without prior sensitisation, NK cells has been accepted as a sub-population of the lymphocytes, which conduct highly regulated killing mechanism. The current understandings of NK cells originate from the models of missing-self and induced-self recognition. These models introduce that NK cells unlike other T or B lymphocytes do not recognise foreign antigens but are initiated by detecting changes in the surface molecules of the cells. Such phenomenon describes that certain cells expressing less major histocompatiblity complex (MHC) class I are the major target of the NK cells. This MHC class I dependent recognition is responsible for the killing of virus-infected or malignant tumour cells with def icient expression of MHC class I are attacked by NK cells, whereas normal healthy cells are not. However, missing-self model does not provide sufficient explanation on how the NK cells recognise their target cells, when those autologous cells that do not express MHC class I molecules, such as erythrocytes are counted. The self-induced hypothesis complements the missing-self model by proposing that NK cell detects cell stress-induced self-ligands that trigger activation of NK cells. Taken together, it is now well established that activation of NK cells depends on a delicate balance between activating and inhibitory signals. Normal autologous cells express inhibitory signals and no activating ligands, whereas malignant or stressed cell actively expresses activating NK ligands with downregulated MHC class I molecules. Upon the activation, NK cell functions can be categorised into three; I. Cytotoxicity: NK cells perform their cytotoxicity by conducting two mechanisms. First, they exocytose cytotoxic granules containing perforin and various granzymes, resulting in the apoptotic death induced by permeated granzymes. Second, they express tumour necrosis factor (TNF) receptor superfamily (TNFRSF) members such as FAS that engage with corresponding ligands to induce apoptosis. Third, NK cells hire TNF-related apoptosis-inducing ligand (TRAIL) as a cytotoxic effector molecule. II. Regulation of immune response through various cytokine production: NK cells produce many cytokines such as IFN-g, IL-3, GM-CSF, TNF-a and chemokines such as MIP-1, RANTES and IL-8 III. Direct contact co-stimulation: Via costimulatory ligands, such as CD40 and OX40L, NK cells induce T cells and B cells stimulation. Recently, Tbox21 transcription factor (T-bet) is emerging as a crucial factor in NK cell development and maturation. Several studies have demonstrated the defected functions of T-bet KO NK cells in the aspect of cytokine production and NK cell mediated killing. How T-b et transcription factor participates in the NK cell development will be discussed in detail in the introduction. NK cell development The interactions between the precursor NK cells and bone marrow stromal cells are pivotal in the development of NK cells. The NK precursor cells in mice showing high expression of CD122, one of the four subunits of IL-2/IL-15 receptor are constantly influenced by IL-15 in the early stages of NK-cell development, especially during the stage of cellular expansion. These immature NK cells that express NK1.1, acquire CD94-NKG2 heterodimer on their surface as well as Ly49 receptor prior to the proliferation phase. After proliferation, terminal maturation is accompanied by the expression of high levels of CD11b and CD43 along with the perfected cytolytic function and IFN-g production. These interaction with various cytokines are not only confined to the mice NK cells, but immature human NK cells are observed to be perform their cytotoxicity in the manner of TNF-related apoptosis-inducing ligand and release type 2 cytokines in their early development. To elucidate the NK cell developme nt, transcription factors are undeniably key elements as several studies already revealed several transcription factors including interferon regulatory factor(IRF)-1, IRF-2, ETS-1 and T-bet regulates the ability and development of NK cells. For instance, those mice missing GATA-3 transcription factor that are important in Th2 cytokine production, produce NK cells with immature phenotype and also influence the production of IFN-g. Additionally, Ets-family member myeloid elves like factor (MEF) also participates in the determination of NK cell function that its deficiency leads to the failure of expressing perforin and have cytotoxicity. Furthermore, the T box 21(T-bet) transcription factor knockout mice are found to carry significantly less number of NK cells expressing reduced levels of CD43 and CD11b and show receded IFN-g production. T-bet transcription factor will be discussed in more depth later. Once fully matured NK cells are emerged, it is interferons and cytokines that af fect expansion and modulate the function of NK cells. As mentioned briefly, IL-15 and IL-15 receptor (CD122) plays pivotal role in the early development of NK cells. Having said that, IL-15 and its receptor also contribute to the survival and maintenance of mature NK cells. However, it seems CD122, the IL-15 receptor is not solely functioned by the interacting with IL-15 as NK cells from the IL-15R KO mice develop as normal when transferred to a host where the non-hematopoietic cells express IL-15R. Rather, the concentration of IL-15 on stromal cells is decisive in the maturation of NK cells. Having said that IL-15 contributes to the early development and maturation of the NK cells, IL-21 rather enhances the cytotoxic ability and production of IFN-g. Yet, the clear role of IL-21 is still controversial, since the earlier results claiming the importance of IL-21 for the differentiation of NK cells conflict with the latter showing the normal development of NK cells regardless neithe r the existence nor the concentration of IL-21. Nevertheless, some studies suggested the antagonistic influence of IL-21 on the cellular expansion of NK cells. Transcription Factors in NK cell development It is now a well-known fact that, as with all cells of the hematopoietic system, NK cells are derived from bone-marrow hematopoietic stem cells. From the previous studies, it has been generally accepted that NK cells development is defined into several stages including precursor NK cell (pNK), immature NK cell (iNK), and mature NK cell (mNK). According to the recent publish by Vosshenrich et al, six stages are suggested (Fig1), taking into account up-to-date studies. The first stage A is the precursor NK cells as defined by Rosmaraki et al that express high CD122 (IL-2Rb) but lack other known NK cell marker including NK1.1, DX5 and Ly49R. The two subsequent stages, B and C are immature NK cells (iNK). At stage B, the cells are yet inadequately cytotoxic, but start to possess exclusive NK markers, NK1.1 and NKG2D (but not DX5 nor Ly49R). Ly49 receptor begins to show in the following stage. Vosshenrich defined the mature NK cells by observing high expression of DX5 which appears from the stage D. Two additional markers that are initially appearing from the stage C are upregulated as a functional markers; CD11b expression is elevated first (stage E), followed by CD 43(stage F). The fully matured NK cells attain the cytotoxic ability and also can pr NK cells are fully functional and also can produce IFN-g after the stimulation. These mNK cells will then migrate to the periphery via blood. Each developmental stage of NK cells is sophisticatedly controlled by diverse transcription factors. These factors can be grouped into three, according to the stages they correlate to (Table 1); the first group includes Ikaros, Id2, Ets-1 and PU.1 and are known to control the population size and maintenance of early precursor NK cells. Additionally, these transcription factors are essential to establish the early expression of CD122 (IL-15Rb), that the previous studies by two different groups, Ikawa et al and Boggs et al both observed the impaired expression of CD122 from Id2 KO and Ikaros KO mice respectively and therefore, reduced population of NK cells along with severely defected cytotoxic ability and cytokine production are observed. The second group of the transcription factors includes Gata-3, IRF-2 and T-bet. These appear to be primarily involved in the maturation of the NK cells specifically the induction of cytotoxic ability as well as migration to the peripheral site. Each deficiency results in the decreased population of NK cells in the liver, spleen and all the peripheral regions respectively, yet the normal NK cell population in the bone marrow is found. Additionally, the peripheral NK cells missing those transcription factors are found to be immature as they adapt low expression of CD11b and CD43. These functionally defected phenotypes are well demonstrated that severely decreased production of IFN-g is resulted from all IRF-2, Gata-3 and T-bet deficient NK cells. However, the defected NK cells from Gata-3 and T-bet KO mice performs essen tially normal cytotoxicity while IRF-2 deficient NK cells exhibit severely reduced cytotoxic ability. This observation support the previous reports that the maturation marker, CD11b and CD43 may not have high correlation with cytotoxic ability, rather DX5 has higher association. The third group of the transcription factors includes myeloid elf factor (MEF), microphthalmia-associated transcription factor(MITF) and CCAAT/enhancer binding protein-g (CEBP-g). These TFs are reported as a regulator of functional development of NK cells as their deficiency results in the significantly reduced cytotoxic ability and to produce cytokines after stimulation. Secretroy lysosome in NK cells and lytic synapses NK cells produce cytolytic granules during their development and maturation. These granules that possess acidic lumens as well as several lysosomal hydrolases and marker proteins hire their own special transport systems that allow highly regulated secretion and fusion with the plasma membrane. Defects in the formation or trafficking of the lysosome result in severe immunodeficiency. Upon the target cell recognition, these granules are trafficking to the site of NK cell-target contact site and fuse with the plasma membrane. Subsequently, several soluble effector molecules and lysosomal hydrolases are secreted into the lytic synapses. Structurally, lytic granules formed in the NK cells are defined as secretory lysosomes that are distinguished from a conventional lysosome due to the specialised transport systems. Nevertheless, these granules have structural similarities to the conventional lysosomes in the manner that both lysosomes are accessible via the endocytic pathway. Convent ional lysosomes usually have multi-vesicular structure, whereas secretory lysosomes have dense cores or multi-laminar appearances. Both conventional and secretory lysosomes possess specialised functional proteins such as lysosomal acid hydrolase, receptor proteins and heat shock 70 family proteins. Each is responsible for degradation of old protein, recognition and delivery respectively. There are also other resident membrane proteins such as Lamp1 and CD63 which are largely used as activation markers of cytolytic cells including CTL and NK cells. Lamp1, also known as CD107a will be discussed in detail later in this chapter. In addition to the common components in both conventional and secretory lysosomes, there are exclusive lytic molecules in the granules secreted by NK cells. These include Granzyme A B, Granulysin, and perforin. The detailed function of these molecules will not be discussed here. Exocytosis of lytic granules in the NK cell involves several stages beginning wi th target cell recognition. Most importantly, NK cell-lytic synapses are constructed in where NK cells directly contact to the target cells. The formation of lytic synapses is defined into a series of distinct steps. The first step is the initiation stages that include establishing a close-cell to cell contacts. NK cells either accidentally or possibly by chemotactic attraction, gets in contact with target cells. This step involves the interaction between NK receptors and various ligands on the surface of the target cells such as NKG2DL. The initial contact could subsequently lead to the adherence of NK cell to the target cell, followed by activation of NK cells. The integrin family of adhesion molecules expressed on NK cells including lymphocyte function-associated antigen 1 (LFA1) and macrophage receptor 1 (MAC1) build the firm adhesion to the target cells that facilitate the maturation of the lytic synapses. Additionally, integrins may also participate in signalling that can full y activate some NK cells. Yet, it may not involve the full achievement of NK cytotoxic abilities. Importantly, these initial steps in the formation of the NK cell lytic synapse probably occur before molecular patterning or polarisation are evident and completed quickly. Whether the NK cell progresses to molecular reorganisation at the synapse seems to depend on the level of signals received through inihibitory receptors such as killer cell immunoglobulin like receptors, which can establish the so-called inhibitory synapses. Such regulation ensures that NK cells effectively carry out their surveillance function by leaving most cells undisturbed while being ready to destroy cells that are diseased. The inhibitory synapses are especially elegant in that it directly interferes with the ability of the lytic synapse to progress past the initiation stage. The subsequent stage is the effector stage. Here, the cruicial steps of the granule exocytosis take place; 1. Formation of an immunol ogical cleft where the granules are secreted. 2. Trafficking of granules towards the synapses, 3.fusion of the granule membrane with plasma membrane for the release of the lytic molecules. Under the condition that synapse initiation has occurred in the absence of the inhibitory signals, reorganisation of the immunological synapse is initiated. This begins from the actin reorganisation that involves the formation of F-actin networks from the cellular pool of monomeric globular actin (G-actin) in association with Wiskott-Aldrich syndrome protein (WASP). The absence of WASP is reported to have decrease in the F-actin accumulation at the synapse and NK cell cytotoxicity. As actin reorganisation is processed, following events occurs; receptor clustering, lipid-raft aggregation, further activation signalling, and lytic granule redistribution. Although the detail of such events is not clarified, it seems that these are essential for both cell adhesion and triggering of cytotoxicity. Especi ally, it has been only shown that although receptor clustering is crucial in the signalling within the NK cells, so far, microclusters has been indentified at supramolecular inhibitory cluster. During the effector stage of lytic synapse, polarisation of lytic granules also takes place. The granule moves along the microtubules towards the microtubules organising centre (MTOC) in association with motor proteins such as Kinesin family proteins; Kinesin protein families have a range of motor proteins that are largely involved in the intracellular trafficking of vesicles and organelles, yet the exact motor used is not revealed. MTOC, in the mean time, begins to polarise towards the immunological synapses, and consequently, the granules move to the NK-target cell contact site. Prior to the fusion of lytic granules to the plasma membrane, there are few final steps of effector stage, which are docking of lysosomes to the synapse and vesicle priming. The vesicle priming is the essential prep aration step for fusion through acquisition of biochemical attributes. After vesicle priming, the lytic granules can fuse with the plasma membrane, releasing the granule contents into the cleft between the cells. the mechanism underlying these steps still needs to be elucidated, but information from CTL may give a clue to this process. In CTLs, docking to the plasma membrane requires members of the RAB family of small GTPases, which are important regulators of vesicle trafficking and compartmentalisation. It undergoes post-translational prenylation to gain hydrophobicity which facilitates its interaction with a target membrane. Rab27a is found in NK cells and shown to interact with Unc13D (also known as Munc13-4) for the vesicle priming. Rab27a-Unc13D complex targets solule N-ethylmaleimide0sensitive0factor accessory-protein receptor (SNARE) family protein acting upon the facilitation the fusion of membranes. SNARE proteins are categorised into two; proteins that can be found on the vesicle membrane is called vSNARE whereas those present on the target membrane is tSNARE. Interaction between tSNARE and vSNARE is required for membrane fusion. So far, Vamp7 is the only identified tSNARE protein that involves in NK cell killing. The Final stage of lytic synapse is the termination stage after the lytic granules are exocytosed. This stage involves a period of inactivity and downmodulation of the accumulated activating receptors followed by detachment of NK cells from the target cells. Once the NK cells exert its cytolytic function, it detaches from the target cell and restore its cytolytic potential by regenerating new lytic granules and re-expresses activating receptors. Activation signal of NK cells. Unlike T and B cells, NK cells do not have antigen specific receptors, instead Dendritic Cell and NK cell interaction There have been significant advances in the understanding of the activation and function of NK cells both in human and mice. Since the report by Fernandez at al in 1999 described the DC-NK interaction, it is now well-understood that NK cells and DCs activate one another during an immune response. DC are crucial regulators of both innate and adaptive immune response. As an antigen presenting cell (APC), DC efficiently process antigens in association with MHC molecules. Yet, their functions are followed by the complete maturation of DC triggered by direct encounter to microbial ligands. Once exposed to the pathogen, DC migrate to the T cell area of lymph nodes at where APC-T cell interaction begins. DC-Mediated NK cell activation: How DC contribute to the activation of the NK cells has been well documented by many different experimental systems using different sources of DC including mouse DC cell lines, mouse bone marrow derived DCs, human monocyte derived DCs and human cord- blood-derived DCs. While these studies generally agree on the key role of DC-derived cytokines and surface molecules in driving NK cell cytotoxicity, the mechanisms involved has been emerged carefully suggested to explain the IFN-g production both in human and mice: 1) Direct cell contact induces the production of IL-12 necessary for NK activation, and other cytokines. 2) DC-derived IL-2 promotes NK cell functions in association with other cytokines.(Fig1). In addition, the maturation state of the DCs might influence their ability to activate NK cells. Several studies have shown that immature DCs require a maturation stimulus to activate NK cells, whereas others have shown that immature and mature DCs are equivalent in their ability to activate NK cells. Nevertheless, it is inevitable to accept that both IL-12 and IL-2 are the key cytokines for the NK cell activation process. IL-12 released after DC-NK direct cell contact appears to be presented to NK cells through the formation of some kind of synapse which greatly increases the efficiency of IL-12 even in the low dose. Many studies suggested the role of IL-12 as a key modulator that induces NK cytotoxicity, yet it is still unclear as there has been a suggestion of possible need of other soluble factors. In addition to IL-12, other cytokines such as type 1 IFN and IL-18 may sustain the functions of NK cells in terms of IFN-g production, migration, cytotoxicity and proliferation. IL-18 appears to be playing central role in promoting the migration of NK cells to secondary lymphoid organs where they encounter DC and produce high level of IFN-g once exposed to IL-12, IL-2 and TNF-a. In addition, NK cells showed enhanced cytotoxicity in association with IL-18. IL-2 is now generally accepted as a key cytokine to generate the NK cell activation both in vivo and in vitro, but it had been excluded from the list of those activating NK cells in vivo as it had been understood as an exclusive product of T cells. Soon as i t was figured that IL-2 was also produced from activated DC during the first hours of stimulation, Zanoni and his colleagues has established a work based on the theory that IL-2 may have a physiological role in activating NK cells. They discovered that TLR-dependent full maturation stimuli drove DC to elicit NK cell activation via IL-2 interaction whereas TLR-independent stimuli did not induce the release of IL-2, neither the activation of NK cells. Moreover, NK cell activation process in human has also been revealed recently by Newman et al that the capacity of human NK cells to produce IFN-g is dependent upon cell contact-dependent and IL-2 mediated signal derived from myeloid DC, and this interaction may be driven via cell-cell direct contact in association with TNF expressed by DC that binds to RNFR2 on the surface of NK cells. Aims Having said that NK cells mediate their cytolytic ability upon their activation via various stimuli, it is an interesting question to address how NK cells will react to the different strength of activating signals. Besides, NK cells can also be activated by both immature and mature DC in vivo. Thus, it is worth to assess the effect of both immature DC and mature DC on NK cell activation using various conditions. The aim of the first part of this study is to investigate the expression level of CD107a on NK cells activated by either different strength of stimuli or different maturation state of DC. This will be done by using mouse splenic NK cells. With these NK cells, it should be able to observe the response of NK cells under different conditions and DC-mediated NK activation. Lastly, there has been many data published on Tbox21 transcription factor (T-bet) as a essential regulator of NK cell development and maturation. It is well-established that T-bet deficient NK cells hav e diminished functional activities such as cytotoxicity or cytokine production. However, it is uncertain whether this diminished cytolytic abilitiy is owing to reduced number of lysosome or due to disturbance in the lysosomal trafficking. Thus, it brings an interesting point to have a look at the underlying mechanism of how T-bet is involved in the NK cell mediated killing. The aim of the second part of this study is to compare the quantity of lysosomal content in both WT and T-bet KO NK cells. Subsequently, RNA level of genes that are likely to be involved in the lysosomal trafficking will be investigated using real-time PCR techniques. This should be able to show the influence of T-bet transcription factor on NK cytolytic abilities, and hopefully identify the genes involved in the trafficking mechanism. Materials Methods Mice and Regents. BALB/c and C57BL/6 mice were obtained from Harland UK and T-bet KO mice from (Taconics, USA). Cell lines and primary cells were maintained in medium containing RPMI supplemented with Penicillin/Streptomycin, 10% FCS (fetal calf serum), L-glutamine, non-essential amino acid, pyruvate, hepes, and 2-mercaptoethanol. Splenectomy The mice were sacrificed by cervical dislocation. Mice were placed on their right side and an incision was made on the left side skin with a pair of scissors. The incision was made approximately 2.5 cm long, from between the last rib and the hip joint. Another incision (1-2 cm) was made in the peritoneal wall and the spleen, a red enlongated bean-shape organ, was pulled onto the exterior wall. The spleen was removed by cutting off the mesentery and connective tissues attaching the spleen to the abdominal cavity. NK Cell Enrichment (DX5 positive enrichment KIT negative enrichment) Splenic NK cells obtained from BALB/c, C57BL/6 a nd T-bet KO mice were enriched using two MACS enrichment protocols; DX5 Positive enrichment and KIT negative enrichment. DX5 positive enrichment: splenocytes were obtained extracted using MACs buffer (Sterile PBS with 2nM EDTA and 10% FCS) and centrifuged for 5min at 1200rpm. Using 10ml of MACs buffer, the cells were resuspended, and then counted with trypan blue dye at 50% concentration. Once the cell number was determined, remaining cells were centrifuged for 10min at 1200rpm and resuspended with MACs buffer (amount varies). Subsequently, DX5 microbeads (Miltenyi) was added to the cell according to the manufacturer instruction and incubated in fridge for 10min. The cells were then washed twice with MACs buffer and resuspended with selected amount of MACs buffer. (Amount varies according to the cell count). Using the LS column (Miltenyi), NK-depleted splenocytes were isolated by MACS magnetic cell sorting (Miltenyi). After three times wash, remaining NK cells in LS columns were collected. KIT negative enrichment: splenocytes were obtained extracted using MACs buffer (Sterile PBS with 2nM EDTA and 10% FCS) and centrifuged for 5min at 1200rpm. Using 10ml of MACs buffer, the cells were resuspended, and then counted with trypan blue dye at 50% concentration. Once the cell number was determined, remaining cells were centrifuged for 10min at 1200rpm and resuspended with MACs buffer (amount varies). According to the manufacturer instruction, biotin antibody cocktail (Miltenyi) was added to the cell and incubated in fridge for 10min, followed by microbeads coated with anti-biotin(Miltenyi) and another 15min incubation. The cells were then washed twice with MACs buffer and resuspended with selected amount of MACs buffer (Amount varies according to the cell count). Using the LS columns (Miltenyi), flow-through containing NK cells were isolated by MACs magnetic cell sorting (Miltenyi). Additional 3ml of MACs buffer was applied to the LS column to collect all NK cells. NK cell Sorting using FACS Aria The splenic NK cells were obtained from WT and T-bet KO mice and enriched using magnetic cell sorting method. The enriched cells were counted with trypan blue dye. Once the cell number was determined, they were stained with either NKp46-Alexa647 or NKP46-biotin followed by Streptavidin-PE, and CD3-APC or CD3-FITC. After the 30min incubation period, enriched NK cells were washed three times with MACs buffer and analysed using FACs Aria II(BD Science) to check the purity. NK cell purity was 60~80% after MACs cell enrichment. Enriched CD3- NKP46+ NK cells were sorted using FACs Aria. Purity after sorting was 97~99%. Sorted cells were directly frozen in Trizol and kept under -80oC. Plate coating and FACs staining of splenic NK cells The high-binding well plate coated with a-NK1.1 and aNKG2D was prepared a night before the NK cell stimulation. To prepare the antibody coated plate, the mastermix was prepared at two different concentrations; 50ug/m l and 10ug/ml for a-NK1.1 and a-NKG2D. Each mastermix contained 100ul of carbonate buffer with 5ul or 1ul of antibodies according to the concentration. As a control mastermix, 1ul of isotype antibody was mixed into 100ul of carbonate buffer. The mastermix was dispensed to the well plates and gently swirled for the even spread-out. The plate was incubated in the fridge over night. Next day, splenic NK cells were obtained from C57BL/6 mice. After the cell enrichment, the NK cells were centrifuged and resuspended with the mastermix containing 4ml of medium, 2.6ul golgi stop and 30ul a-CD107a Alexa488 (The amount was determined according to the manufacturer instruction). Prior to the dispense of NK cells onto the well plate, the plate was washed three times with medium. In each well, 200ul of cells were applied and spun for 5min at 800rpm. Subsequently, it was incubated for 4hrs at 37oC. After the incubation period, the cells were transferred to the regular 96 well plate and washed thre e times with medium. Flow cytometry The second mastermix were prepared according to the manufacturer instudction. It contains ;1) 1.25ml of MACs buffer with 10ul CD3-APC and 20ul NKP46-biotin 2) 1.25ml of MACs buffer with 8ul streptavidin-PE. NK cells were resuspended with the mastermix containing CD3-APC and NKP46-biotin and incubated on ice for 30min. Next, it was washed three times with MACs buffer and stained with the second mastermix with streptavidin-PE. After the incubation, the cells were washed three times and analysed with LSRII (BD science). Propidium Iodide was added just before the analysis in order to recognise the dead cell population. DC culture NK stimulation After removing all muscle tissues with gauze from the femurs and tibias, the bones were placed in a 60-ram dish with 70% alcohol for 1 min, washed twice with PBS and transferred into a fresh dish with RPMI 1640. Both ends of the bones were cut with scissors in the dish, and then the marrow was flus hed out using 2 ml of RPMI 1640 with a syringe and gauge needle. The tissue was suspended, passed through nylon mesh to remove small pieces of bone and debris, and red cells were lysed with lysis buffer. After washing, lymphocytes and Ig positive cells were killed with a cocktail of mAbs and rabbit complement for 60 min at 37oC. The mAbs were GK 1.5 anti-CD4, HO 2.2 anti-CDS, B21-2 anti-Ig, and RA3-3A1/6.1 anti-B220/CD45R. 7.5-10 x 10 s cells were placed in 24-well plates in 1 ml of medium supplemented with 500-1,000 U/ml GM-CSF. The cultures were usually fed every 2 d by gently swirling the plates, aspirating 75% of the medium, and adding back fresh medium with GM-CSF. An object of these washes was to remove non-adherent granulocytes without dislodging clusters of developing dendritic cells that were loosely attached to firmly adherent macrophages. DC maturation was induced by incubating the cell overnight with 0.5ug/m; of LPS. DC was co-cultured with splenic NK cells negatively sorted by NK cell isolation KIT. After 4 hr incubation, NK cells were labelled with CD3-APC, NKP46-Biotin, Streptavidin-PE and analysed by flow cytometry. RNA isolation 4106 WT NK cells and 2.3106 T-bet KO NK cells were kept in trizol under -80oC. 0.2ml of chloroform was added to the sample and centrifuged at 12,000g for 15min. The Top layer containing RNA was extracted and mixed with 500ul of isopropanol. Subsequently, the samples were centrifuged at 12,000g for 10min and 1ml of 75% ethanol was added. Samples were centrifuged at 74,000g for 5min and excess ethanol was carefully removed. Samples were allowed open in air to dry any remaining enthanol. 20ul of nuclease free water was added. Nanodrop (spectrometer) was used to analyse the samples. The amount of RNA from WT and T-bet KO NK cells were 71.5 and 38.6ng/uL respectively. Reverse Transcription PCR. cDNA was synthesised using the RNA obtained from WT and T-bet KO splenic NK cells. Mastermix was prepared containing Oligo dT(2ul), PCR Mastermix (4ul), Reverse transcriptase (1ul for RT+) and nuclease free water. 4 PCR eppendorf tubes were labeled as WT+ WT- T-bet+ and T-bet- (negative sign indicates RT- tubes for control). The equal amount of RNA was applied to each tube, followed by the mastermix prepared. The samples were placed in the PCR machine and amplified as the program instructed: 90min 42oC, 5min 85oC. Real-Time PCR The amount of RNA of interest in splenic NK cells was determined using real-time PCR. The mastermix was prepared containing Taqman mastermix, Beta-actin control, Gene expression assay and cDNA prepared. As instructed, duplex PCR was performed using total volume of 10ul. On the 384 well plate, 5ul of Taqman mastermix, 0.5ul of gene expression assay, 0.5ul of ß-actin control, 3ul of nuclease-free water and 1ul of sample was applied to each well. Once prepared, the 384 well plate was placed in the real-time PCR machine and run as instructed. Results CD107a expression is elevated on NK cells following the stimulation. To investigate whether the activation of NK cells is influenced by intensity of stimulus, the CD107a expression was examined using two different conditions. As previously reported, CD107a is a marker for cytotoxic ability of NK cells and CD8+ cytotoxic T cells as it is expressed at high levels upon both cells being stimulated. Two different concentrations of either a-NK1.1 or a-NKG2D were harboured. Both NK1.1 and NKG2D are reported as receptors of activation of which initiate transmembrane signals that activate cytotoxicity. Freshly isolated splenic NK cells from C57/BL6 mice were incubated on the high-binding 96 well plate coated with a-NK1.1 and a-NKG2D. Each antibody was coated at two different concentrations of 10ug/ml or 50ug/ml. After 4hr incubation, NK cells were stained for CD3 and NKP46. Representative data from one subject is shown in Fig. 1. Surface expression of CD107a was low in unstimulated NK ce lls (0.32%) (Fig.1A). Unpurified spleen NK cells stimulated by a-NK1.1 at two different concentrations expressed CD107a at much elevated levels (9.35% and 9.31% for 50ug and 10ug concentration respectively) (Fig.1C,D). The unpurified spleen NK cells stimulated with a-NKG2D at 50ug/ml and 10ug/ml both showed increased expression (8.28% and 8.45% respectively), yet it is faintly lower than those stimulated with a-NK1.1. Compared to the unpurified splenocytes, the negatively sorted spleen NK cells by NK cell isolation KIT showed inconsistency that, although the expression of CD107a was all increased on NK cells stimulated with NK1.1 or NKG2D at both concentrations, the degree of elevation was minor except those stimulated with NK1.1 at 50ug/ml concentration (7.32%). Other three showed 4.28%, 3.92%, 3.3% at NK1.1 10ug/ml, NKG2D 50ug/ml, and 10ug/ml respectively. Taken together, these data demonstrate that CD107a is highly upregulated on the surface of NK cells following stimulation, but the intensity of stimuli did not greatly affect on the activation level. Having said that the CD107a expression was upregulated following the stimulation using a-NK1.1 and a-NKG2D, DC-mediated NK cell activation was subsequently examined, as DC is a source of cytokines required to activate NK cells in vivo. To assess whether the DC-mediated NK cell activation is regardless the maturation of DC, bone-marrow DC from C57BL6 mice (approximately 80% purity) was subdivided into two populations; one incubated overnight with LPS for the stimulation, and the other left untreated as a immature DC. Negatively sorted splenic NK cells by NK cell isolation KIT was co-cultured with DC in the well plate and incubated for 4hrs. Next, NK cells were stained for CD3 and NKP46. As a control, purified NK cells with no culture were hired to compare the CD107a expression. In a control group, hardly detectable expression of CD107a (0.02%) was observed, indicating most of the NK cells were left inactivat ed as expected (Fig.2.A). The frequency of activated NK cells co-cultured with iDC was appeared to have higher; 3.05% and 2.31% for iDC co-culture at 1:1 and 1:5 ratio (NK:DC) respectively. It clearly demonstrates that iDC can induce the activation of NK cells. However, it should be noted that the LPS-stimulated DC co-culture did not induce the activation of NK cells so efficiently as the immature DC as expected (0.8%, 1.77% and this conflicts with other data published by others that the mature DC elevates the NK functions in all manners. This may be due to the ratio adopted here that NK cells possibly induce their cytotoxic ability upon DC when there is too large number of DC. To assess such an issue, further investigation is required by adopting various environments of DC stimulation such as IFN-a or CD40L. However, although the expression of CD107a on NK cells simulated by LPS-stimulated DC was beyond the expectation, these results demonstrated DC can mediate NK cell activations regardless their maturation. Taken together, CD107a is upregulated upon the activated NK cells stimulated by either a-NK1.1 and/or a-NKG2D or bone-marrow DC, indicating that NK cells initiates the production of cytolytic molecules. Both results may indicate that the activation of NK cells is regardless to the intensity of stimulation since different concentration of a-NK1.1 or a-NKG2D, or different ratio of DC:NK does not produce noticeable difference in the CD107a expression. However, further investigation is evitable as these data were obtained from only limited number of mice, and repeated procedures shall be cruicial in order to reveal more precise correlation between intensity of stimuli and NK cell activation. T-bet/- NK cells has higher RNA level of CD107a than WT NK cells Following the demonstration that NK cells mediate their cytolytic functions upon the activation, I subsequently assessed to determine whether T-bet transcription factor influence the CD107a express ion by looking at the RNA levels in both WT and T-bet. From the previous findings, it is now a known fact that T-bet-/- NK cells mediate less functional activities in both cytotoxicity and cytokine production. However, it is not revealed whether T-bet deficiency results in reduced amount of cytolytic vesicles or disturbance in the trafficking of the lysosomes. In order to address this remaining question, RNA extent of CD107a was measured in both C57/BL6 WT mice and T-bet KO transgenic mice by real-time PCR technique. Splenic NK cells were extracted from WT and T-bet KO transgenic mice and purified by NK isolation KIT. Subsequently, NK cells were stained for CD3 and NKP46 and sorted using FACs AriaII. Purity after sorting on NK cells from both sources were upto 98%. NK cells were kept in the trizol under -80oC. Next, RNA was separated from the cells using chloroform, isopropanol and ethanol and kept frozen under -80oC. The extracted RNA was hired to synthesise complementary DNA stran d by reverse transcription PCR technique and readied to perform the real-time PCR. Gene expression assays were purchased from Applied Biosystem according to the genes of interest. These genes are as follow: CD107a, Rab27a, Unc13d, Vamp7, Wipf1, Dnm2. Additionally, gene expression assays for IFN-g and T-bet were also taken into account to examine the corresponding RNA levels. Using the Taqman PCR mastermix, ß-actin, and gene exression assay, the real-time PCR was performed on those eight genes of interest. Subsequently, Delta-delta CT method was hired to calculate the RT-PCR results. As the PCR was repeated several times, the ratio between delta CT values from WT and T-bet-/- NK cells was calculated and averaged. The representative data from one subject is shown in Fig.3. As a control, T-bet was examined on both WT and T-bet-/-NK cells. As expected, T-bet was not found in the T-bet deficient cells whereas WT NK cells have much higher levels. By looking at the results, the RN A level of CD107a was observed repeatedly to be higher in T-bet deficient NK cells than WT NK cells (Supplementary data 1). The averaged ratio indicated that CD107a RNA was 1.7 times higher in T-bet-/- cells. This result directly conflicts with the phenotype of T-bet-/- NK cells to have reduced ability to killing. Other possible explanation can be that the capability of cytolytic vesicle production may not be defected, but the trafficking of such vesicles could be diminished. To address the following question, RT-PCR was performed on the genes possibly involved in the lysosomal trafficking. First, Member RAS oncogene family 27a (Rab27a) was tested, which belongs to the small GTPase family, RAS family that are important regulators of vesicle trafficking and compartmentalisation. According to the RT-PCR results, T-bet deficient NK cells produce a reduced amount of Rab27a in terms of its RNA extent that the mean ratio of T-bet KO cells was 85% compared to the WT. Considering that Rab27 a participate in the lysosomal trafficking, it is possible that reduced cytotoxic ability is due to the defect in the trafficking, not the amount of lysosomal vesicles. Subsequently, dynamin 2(Dnm2) was examined. This gene is the isoform of the conventional dynamin family of large GTPase that participate in the receptor mediated endocytosis and membrane remodelling. Additionally, it also localises with lytic vesicle and regulates the fusion to the plasma membrane. By looking at the RNA levels, it was elevated in T-bet deficient NK cells unlike Rab27a. Average, it was one half folds higher than WT NK cells, indicating that defect in cytotoxic ability is not owing to the final fusion of the vesicles to the plasma membranes. Next, Wipf1 belonged to Wiskott-Aldrich syndrome (WAS)/WASL interacting family was examined. Wipf1 is known to be involved in the initiation step of NK cell lytic-synapse formation. From the previous findings, the phenotype of defect in this gene shows NK cells wit h decreased cytolytic ability, nevertheless, overexpression in Wipf1 results in the enhanced ability. Taken into account that T-bet deficiency brings defected ability of cell killing, it was a surprise to see that Wipf1, like CD107a or Dnm2, was more produced in terms of its RNA level in T-bet-/- NK cells. The RT-PCR result revealed that it was almost two fold higher than WT (1:1:97 in WT : T-bet KO). This demonstrates that the formation of lytic synapses is not manipulated by T-bet transcription factor, at least not by Wipf1. Following the Wipf1, the next gene tested was Unc13d (also known as MUNC13-4). This gene belongs to the Unc13 family, which is known to involve in the vesicle maturation and regulation of cytolytic vesicle release. It interacts with Rab27a and primes the lytic vesicles for fusion with plasma membrane at lytic-synapse. Again, T-bet deficient NK cells had higher RNA extent of Unc13D (1:1.56 to T-bet KO). Lastly, Vamp7 was tested. In fact, Vamp7 was the most high ly expected gene to test, because previous findings describe its essential role in target cell killing. To be brief, Vamp7 is known as Vesicle Associated Membrane Protein 7 that is found to be colocalise with CD107a molecules and participate in the fusion of transport vesicle to the target membrane. Although any trace of Vamp7 RNA was not found in both WT and T-bet-/- NK cells, it was not surprising fact, because Vamp7 is the soluble N-ethylmaleimide-sensitive-factor accessory-protein receptor (SNARE) among which present on target membranes (t-SNARE). As a matter of fact, this can explain how NK cells can be protected from their own lysosomal vesicles. In the absence of t-SNARE proteins, lytic synapses cannot be constructed, therefore for those NK cells missing Vamp7 is safe from its own vesicles. Following the investigation on genes of which participate in the lysosomal trafficking, level of IFN-g was also quantitated. Although the RNA level does not directly reflect the IFN-g p roduction, it is worth have a look to assess whether T-bet deficiency influences the RNA transcription. As a matter of fact, T-bet deficient NK cells seem to have a higher IFN-g RNA level than WT that it was approximately 37% elevated. Based upon the previous studies, IFN-g production is diminished in the absence of T-bet, yet its RNA level indicates T-bet is not directly acting on the RNA production but on the translation pathways. Taken together, how T-bet deficiency affects on the lysosomal trafficking was tracked by looking at the RNA levels of genes supposed to be involved in the formation of lytic-synapses. Except Rab27a, other four genes were shown to be elevated in the absence of T-bet deficiency whereas Vamp7 was not found on both types of cells. This indicates that Rab27a is involved in unknown mechanism of lysosomal trafficking, which results in the functional defects in T-bet-/- NK cells. Discussion The findings presented here have demonstrated the activation of NK cells in different environments and the effect of T-bet deficiency on the genes believed to be involved in lysosomal trafficking. Resting NK cells show minimal functional activities in order to prevent unnecessary cell killing, yet once stimuli is detected, it rapidly induces its lytic functions against the target cells. However, the question I tried to address was whether the different strength of stimuli would make any difference in the NK cell activation. Considering that NK cells mainly target stressed or virus-infected cells, they should be highly sensitive enough to detect any small stimulus, otherwise it may result in the massive infection or even worse, cancer. This study has shown that the stimulation of resting NK cells boosts up the expression of CD107a, yet the intensity of expression may not be proportional to the strength of stimuli given. CD107a, which is the lysosomal associated protein representi ng approximately 50% of the proteins embedded on the lysosomal membrane, has been hired as a functional marker for the NK cell activity. Given that CD107a reflects the cytolytic activity of NK cells, it seems that activation of NK cells is irrelevant to the strength of stimuli. By using the different concentration of a-NK1.1 and a-NKG2D, those provide direct activation signals, this study demonstrated that the CD107a was upregulated on NK cells following the stimulation, indicating the production of lysosomes of NK cells. Moreover, the level of its expression was not noticeably varied when different concentration of stimulatory antibodies was used so that shows NK cells can mediate their cytolytic ability even with small stimulus detected-thereby provide rapid response and protection against any danger signal. Dendritic cells have been shown to be involved in the activation of resting NK cells once they have been matured after the exposure to the antigen. DC produces various cyto kines necessary for NK activation, such as IL-2, IL-12, IL-18, IFN-a/b, as a response, NK cells achieve their cytolytic functions as well as cytokine productions. Here, immature DC and LPS-stimulated DC were used at different ration to NK cells to observe change in the expression of CD107a. As expected, LPS-stimulated DC elevated the CD107a expression and this result explains the key role DC during the early events of immune response, that once DC encounter with a pathogen, they release various cytokines that ;IL-2 and IL-15 to induce activation and/or proliferation of NK cells, IL-12 for induction of IFN-g by NK cells, IL-18 to direct NK cells capable of migrating to secondary lymphoid organs where they can interact with DC. However, albeit elevation of CD107a in both ratio used, it was noticeably increased when the DC ratio to NK was higher. Once DC is matured, IL-1b is produced which, along with high mobility group B1 (HMGB1) factor, protect DC from NK cell mediated killing. Thus , DC can keep mediate NK cell activation without themselves being killed. Additionally, sufficient DC in the environment would provide more cytokines, thus resulting in the higher activation rate. Immature DC, however, have shown the opposite results that the low ratio in NK:iDC mediated more expression of CD107a. Although the underlying mechanisms are elusive so far, iDC has been reported to be capable of inducing NK activation. In fact, NK cells interact with iDC to polarise and secrete IL-18 through synaptic cleft, thus initiate their activation. In turn, activated NK cells produce HMGB1 that induce DC maturation as well as protection against NK-mediated DC killing. Thus it is natural to see the elevated expression of CD107a in the co-culture with iDC. Then why the expression is low in the high NK:iDC ratio? Study by Piccoli et al has proposed the killing of DC by NK cells in infected tissues to amplify the stranger/danger signal derived from the invading pathogens. Although D C can secrete lysosomal hydrolases, this can be defeated by high NK/DC ratio. Thus, high NK/DC ratio rather kills DC rather than maturation and consequently, this results in the reduced number of DC in the NK-DC co-culture, followed by less activation of NK cells. Only those cells received HMGB1 survives and produce cytokines to activate remaining resting NK cells. Despite the fact that these results all successfully demonstrated the activation of NK cells in various environments, it still leaves few laments for the further investigations. First, due to the limited resources and time, these experimentations were carried out once and thus there was no sufficient data to confirm the analysis in depth. Repetition on these investigations is inevitable to construct more firm and detailed analysis that are more feasible with other findings. Second, what I intended to assess through the project is the functional aspect of NK cells in relation to T-bet transcription factor. Thus, further investigation is required on looking at CD107a expression on both WT and T-bet -/- and this will advance the understandings on NK cells and its function. It is now well-established that T-bet deficient NK cells have imperfect functions in every aspect, that they show decreased cytotoxicity and cytokine productions. Given that T-bet transcription factor is involved in the early development of NK cells, it is not so surprising fact to have such a consequence. However, the underlying mechanism on diminished cytolytic function is so far elusive and therefore it is still not clear whether T-bet deficient NK cells produce less lytic vesicles or it is due to the disturbance in the lysosomal trafficking. Based upon the preliminary results from Dr.A Martin-Fontecha,which demonstrate that the lysosomal content of T-bet deficient NK cell is reduced compared to the WT NK cells, real-time PCR was performed on those genes that may participate in the lysosomal trafficking to determine whether both results agrees upon each other. By targeting the five genes, CD107a, Rab27a, Unc13D, Vamp7, Dnm2 and Wipf1, I have shown that T-bet transcription factor contributes on all genes except Vamp7 that had no trace on both WT and T-bet deficient NK cells. CD107a encodes the highly glycosylated protein that contributes half of the proteins on the lysosomal membrane. Thus, it has been used as a NK degranulation marker. This study has shown that mRNA content of CD107a was consistently higher in T-bet deficient NK cells than the control. This result implies that there may be defective formation of cytolytic vesicles as the lysosomal content was found to be reduced. The vesicles may simply possess more CD107a protein or possibly their size may vary, yet any of this has not been determined. CD107a closely interacts with Unc13D protein (also known as Munc 13-4), the member of Munc13 family of proteins, and it is probably responsible for vesicle priming. The targets of Unc13D are members of the SNARE family which can be defined into vSNARE and tSNARE; vSNARE are the SNARE proteins found on the vesicle and tSNARE are those present on target membrane. SNARE proteins interact in a coordinated manner to facilitate the fusion of two distinct membrane. Unc13D has been suggested to interact with SNARE protein and mediate the membrane fusion. Moreover, it has been found to be closely related to CD107a molecules that previous finding has demonstrated CD107a expression is significantly decreased when Unc13D has been mutated, as well as NK cytolytic ability. Often, their close interaction allows using the expression of CD107a as a screen tool of familial hemophagocytic lymphohistiocytosis (FHL), the defective genetic disorder on NK cells by mutated Unc13D. In this study, I have demonstrated that T-bet deficient NK cells possess higher content of Unc13D mRNA, which is consistent to the CD107a mRNA level that has also been found to be upregulated in T-bet deficient NK cells. The se two positively regulated genes in the absence of T-bet transcription factor may suggest that lytic vesicles in the cell can possibly be primed and fuse to the target membrane more than the normal level. As a matter of fact, overexpression of Unc13D enhances the degranulation of lysosomes. Thus, at the baseline, this may explain how T-bet deficient NK cells can still induce cytotoxicity even if reduced content of lytic vesicle is synthesised. In addition to CD107a and Unc13D, Rab27a is a member of Rab family of small GTPases that are important regulators of vesicle trafficking and compartmentalisation. The protein encoded by Rab27a gene undergoes post-translational prenylation to gain hydrophobicity, which facilitate the interaction with target membrane. Consequently, the interaction mediates the docking of lytic granules. In addition to that, Rab27a is a direct partner of Unc13D that both proteins are normally found in not only NK cells but CTL and mast cells where these two c olocalise on the lytic vesicles. Unc13D protein binds to the Rab27a, forming a Rab27a/Unc13D complex that plays a essential role as a SNARE complex regulator. Thus, defects in Rab27a would prevent formation of the complex therefore lead to the functional inability to secrete the lysosomal content. I have shown that T-bet deficiency reduced the mRNA level of Rab27a, the only gene showing reduced level among the genes tested. This may suggest the reason why T-bet deficient NK cells has attenuated cytotoxicity, that reduced level of Rab27a leads to downregulated expression of the protein and therefore, Rab27a/Unc13D complex was not fully constructed as in the WT NK cells. Taken together, diminished cytolytic ability in the absence of T-bet transcription factor is not only owing to the decrease in lysosomal content, but the trafficking of lysosomes was also disrupted by less number of SNARE complex. Nevertheless, it should be noted that this defective phenotype can be reversed by cultur ing NK cells in the presence of IL-2. It may not induce increased production of Rab27a in both RNA or protein level, but would trigger alternative pathway for Rab27a function. Further investigation is needed to determine whether T-bet deficient NK cells recover their functional activity in the presence of IL-2, and this would clarify the role of Rab27a. As like those genes showing elevated mRNA levels, Wipf1 was also higher in the absence of T-bet deficiency. Considering the function of protein encoded by Wipf1, it may not be the main cause of the reduced ability of NK cells. Wipf1 encoded protein, WIP is known to be important in the NK cell cytotoxicity as previous finding demonstrated WIP knockdown completely deters cytolytic function whereas overexpression enhances the killing. Its initial role is to bind to a region of WAS protein and stabilises filamentous actin and consequently, regulate the cytoskeleton rearrangement. This can be also found in the macrophage for the podoso me formation, but in the NK cells, it is crucial for immune synapse dynamics. Moreover, it associates with lytic vesicles in the NK cells and participate in the lysosomal polarisation to the lytic-synapse. Altogether, deficiency in T-bet transcription factor elevates the Wipf1 mRNA level, but this may not be directly responsible for the reduced functional activity of T-bet deficient NK cells, since its elevation would increase the functional activity. At the baseline, it suggests that formation of lytic-synapse is not directly regulated by T-bet transcription factor. Lastly, this study has shown the complete absence of Vamp7 in both WT and T-bet deficient NK cells. This was an unexpected result as many studies previously demonstrated that Vamp7 deficient NK cells show significant decrease in their cytolytic ability. Despite repeated RT-PCR was performed, Vamp7 was not found in any results. This may be due to the systematic errors during the performance, yet, it is still not easy to understand why Vamp7 did not produce any results or show extremely low level while other genes tested together worked fine. Possible reasons can be either problem with sample NK cells used or gene expression assay. Nevertheless, it is also hard to believe WT and T-bet deficient NK cells both do not produce any Vamp7 RNA. It is very likely it is the problem with Vamp7 gene expression assay, but to clarify, further investigation is required. Taken together, this study have demonstrated the activation of NK cells in the various environments and RNA levels of genes believed to be involved in the lysosomal trafficking. NK cells, as a pivotal player in the immune response, it induces its cytolytic effects upon its stimulation by various types of stimuli. However, its activation may not totally depend on the intensity of the stimuli. There can be other possible underlying mechanisms in the activation of NK cells. Through this study, immature DC shows its ability to induce the activat ion of NK cells just like mature DC. However, as iDC express low level of MHC class I molecules, it easily becomes the target of NK cell mediated killing, and as a consequence, high NK:DC ratio may kill iDC rather than maturation. On the other side, mature DC can protect themselves from NK cells, thus stably can mediate activation of NK cells. Lastly, T-bet deficiency was shown to affect RNA levels of many genes involved in the lysosomal trafficking. Especially, mRNA content of Rab27a was noticeably reduced while other genes of interest increased. This suggests that reduced amount of Rab27a protein leads to the less SNARE complex and therefore, disturbs the overall lysosomal trafficking.

Teaching Competency of English Language Teachers Free Essays

COMMUNICATION AS AN IMPORTANT SOFT SKILL IN LANGUAGE TEACHING Mrs. N. Mahalakshmi D. We will write a custom essay sample on Teaching Competency of English Language Teachers or any similar topic only for you Order Now T. Ed. , M. A. , M. Ed. , NET. , PGDACE. Research Scholar Department of Education Annamalai University ————————————————- prusothmaha@gmail. com Abstract ————————————————- English is being taught as a second language in our Indian schools. As it is our national language, much importance is given to this language in our education system. The language teachers are expected to be more competent to develop the basic skills of the language so as to develop the communicative competence of the learners. Now-a-days, soft skills are considered as another important aspect of the teachers for efficient teaching. Regarding the soft skills, communication skill is the most important one that is needed by the language teachers to optimize the learning experience of the students. This paper tries to reveal the need of Effective Communication Skill as one of the important soft skill for the language teachers. The concept of soft skills Soft skills can be said to incorporate all aspects of generic skills that include the cognitive elements associated with non-academic skills. Soft skills are identified to be the most critical skills in the current global education and the era of technology. The reorientation of education for sustainability also relates the importance of these soft skills. Soft skills in Education Vast research and expert opinions have been sought in the effort to determine the specific soft skills to be implemented and used in higher institutions of learning. Based on the research findings obtained, seven soft skills have been identified and chosen to be implemented in higher education as: * Communicative skills * Thinking skills and problem solving skills * Team work force Life-long learning and information management * Entrepreneur skill * Ethics, moral and professionalism and * Leadership skill The important soft skill needed for the language teacher Communication is as important aspect of language teaching. Effective communication skills are required for effective language teaching. Teachers of English are expected to have good comman d over the language and possess excellent communication skills. Communication skills include – using the target language effectively, the way of speaking, body language and facial expressions, pitch and tone of voice and interpersonal skills. It is possible that they have some presuppositions about communication and communication skills which are considered to be one major factor in becoming an effective teacher. According to Dettmer, Thurston, and Dyck (1996), West and Cannon (1988), and Carl Rogers (1962) communication is among the most important skills for educators to possess. The role of communication is emphasized also by Lunenburg Ornstein (1996, p. 176) as: â€Å"Communication is the lifeblood of the school; it is a process that links the individual, the group, and the organization†. A gap in meaning between the intended and the received message can cause problems in the outcome of even the best teaching decision. Poor listening skills, ambiguous use of verbal and nonverbal language, poor semantics, and differing values are all items that can distort a message. To become effective communicators, educators must be aware of these potential problems and consciously work to eliminate them from their classroom interactions. They must also become knowledgeable about the importance of language in the learning process which gives a vital role to language teachers. Body language of the teacher In the communication skill, the body language and the facial expression of the teacher is of much importance which arrests students’ participation. The ‘presence’ that a teacher has in the classroom is crucial in determining ‘how much’ learning takes place and ‘how well’ learning takes place. A tension free atmosphere is extremely important in language learning classroom. More than what behaviour reveals, it is the non-verbal behaviour that is of significance. Self respect, confident behaviour and tone and eye contact are some positive indicators. Some of the ways in which body language can improve the desired atmosphere within the class are: * Keeping eye contact with the student you are talking to, and with every student in the class; * Standing ‘tall’ and walking in with head held high, instead of shuffling in, head bowed; * Having a calm, relaxed face – smiling and laughing easily; * Using facial expressions that show you are listening and responding to what the student is saying; * Smiling and nodding when a student is saying something; Linguistic competence versus Communicative competence Language is a tool of communication. One can communicate ideas, thoughts, feelings, opinions, attitudes, information and even misinformation through language. Different people express the same idea in different words. Language is a tool serving four main functions. These important functions are important for effective communication in the language classroom. The important functions of the language are: * Social function * Informative function * Expressive function * Directive function Keeping in mind these four important functions of language, let’s examine if our students are effective communicators in English. Most of our graduates are good at writing beautiful and very literary answers to questions on Shakespeare, Wordsworth and other great writers. However, their literary competence isn’t enough for them to be able to communicate effectively and efficiently in everyday situations. The ability to communicate requires us to use language to perform interpersonal functions such as starting a conversation, joining and leaving a conversation, making the hearer feel comfortable, giving options, and so on. Mere linguistic competence isn’t sufficient. Of course, there’s no denying the value of linguistic mastery, which is the basis for communicative competence. Without words and grammar patterns, one can’t think of building communicative competence. However, rules of use are more essential than rules of grammar. Many graduates don’t know how to introduce themselves and how to introduce others; they don’t know how to ask for information politely, how to disagree tactfully, how to offer suggestions, etc. This is one very significant aspect that we need to pay attention to. Secondly, their English is bookish. They don’t know that choice of syntax and vocabulary depends on the topic, the occasion, and the relationship between the speaker and the listener. It’s important to know what to say, when, to whom and how. Thirdly, the students need to be told that the vocabulary and syntax of spoken English are different from vocabulary and grammar of written English. They seem to be unaware of the fact that the words and grammar of spoken English are simpler than those of written English. As a result, they don’t use contracted forms and question tags while conversing and their English sounds bookish. Developing the communication skills of the learners In language teaching developing the skill of listening, speaking, reading and writing  skills should be given importance. These language skills are the foundation of communication skills. A good communicator is a keen and interested listener. Even a good listener cannot be an effective speaker. In order to be a good speaker, one has to master the accent, the rhythm and the intonation of the English language. Also one has to mind the tone of voice and make an effective use of facial expressions, gestures, eye contact, and posture. An excellent communicator uses verbal and non-verbal language to achieve the best effect. In order to develop good communication skills of the students, the language teacher need to * develop the listening, speaking, reading and writing skills * to be able to use language to perform various functions * master the rhythm, accent and intonation of the language * understand the differences between spoken and written language * remember the difference between meanings and messages Conclusion To remedy this situation we need to connect literature teaching with life outside. In language teaching, the academic world and the real world should not stand apart as islands. From the standpoint of the learner, the great waste in the school comes from the learner’s inability to utilize the experiences he gets outside the school. To fill up this gap, the communication skill should be given importance in language teaching. Developing communication skills of the learners requires the efficiency of language teachers. So, the communication skill should be given primary importance both at the pre-service and in-service level of the language teaching. REFERENCE * Applbaum, L. et. al. , 1973, Fundamental Concepts in Human Communication, Confield Press, London * Brown, H. D. 1981, Principles of Language Learning Teaching, Prentice Hall, Enlewood Cligts. * Corner, J. et. al. , 1993, Communication Studies:An Introductory Reader, Edward Arnold, London. * Dickinsen L. and Carver D. J. 1980. Steps Towards Self-direction in Foreign Language Learning in Schools. ELT. Vol. 35:1-7. * Dickinsen L. 1987. Self-instruction in Language Learning. Cambridge, Cambridge University Press. How to cite Teaching Competency of English Language Teachers, Essay examples

Take a Stroll Down the Customer’s Journey

TAKE A STROLL DOWN THE CUSTOMER’S JOURNEY Recent studies done by Word Stream show that the average conversion rate for pay-per-click advertisers is 2.35%, which is about the same chance some have of becoming a millionaire. What happens between the click and the customer being converted or not? What sort of journey does the customer go through, and how can companies make sure that each customer stays on the path to a purchase? If marketers zoom out and take a look at what a potential customer’s journey might look like, they can fill in the gaps between what a customer wants that experience to be and what kind of experience you would like to give them. Visually mapping that journey, gathering all of the data points, the analytics, the emotions, and strategies involved is one of the best ways marketers can begin to understand their customer. We spoke with two Houston marketing experts to get some insights on how they are using the customer journey map to help their clients. Graph showing search conversation rate distribution (Image from Word Stream) But What Exactly is a Customer Journey Map? It’s easy to get lost in marketing and advertising jargon, but the core of what a customer journey map is really quite simple. It tells the story of what a company’s customer would go through to buy their product or service, or whatever the company’s particular end goal is. The journey a customer goes through could lead down a hundred different paths. A map could focus on the web journey, an experience at a traditional brick-and-mortar store, a call experience, or the map could encompass every route. More touchpoints mean a more complicated map, but it is the important or key ones that are crucial to get down. Source: Behance The experience a customer has with a company is already on its way to overtaking importance in price and product, and according to a recent report, by 2020 it will have succeeded. Experience will be 16% more important than price. Beau Pedraza, the SEO Lead at Forthea, a Houston-based internet marketing company, agrees. The ultimate goal is not just to satisfy the end goal by having a customer purchase a product, or engage with a call to action. It’s to make it easy and intuitive for anybody to reach that end goal and then come back because we made their experience easy and seamless. There’s the human side of it that I think a lot of people in marketing, especially digital marketing, forget about. With   two-thirds of customers willing to spend more with a company that they believe has better customer service, it is definitely worth the time spent to make a customer journey map. How Do You Track the Customer Journey? The most important part of beginning to map the customer journey is the customer or user themselves. Brandi Lalanne, the Senior Digital Strategist at The Black Sheep Agency, a cause-driven Houston marketing firm, said it best. You need a user in order to map them down the path. Designing CX, a website dedicated to helping customer experience innovators and change agents, laid out some fairly simple steps in a course on creating a customer experience map, and it starts with selecting a user. Select a user. Do you want to map the journey of a stay-at-home mom? Or maybe the person you want to map works 40 hours or more a week. Or theyre a student in college. Or maybe, you need to map them all. Getting to know the user and what route they might take is important in creation of the map. Map out a user’s step-by-step experience. This is where marketers can make sure that their customer is going to have a good experience and set them off on the right path. The Designing CX course suggests working from point A to point B, starting with assumptions and then gathering hard data. Does your user go straight to a phone call, or maybe to the website first? After the phone call, what happens? Is there a research phase, a waiting phase, a purchase phase? You have to keep asking yourself, â€Å"What happens next?† Map the answers to this question, but remember, everything is from the customer’s point of view. Map touch points and systems â€Å"on stage† and â€Å"backstage.†Identifying the touchpoints, every physical or virtual interaction, for each step of the map is what is important here. This could be where the hard data comes in. Pedraza explains.We use an assortment of methods of the digital side ranging from Google AdWords to Google Analytics, and other analytics that are at our disposal. We also use call tracking; we use ad data; we use all the granular offerings that each one provides. They tell us how the customer engages, [how they] use the site, how they flow through a website; if they go to the home page, what’s the next page they go to. If they arrive on a landing page, where are they ultimately trying to go?If your data tells you that the average phone wait time before a customer will hang up is 12 minutes, then you will want to make sure that your customer’s experience reflects that. Does it take a customer 2 hours or 2 days to decide to purchase? Analytics can show you where you need to make improvements, changes, or if there is redundancy in your touchpoints.Google Analytics has proven paramount in tracking customer experience. Ariat, one of the nation’s leaders in equestrian footwear and apparel, used analytics in their website relaunch. The measurements and data allowed the company to see where improvements were needed, ultimately leading to a 14% increase in conversion. Add customer attitudes and needs. On the other side of hard data is the emotional side. Empathy mapping is a huge part of the customer experience, and that is the fourth step in the Designing CX course. Pedraza believes that this step is of the utmost importance. We like to see what people think about our clients, and that information should never be discounted it’s [the end user] that is the engine that drives it all. They are the ones that our clients are interested in, and we should be interested in them as well. To find out the emotions, Pedraza suggests asking for reviews or surveys from past customers, using the analytics, asking yourself what your customers are saying about you, or just using a little guesswork.Tracking emotions and empathy can be difficult, because, as Lalanne states, â€Å"You can’t track joy as a metric.† However, as the below image shows, the emotional experience is an important part of the map. Human emotions by nature are on a consta nt roller coaster, but companies will want to keep their customer as stable as possible.By knowing how the customer feels and is treated at each touch point on the map, companies can glean valuable information to help direct the customer to a purchase. Identify problems and opportunities.The last step in the course might be what marketers have the most trouble with. Once the map is finished, some marketers might find themselves asking, â€Å"now what?† Lalanne laughs about this, reiterating how important it is to refer back to the map. It’s continuously living and breathing, if you refer back to it, you’ll be fine.It is understanding the customer’s journey that can lead to improvement.Sometimes, that is precisely what the map helps you do. Such was the case with Pedraza. Assuming the end user for a particular project wouldnt utilize a mobile site very much, Pedraza left off the mobile part of the journey.I felt like in the map that we created initially we were missing these big segments on mobile, and when we started to cater to mobile, we saw these massive uptakes from 10 to 15% in traffic in about two months†¦That was the biggest thing that really got me on customer journey mapping†¦ that esp ecially in our line of work, that we have all these assumptions of what people might do or what people might think†¦but you need to be willing to test your own theories and willing to accept that you just might be wrong.After your map is finished, it will also show you whether your brand ideas and promises match up with your actual customer experience. Does your company promise fast service, while your map suggests otherwise? Why exactly do your customers emotions take a turn for the negative during the purchase process?Designing CX suggests redesigning the experience as needed to influence attitudes. If your map isn’t working, it’s OK to change it. Keep referencing the map. Keep it alive. A customer’s journey never ends, and neither should your map. Why is the Customer Journey Important? We’ve already noted that customer experience is on its way to becoming the most important factor in a customer’s decision making, and word of mouth is playing a huge roll in its rise. Esteban Kolsky notes that 13% of unsatisfied customers will tell at least 15 or more people about their experience; adversely, 72% of satisfied customers will share their experience with six or more people. This year, 86% of companies expect to compete mainly on the experience of a customer, a 50% increase in the last four years alone. In a world full of consumers, paying close attention to the customer is possibly the best strategy a marketer could have. Before hanging up, Lalanne left off by stressing this point. I think everyone should [make a] customer journey map. If you really care about your users and your audience, then you should take the time to understand them as if they’re an actual person and not just a data point.