Friday, April 5, 2019
Jean Piaget: The Four Stages Of Cognitive Theory
Jean Piaget The Four pegs Of Cognitive TheoryCognitive surmise of Jean Piaget includes intravenous feeding stagecoachs of learnment that tikeren dissemble through and through during which the explanatory behaviors of infants transform into the move up, logical intelligence of grownhood. in that respect argon three in all important(p) specific characteristics of Piagets theory of which the start-off 1 is be a popular theory, that is, cognitions all aspects undergo a similar course of change. A nonher characteristic is that churlren move through the stages in an invariant sequence. Piaget believed that there is a comparable place that fryren follow. Third, the stages atomic number 18 universal. Stages in cognitive theory fas decennium on the theory to include all fryren everywhere (Berk, 2003). Biological concepts argon employmentd in a particular(a) commission in Piagets theory. However, he put forwardd the sizeableness of genic and environ noetic facto rs on the way that kidskinren move through the stages (Crain, 2005). He emphasised that the speed of baberen plot passing those stages is affected by differences in genetic and environ ami competent factors.Jean Piaget use the stipulation contrivance while explaining human race beings organized way of qualification sense of experience (Mark, 1969). Traill (2008) explains that the term scheme used by Piaget is variant from peoples everyday usage of scheme. The term can be any pattern for exploring and learning from the milieu and it has three contrastive understanding structures. Piaget calls world-class intellectual structures to emerge as behavioral schemes, ones that appear after 2 course of instructions as emblematical schemes, and structures that appear after 7 years as operational schemes (Piaget, 1972, as cited in Traill 2008). For instance, dropping scheme of an 8 month old baby and a 25 months of allow for non be the same, as sooner it allow for produce more than(prenominal) deliberate and creative. Toddlers, different from infants, begin to turn over forrader performing and Piaget identifies that transition from sensorimotor to cognitive approach to the humanity which depends on psychogenic prototypes. (Piaget, 1926, as cited in Berk 2003) Images and concepts argon the twain powerful mental representations.E superfluously, the shift from sensorimotor to cognitive approach is accounted for two processes adaptation, consisting assimilation and modification, and organization. interpretation of new structures into already existing schemes is called as assimilation and modification of existing schemes into adaptation of new experiences is called as accommodation. Cognitive adaptation aims to adjust to the environment and is a result of the equilibrium between assimilation and accommodation (Block, 1982). While nerve-wracking to grasp an object, a baby is experiencing the assimilation process, while removing an obstacle and grasping an object, a baby now accommodates the scheme (Crain, 2005). During the organization process more complex intellectual structures atomic number 18 need with existing schemes by children. For instance, after the baby experienced and covered dropping movement, past he/she will relate it with throwing movement as rise up as understanding the concepts of near and far (Berk, 2003).The Sensorimotor Stage (Birth to 2 Years)Jean Piaget observed his children during their developmental flow and constructs the stages establish on his observations. His books mostly involve more instances from his dialogues and interactions with his children. The sensorimotor stage consists of six substages.(Santrock, 2004)That stage starts with the use of reflexes from birth to 1 month. Newborn reflexes take important place in sensorimotor stage. According to Piaget inborn reflexes are consisted from first schemes. He states that as children use inborn reflexes and experience assimilation, they desire to put them to active use (Crain, 2005).After one month, children begin to recur their chance behaviors and primary philippic reactions period (one to four months) starts. A baby experiences the thumb sucking by bringing her hand to her mouth by a chance, when the hand falls she wants to get it back and experiences umteen failures until she gets it back (Crain, 2005). At that example the child organizes the hand movement and sucking which is a kind of flier reaction. Piaget also states that children at that period signal the first efforts at imitation (Berk, 2003).The next substage is utility(prenominal) circular reactions and is observed between fourth and eighth months. Infants start to experience motor winments that encourage them to play worry to their environment. Infants begin to get enjoyment from the response of the environment to their attempts and they repeat their movements that get reaction from their surrounding (Santrock, 2004).Coordination of succorar y schemes substage takes place during eight to twelve months. At this stage infants begin to coordinate tow or more actions to achieve simple objectives. In sumition with an intentional purpose, babies try to imitate behaviors after watching a person. One may be up to(p) to observe a baby at this stage trying to stir with a spoon. In addition, a baby may begin to cry when she sees her mother exhausting her coat in order to stop her mother leaving (Berk, 2003).In substage 5, tertiary circular reactions (twelve to 18 months), children are enkindle with different outcomes. Piaget had observed one of his children hitting on a table at different rates in order to listen different sounds that he creates (Crain, 2005). It should be noted that all experiences are results of childrens congenital curiosity round the environment around them that Piaget empha sizes within his cognitive development theory.The last substage of the sensorimotor period is named as beginnings of horizon or internalization of schemes lasting from eighteen to twenty months. During that substage children chip in the capacity to remember the behaviors that are not present (deferred imitation). Their efforts on imitation also indicate collide with and they experiment with actions inside their heads. Besides, children can be observed to engage in diagnose-believe play during that period (Santrock, 2004).Object PermanencePiaget and umpteen tecs concluded that infants appreciate concepts of permanence objects. Up to four months, children do not make any attempt to an object leaving in front of their eyes. During secondary circular reactions stage children are more able to explore their surrounding and they have a repair sense of permanence of objects. At stage four children have the ability to find the hidden objects. If an adult takes a act as behind a box, the baby will look at the behind of the box and find the toy. During the stages cardinal and six children are able to follow di splacements and follow invisible shifts (Crain, 2005).Beginnings of compartmentalizationBefore the content of mental representation children are not able to categorize objects. During the first year of their life, children experience perceptual categorization. For example they can categorize the legs of an animal. Conceptual categorization begins with the end of first year they are now able to categorize similar characteristics and behaviors. Active categorization period starts with the beginning of the second year. It is stated that sorting objects into two classes can be observed in eighteen months babies. In the second year babies can collection two different kinds of objects without grasping them (Berk, 2003).When the observed milestones of research and the description of substages of Piaget are compared from birth to two years, two similarities and differences are seen. in that respect are points that seem to occur earlier than Piaget accepted such as categorization, deferr ed imitation, and figurative paradox solving. Those differences are explained otherwise from galore(postnominal) researchers. Some of the surveys indicate that some children born with different intellectual capacities and some of them with a set off limits which causes those differences. The latter argue the theory of Piaget in equipment casualty of biologic considerations.The Preoperational Stage (2 to 7 Years)Preoperational stage is lasting from two to seven ages in which the child is more fit while dealing with the environment. Although the backgrounding of child is still disorganized and illogical, that is the period that children begin to use symbols and quickly develop representation. One of the important symbols that indicate increase during that period is language (Santrock, 2004). Piaget believed that experience of internal images occurs before labeling words and he did not take language as an important tool in cognitive development of children. Berk (2003) argues that Piaget had misadjusted the role of language in early intellectual development. She proposes that conceptual abilities of children are exaltedly affected from the dialogues of children with adults. Moreover, there are many another(prenominal) psychologists that believe as children develop their language ability, they begin to think more logically.Children experience transductive reasoning during that stage which means shifting from one particular to another. Children place two un cerebrate situations into the same case as if they have a dealinghip. One of Piagets children had concluded that she hadnt had her nap yet so it wasnt afternoon (Piaget, 1924). Piaget (1924) explains that statement as an example of transductive reasoning, because the child did not look on the understanding that afternoons include many different events and having nap is only one of them.An important milestone of the increase in mental representation is make-believe play during preoperational stage. The differences in make-believe play between sensorimotor and preoperational stage can be clearly observed. By the middle of preoperational stage make-believe play of children indicate real life conditions. In addition, by preoperational stage children begin to engage in sociodramatic play, they coordinate variety of roles and story lines during their play.One of the criticized points of cognitive theory of Piaget is based on the belief of Piaget that play reflects childrens cognitive and social skills, notwithstanding there are many recent studies indicating the contri entirelyion of play on those skills. Especially during sociodramatic play, children interact with their peers longer and they are more cooperative. more psychologists believed the role of streng henceing of make believe play on a wide scat of mental abilities and logical reasoning (Berk, 2003).EgocentrismPiaget stated that children look at their surrounding from their own vantage point and they ignore perspective s of others. Three-mountains study is one of the famous observations of Piaget explaining egocentric behavior of children at preoperational stage. He had used a model of three mountains and taken a child for a walk around the model in order to project opportunity for the child to look at the model from different lot. Piaget had placed the child from one point of the model and placed a toy to another place. The child had been asked what he/she proverb while feel at the model and what the toy would be seen while looking at it. wholly the children could correctly explain what they were seeing, however children at preoperational stage gave the same answer with their own view (Crain, 2005).Studies emphasize on the relation between opportunism and social communication. Children at preoperational stage, according to Piaget, fail to recognize the take of their peers during verbal interaction (Rubin, 1973). As they look only from their own view, they are able to understand view of th e person inter acting with them. They think that they can be seen from everywhere, everybody see and hear them. An adult may observe a child at this period telling that nobody could see him/her while closing his/her eyes with hands.AnimismPiaget (1951) proposes that the child recognizes no limits between himself and the external world and it is expected that the child would see many nonliving and non acting things as living and conscious and he explains this phenomenon as animism. In his book The Childs Conception of World, 1951, he identifies the reason for him to use the term animisim. He accepts that animism was term used for primitive human beings and responds the review articles by telling that he had used that term as a generic term and emphasizing on the different types of animism in psychological origins (Piaget, 1951).Children at preoperational stage have a belief that objects are alive because they move and grow. For example, a child may tell that there are not any cars o n the road, because they are sleeping. Piaget depict animism inside four stages. Initially children accepted useful things as living. At this first stage lowly or damaged objects were not alive for them. At the second stage, moving objects, whether are moved by an external factor or by themselves, were considered as alive. In stage three, to be categorized as living, things should move by themselves. Lastly, at the fourth stage, adults know that plants and animals are living things only (Moriarty, 2005).IrreversibilityGoing through a series of steps and after changing burster is difficult for children at preoperational stage. Another well know experiment of Piaget indicates that problem in a way that there are children shown 16 boxes, 6 of which are yellow and 10 of which are red. When children are asked whether red boxes are more or boxes, children at this stage responds as red boxes and fails to be certified of that both yellow and red boxes are boxes. In his book The Childs C onception of World, 1951, Piaget gives examples about irreversibility. There are dialogues indicating their inability such as, asking a child about her sister, the child responds that she has a sister named A, then Piaget asks the child whether A has a sister or not, the child responds that A has not a sister. (Piaget, 1951)Inability to ConservePiaget propounds preoperational childs lack of preservation by applying experiments of liquids and number. He shows two same size glasses to the children and fulls the glasses with water. He asks children which of the water was more. All the children respond that they were adapted in amount. Then he puts the water in one of the glasses into a different size glass (wider or taller) and repeats his question. Children at preoperational stage tell that they are now different. They have not the capability to perceive that certain physical features of objects remain same, even their physical appearance changes (Santrock, 2004)Based on experiments of Piaget, at the beginning of seven children begin to give the correct answer to the conservation bear witnesss. Before that age children indicates at conservation but not totally achieve it. They give answers like one is more because it is taller and then change their answers the other one is more because it is wider.Besides, irreversibility of the child can be concluded based on the conservation of liquid experiment. The child cannot understand the end result as a reverse of the sure one.Jean Piaget also had thought about the failures of children from the linguistic point. Terms such as taller, more, wider takes cadence to be understood. He suggests ways to overcome that problem and tells adults to apply experiments by development different sentences and establishing questions by using different words within a particular case (Berk, 2003).Piaget experimented conservation of children also with using number. A row of egg cups and a bunch of eggs are given to children and they a re asked to take equal eggs to fill the cups. At the first half of preoperational stage children ignored the number of eggs in the cups and they made an equal length with the rows. Children at the second half of preoperational stage made one-to-one correspondence. However, those children failed in the second question. Piaget then brought together the eggs and asked which of them was more and they could not respond that they are still same. just about of them thought that the longer was more in number. Piaget explains that phenomenon as been influenced by their sudden perceptions than by logic (Crain, 2005).CategorizationIn coordinating and extending knowledge in cognitive development theory of Piaget, categorization takes an important place. Researchers state that children begin to make similar categorizations and form some categories with the age of two such as animals, plants, and vehicles (Scholnick, Nelson, Gelman, Miller, 2008). Things or objects in all of those categories ha ve differences in perceptual characteristics, so a conflict emerges with the main psyche of Piaget that childrens reasoning is governed by the way they see and objects appear. By the age of three children become able to make trenchantions between basic and general categories such as furniture versus tables. However, children at preoperational stage are not able to organize objects into classes and subclasses based on their basic similarities and differences (Meadows, 1986).It is argued that Piaget was partly right and partly wrong with his closes of preoperational stage. Researchers give simplified tasks for children and preschoolers indicate the beginnings of logical operations, however there are important differences between their reasoning and children at school age. Difficulties were observed in conservation establishs, three mountains tests and appearance-reality tests. As opposed to perceptual approaches to solve problems, children intrust on increasing effective mental. In order to give an example, research indicate that children who have not the capability to use counting while comparing two groups of objects, do not also conserves number. They begin to find effective solution ways for proms with more objects when they become undetermined of counting. Piaget states that, as many other psychologists accept, children move through variety of stages of understating, although they do not totally handle conservation up to school years.The Concrete Operational Stage (7 to 11 years)The beginning of cover operational stage is supply by logical reasoning. That period is accepted as a major turning point in cognitive development. The time that a child begins to mentally resemble an adult starts with attaining concrete operational stage. A school-aged childs cognitive performance is more evident in terms of concrete operations. For example an eight year of childs response to conservation of liquid test is that the amount has not changed and they are still same. This explanation of the child also indicates the reversibility of the reasoning that the child has now the capability to understand that the reverse is the same with the original. Their ability to achieve conservation tasks indicate their logical thinking (Richardson, 2003).Between ages seven and ten, children take attention on dealing between a general and two specific categories within the same case. Their awareness of classification is acquire developed. In middle childhood period many children are interested in collections such as coins, rocks, and stamps which show their level of classification.Children in the concrete operational stage are fairly good at the use of inductive reasoning which involves going from a specific experience to a general principle, whereas children at this age have difficulty with using deductive reasoning. Deductive reasoning involves using a general principle to determine the outcome of a specific event.SeriationSeriation refers to the capabi lity to arrange objects along a three-figure dimension. Ordering raise ups from different lengths is one of Piagets tests. Children at five and six years could create series but they made many errors. However, children at concrete operation stage, by scratch the shortest and putting the next, indicate a perfect arrangement with an orderly plan.Children between ages seven to eleven have an ability called transitive inference which refers to mentally arrangement of items. Piaget had showed children three stick with different colors. Children had observed that first stick is longer than second stick and second stick is longer than third stick and they had made the inference that first stick is longer than third stick. Children at concrete operation stage mentally integrate three relations at once. On the other hand, preoperational stage children can achieve analogical reasoning by cost increase of an adult (Meadows, 1989).Spatial ReasoningThe ability of understanding billet is mor e developed in concrete operational children. School age children begin to give directions as they gain more advanced space understanding. Children at five or six years indicate difference while representing an object on a persons, in front of them, left or right. Starting from seven age children achieve mental rotations, that is, they can recognize their own frame and of a person in a different direction (Santrock, 2004).While drawing large crustal plate areas their performance indicate important changes representing their development of cognitive skills. In the middle period of concrete operational stage children achieve to place notes showing the location of objects in their classroom. Their capability to use a rotated map is improving and also their map drawing become more accurate.Research indicate that concrete operational thinking has a limitation that while children are reasoning in a logical manner, they are always dealing with concrete knowledge. With abstract information , they cannot catch the achievement (Crain, 2005). When they are given ideas that do not appear in the real world they are not able to indicate the same responds with concrete ones. That can be easily observed from transitional inference that when children are shown the sticks they give the correct responds whether which one is littlerer or taller than the others. However, when the questions turned to a more concrete version such as As hair is longer than B, B2 hair is longer than C. Who has the long-lasting hair?, up to eleven years children cannot give the right answer (Richardson, 2003).Jean Piaget believed the importance of recondite and appropriate environment for the speed of concrete operational cognitive skills of children. Many research done in small towns or villages represent the lack of generative environment and its consequences on children. Even the easiest conservation tasks are not easily achieved until the age of eleven. On the contrary, many children living in big cities, especially westward nations, have many opportunities in terms of environment and from very young periods of childhood they meet with rich and varied materials. Hence, they easily understand the conservation tasks. The same comparison had done between children receiving a sea captain early childhood education and the ones that spend their early period at home or path. The conclusions indicate better results of the children continuing a preschool program (Berk, 2003).A survey had been made between Brazilian street vendors and Brazilian economically advantaged children on informal version and Piagetian version of class inclusion test of Piaget. In the informal one, the researcher asks children for the price of two chewing gums which are different. In the Piagetian one, four units of one type (mint) chewing gum and two units of the other (strawberry) one is set aside and the researcher asks In which one do you get more money, whether you sell me the chewing gum with mint or all the chewing gum? The success of two groups children indicates difference that on the informal version of the conservation task street vendors performed better and on the other version economically advantaged children performed well (Ceci Roazzi 1994, as cited in Berk 2003).The Formal Operational Stage (11 Years and Older)The child in sensorimotor period can do things and play with objects, the child in sensorimotor period and concrete operational period can think and make reasoning about those objects. Thinking about propositions and relations different from objects and events is another period. Now human beings are bale to make coordinations of coordinations, in other words, they can combine two different groupings of concrete operations within reversibility by reciprocity (Richardson, 2003). In formal operational stage adolescents do not require concrete things as objects of thought.Adolescents at this stage are capable of deductive reasoning. In order to overcome a proble m, they start more generally and think evaluate all possible factors and then proceed to more specific predictions.Piaget used pendulum problem in that stage in which he had showed many strings with different lengths, objects differing in weight, and in order to hang the strings a bar to children. He asked the factors that will affect the speed with which a pendulum swings. Children in concrete operational level made an unsystematic experimentation. The influences of each variable could not be distinguished by them. Adolescents of formal operational stage evidence the factors that have the possibility to affect the speed into four groups strings length, objects weight, the level of force when pushing the object, and how high the object is raised before it is released. At the end they come up to a right conclusion that the length of the string affects the speed (Berk, 2003). They work systematically in terms of all possibilities as a scientist.Cognitive development theory of Piaget is mostly depending on mathematical and scientific reasoning however he has some speculations on social life of formal operational stage adolescents. Piaget thinks that children between seven and ten live in here and now, whereas at the formal operational stage they start to think about long-term periods. They handle abstract ideas such as justice and love and they start to construct theories for better living world (Inhelder Piaget, 1955, as cited in Crain 2005).Piaget identifies egocentrism again and believes that a new kind of egocentrism is carried by utopian during that period. Piaget takes into account and repeat the forms of egocentrism from beginning to later periodsAt the beginning, sensorimotor period, infants have no idea bout the environment around them and they are egocentric, still the objects that they cannot see have no existence on their own.At the next level, preoperational thought, children enter a representative world in terms of language and symbols. They indic ate difficulty more than their own view. After a time they become aware of other perspectives and they start to think about concrete objects.Finally, adolescents, formal operational period, meet with a world full of problems and possibilities. Adolescents feel unlimited power in themselves for their thoughts and egocentrism reappears and they are now not able to distinguish self and others abstract views.(Crain, 2005). Teenagers feel that they are at the centre of their environment and they assume that everyone is looking at them. They spend many hours in front of a mirror feeling anxiety for the peoples criticism about them. This is called as imaginary audience. Then with having a thought that everyone is notice them, they start to feel themselves special and unique and this is named as personal fable.Propositional ThoughtAdolescents do not need to refer real-life conditions and they are capable of evaluating the logic of propositions. Previously, they were evaluating statements w hen they were concrete in the real world. Piaget did not give an important role for language in childhood period, but he thought that it was more significant during adolescence. So, verbal reasoning about abstract situations and events is one of the important concepts in formal operational thought. Adolescents thoughts and opinions about abstract conditions and their capacity while expressing those views are demonstration of their propositional thought. Theory and research of Piaget have greatly changed the view at infant action and thought and provided a new way of looking at children (Fischer Heneke, 1996).Evaluation of Piagets TheoryJean Piaget has contributed to the field of child development than many other theorists, as many things he expressed were the first for his time. He was the first to tell that children were different from adults and they are curious and active learners (Berk, 2003). Piaget is the founder of discipline of cognitive development. He was interested in re asoning of children and that ensured the idea of that field would be developmental and should be different from the study of adult thinking. Piagets considerations through the idea about active roles of children on their development can be seen as distinct recently, but it was innovative for his time (Shaffer Kipp, 2001).Piaget tried to explain the process development and from that part he was one of the firsts who not only described the theory. Piagets explanations had a major impact about social and emotional developmental theories. By asking many questions about intellectual development of children and creating thousands of resource for that field, he had important contributions for the educators and researchers (Shaffer Kipp, 2001).Many studies criticize Piaget for underestimating mental capabilities of children due to his concern with defining the underlying cognitive structure which considered performance of children on a conservation task. Piaget is criticized as assuming that child making mistake in one problem will lack the underlying concepts he was testing. Validity of this assumption is refuted by stating that performance of a child may be influenced by many other factors instead of lack in cognitive skills (Scholnick et all, 2008).Researchers have challenged stage theory of Piaget by asking if intellectual development is at all stagelike. It is proposed that transitions in mental gradually occur and the consistency of a performance of a child in a test would be very little. For instance, problems of concrete operations and problems of formal operations may be achieved in different orders by many children with different abilities. Many theorists believe that cognitive functions are so complex that would be evaluated within a stage manner. For instance, a nine years old child may do well on verbal reasoning tests if he likes to deal with word puzzles and play verbal games, however he may not do well in mathematical reasoning (Shaffer Kipp, 2001) .Besides, Piaget ignores cultural differences while claiming that his stages move on the same sequence in all cultures. Children from different cultures are educated differently and a s environment has a direct interaction with cognitive development, cultural differences should be taken into account to get a valid conclusion.Implications for EducationPiaget did influence much from Montessori and Rousseau and emphasized on the importance of active learning of children. He believed that learning is a process of active discovery and should be related with the level of the child. The role of educator at giving the child appropriate and rich environment based on childs interests and modes of learning takes significant place in terms of encouraging innate curiosity of children. An environment with rich, variety, and interesting materials will encourage children to discover and become active learners (Crain, 2005).According to Piaget educators should not teach children in a direct way, chi ldren should be allowed to construct their own knowledge through experience. Children should be given the opportunities to make mistakes and learn through those mistakes, and look for solution ways. Piaget helps educators to be aware of that, meaningful interactions of children with environment and real activities will enable children to learn. All of those factors are the roots of constructivism which is aApoptosis Techniques for metre and ObservingApoptosis Techniques for Measuring and ObservingINTRODUCTIONThe number of cells within an organism is tightly regulated- not simply by peremptory the rate of cell division, but also by controlling the rate of cell final stage. If cells are no longer needed, they commit suicide by activating an intracellular death program. (Liao, 2008)Apoptosis, is defined by distinct morphological and biochemical changes mediated by a family of cysteine aspartic acid-specific proteases (caspases), which are expressed as inactive precursors or zymogen s (pro-caspases) and are proteolytically processed to an active state following an apoptotic stimulus. (Liao, 2008)The aim of this paper is to discuss the techniques for measuring and observing caspase-mediated cell death, whilst commenting on any limitations reported to date.DISCUSSIONThe intracellular machinery responsible for apoptosisKerr, Wylie and Currie (1972) observed controlled cell death that was distinct from uncontrolled necrotic death. They noticed a characteristic, identical sequence of events in many different types of cells and published their observations in 1972 and used the term apoptosis. (Alberts, 2007 Cancer, 1972)In apoptosis, cell shrinkage and tissue layer ruffling (blebbing) occur, and the cell disintegrates into small membrane-bound apoptopic bodies. Inside the cell chromatin condensation and nuclear fragmentation occur, which are accompanied by crack-up of the DNA into regular size fragments. On the surface of the cell lipids are rearranged in the bilay er of the plasma membrane with the lipid phosphatidylserine becoming exposed to the outside. (Hancock, 2005)Measuring and observing apoptosisApoptosis is essentially a morphological state arrived at by a whole variety of different biochemical streets. Some routes may result in the expression or loss of an antigen but there is no assurance that the same biochemical alterations occur in every cell. The identification of protein markers permits ready detection by conventional immunohistochemistry, which facilitates fast and confident assessment of apoptosis. (Harrison, 1996)The in situ end labelling and in situ nick translation techniques rely on the straw man of DNA strand breaks characteristic of the nuclear implosion and fragmentation seen in apoptosis. Thus, labelling with biotinylated nucleotides and subsequent immunodetection can be used to identity sensitively cells with strand breakage. (Harrison, 1996)Activation of the caspase-3 pathway is a hallmark of apoptosis and can b e used in cellular assays to quantify activators and inhibitors of the death cascade by molecular identification. The response is both time and concentration dependent suggesting that multiple pathways play a role in triggering the caspase-3 activation. (Bio Medicine, 2008)TUNEL assay is a technique used for observing activation of the caspase-3 pathway by biochemical verification. The enzyme TdT is able to add nucleotides to the ends of DNA fragments most commonly, biotin-labelled nucleotides (usually dUTP) are added. The biotinylated DNA can be detected by using streptavidin, which binds to biotin, match to enzymes that convert a colourless substrate into a coloured insoluble product. Cells stained in this way can be detected by light microscopy. (Janeway, 2001)The cells with unsuccessful DNA repair may undergo apoptosis in some cases, chimerical positive labelling may result. TUNEL staining has been reported to give false positive staining in the kidneys of nude and BALB/c mice . In situ localization of activated or cleaved caspase-3 is starting to replace TUNEL as the molecular verification of the apoptotic process. (Hughes, 2007)Extrinsic and Intrinsic signal activationThe extrinsic pathway is initiated by ligation of transmembrane death receptors (DR) with their respective to activate membrane-proximal caspases, which in turn cleave and activate effector caspases. A killer lymphocyte carrying the Fas ligand binds and activates Fas proteins on the surface of the target cell. Adaptor proteins bind to the intracellular region of aggregated Fas proteins, causing the aggregation of procaspase-8 molecules. These then cleave one another to initiate the caspase cascade. This pathway can be regulated by c-FLIP, which inhibits upstream initiator caspases, and inhibitor of apoptosis proteins (IAPs), which affect both initiator and effector caspases. (Alberts, 2007 Liao, 2008)The intrinsic pathway requires disruption of the mitochondrial membrane and the release o f mitochondrial proteins, such as cytochrome c. Cytochrome c, which binds to and causes the aggregation of the adaptor protein Apaf-1. Apaf-1 binds and aggregates procaspase-9 molecules, which leads to the cleavage of these molecules and the triggering of a caspase cascade. (Alberts, 2007, Liao, 2008)The primary regulatory step for mitochondrial-mediated caspase activation might be at the level of cytochrome c release. The known regulators of cytochrome c release are Bcl-2 family proteins. Members of this family are divided into two main groups, the anti-apoptotic proteins with Bcl-2 and Bcl-Xl as archetypes and the pro-apoptotic proteins such as Bax. The ratio between these different proteins determines the sensitivity of the cell to apoptosis antiapoptotic proteins inhibit apoptosis by counteracting Bax and Bak, and BH3-only proteins either trigger apoptosis through direct interaction with Bax or sensitize cells to death by inactivating Bcl-2 or Bcl-Xl. (Cartron, 2003 Liao, 2008) RT-PCR primer sets that are specific for genes involved in inducing and regulating the apoptotic response. These primers are specific for genes encryption proteins from the Fas and Fas ligand, Bcl-2 and ICE protein families. The RT-PCR primer sets for studying the apoptotic response are designed to meet several criteria. The primers sets, based on known genomic sequences, amplify a region that spans at least one intron. To distinguish the amplification products from genomic sequences, which are longer than the cDNA products, the primer sets amplify PCR products that are 400 bp to 650 bp in length. The primers are synthesized as 18- to 27-mer oligonucleotides. distributively set of primers amplifies only a specific target. (Biomedicine, 2008)Direct measurement using fluorescence resonance energy ecstasy has shown the interaction between Bax and Bcl-2. (Cartron, 2003)Light microscopy and electron microscopy, including staining, are frequently used to observe the morphological change s of the cells undergoing apoptosis. There are many staining protocols used for identification of apoptotic cells, and the choice varies subject to the laboratory and the tissue being studied. (Wang, 2008)The advantages of Near-Field Scanning Optical Microscope (NSOM) are observing in normal environment, observing in nanometre scale resolution, and observing in non-contact mode. (Wang, 2008)CONCLUSIONUsing morphology, biochemical or molecular methods to identify, localize and quantify apoptosis gives strength to many research studies. The measurement of the level of apoptosis within tissue sections represents only a snapshot of one time point during a developmental, physiological or pathological process. Given the rapid nature of apoptosis and its cryptic nature in tissue sections, these measurements may often be underestimates of the authentic extent of apoptosis. The ability to measure the levels of apoptosis within living organisms, including humans, non-invasively at repeated i ntervals over time would facilitate the analysis of apoptosis in many organs as well as tumours. (Hughes, 2007)These investigatory techniques have broadened the understanding of disease development and will undoubtedly present real opportunities for novel remedy intervention. For example, the gene-driven nature of apoptosis and its modulation by various controlling molecules have provided a basis to develop therapies for selectively protecting or deleting cell populations. (Hughes, 2007)
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