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Capital Punishment

In: Computers and Technology

Submitted By siddiqui1990
Words 4651
Pages 19
Abstract:

Neuropathic pain is chronically increased pain and heat sensitivity resulting from damage to the central or peripheral nervous system. Neuropathic pain is constant, and difficult to treat, because the pain persists even after the injury has healed. Current drug treatments are ineffective, have short durations, and have unpleasant side effects. The SIP-30 gene is a candidate gene for involvement in neuropathic pain. When neuropathic pain is present, SIP-30 levels rise indicating some cause and effect relationship. Transposons are mobile genetic elements that move around within a genome and integrate randomly, in a mechanism similar to some viruses. The PiggyBac transposon, derived from the cabbage looper moth Trichoplusa ni, is a particularly useful transposon, with many features that make it an ideal vector for gene therapy research. Plasmid vectors will be constructed, one carrying the SIP-30 gene, and another carrying antisense SIP-30 in the PiggyBac vector, in order to deliver the genes into mouse nerve cells. This is hypothesized to increase and decrease the expression of neuropathic pain respectively. The gene will be delivered through intrathecal injections into the mouse intrathecal fluid of the spinal cord. If successful, this research can have serious implications for future human gene therapy to treat neurological disorders, using the PiggyBac vector as the gene delivery system.

Introduction:
Features of the PiggyBac Transposon: Transposons are mobile genetic elements that move around within a genome and integrate randomly, in a mechanism similar to some viruses. The PiggyBac transposon, derived from the cabbage looper moth Trichoplusa ni, is a particularly useful transposon, with many features that make it an ideal vector for gene therapy research. The PiggyBac transposon can carry very large sequences; sequences can be as long as 9.1 kb without any noticeable change in efficiency, and sequences as large as 14.3kb have been observed to transpose in mammalian cells. Because the PiggyBac transposon integrates randomly, it integrates into introns and other non-coding sequences 97% of the time, and therefore does not usually disrupt functional genes. As a result, PiggyBac has widespread integration throughout the genome. Furthermore, PiggyBac does not produce the excision footprint mutation that is common to many transposons. Collectively, these features make PiggyBac an ideal vector system for gene therapy research, especially in terminally differentiated cells, such as neurons in the Central Nervous System.

Application to Neuropathic Pain: In my particular project, I will use the PiggyBac vector to test its possible use in the gene therapy of neuropathic pain. Neuropathic pain is chronically increased pain and heat sensitivity resulting from damage to the central or peripheral nervous system. My study will focus on sciatic nerve damage-induced neuropathic pain, which results in gene expression malfunction in the spinal cord.
Neuropathic pain is constant, and difficult to treat, because the pain persists even after the injury has healed. Current drug treatments are ineffective, have short durations, and have unpleasant side effects. The SIP-30 gene is a candidate gene for involvement in neuropathic pain. When neuropathic pain is present, SIP-30 levels rise indicating some cause and effect relationship. SIP-30 is hypothesized to function in the regulation of vesicle exocytosis, though its cellular function remains unknown. I will use the SIP-30 gene in this study in order to target neuropathic pain in a mouse model. This project focuses on the treatment of neuropathic pain, which results from peripheral nerve-induced molecular malfunction in the spinal cord. Since the cells involved in the spinal cord are part of the central nervous system, they do not replicate. This makes gene therapy treatments more difficult, since the cells do not replicate and amplify the genes that they are treated with. The PiggyBac transposon integrates into the host cell genome, therefore allowing itself and any gene it carries to be expressed at moderate but stable levels. This makes it possible for SIP-30 to be expressed to test its role in mediating neuropathic pain. It is our hypothesis that this will allow PiggyBac to be a particularly effective vector for gene expression in nerve cells.

Materials and Methods:
Plasmid Vector The plasmid vector, which will be used in the Intrathecal injections, will consist of the PiggyBac backbone and the beta-galactosidase gene. The PiggyBac backbone is 5.2kb long, and has the BamHI and HindIII restriction sites, which will be important when it comes time to ligate the backbone to the lac Z gene. In addition, the PiggyBac backbone has a sequence for the Act-RFP gene which codes for the red florescent protein marker. This gene will be removed and replaced by the lac Z gene, which will be used as the reporter gene instead. The lac Z gene comes from the beta-galactosidase vector. This vector is 4.1kb long and contains the EcoRI and BamHI restriction sites around the lac Z gene. Subsequently, the SIP 30 gene and RNAi will replace the lac Z gene.

Transformation of cells In order to amplify the PiggyBac plasmids, the DNA must be transformed into bacteria, which will replicate the plasmid DNA through their own replication. The cells used in the transformation were NEB B10Beta E. coli cells. These cells were thawed on ice for 10 minutes. The process is done gradually, in order to protect the transformation efficiency. The undiluted PiggyBac plasmid was added to 50 µl of the cells at volumes ranging from 2 µl to 10µl. The cells were then incubated on ice for 30 minutes to absorb the DNA. The cells were heat shocked for 30 seconds at 42 degrees Celsius, and then allowed to recover on ice for 1-2 minutes. 800 µl of SOC medium was added to these cells, followed by a 37 degree Celsius water bath for 1 minute. These cells were then incubated at 37 degrees Celsius with gentle shaking for 45 minutes. When these cells were done incubating, 200 µl of them were plated on ampicillin and SOB medium Petri dishes and placed in the 37 degree Celsius incubator overnight. The following morning, the plates were taken out of the incubator, the colonies were counted, and the plates were stored in the 4 degree Celsius freezer for future use.

Plasmid Minipreparation by the Alkaline Lysis Method Once colonies for all the PiggyBac plasmids were obtained, the next step was to isolate the plasmid DNA from the bacterial cells. This is done through a miniprep. The first method I used for the miniprep was the Alkaline Lysis method. The first step is to pick a colony from the plates, and grow it in Lb medium with ampicillin, and incubate it overnight at 37 degrees Celsius with vigorous shaking. The following morning, 1000 µl of these cultures were placed in an Eppendorf tube, which was placed in the microcentrifuge and spun for 1 minute at the maximum speed, 15,000 rpm. Afterwards, the supernatant was discarded, and 250 µl of the Qiagen Minikit P1 buffer was added to the cell pellet. This buffer resuspends the cells. These cells are then lysed by adding 250 µl of Buffer P2. When the cells are lysed, the solution turns to a dark blue color. The solution is then neutralized using 350 µl of Buffer N3. This causes a white precipitate to form. These cells are then microfuged for 5 minutes at maximum speed. This forms a pellet of cell debris, and a supernatant containing the DNA. This DNA can late be purified by several methods.

Plasmid Minipreparation by Boiling Method Alternatively, the overnight culture containing the PiggyBac plasmids can be isolated through the boiling method. The first step is to resuspend the pellet in 100 µl of STELT buffer (95 % STET Buffer and 5% Lysozyme). These cells are then boiled for 60 seconds in a water bath. Following this, the cells are microfuged at maximum speed for 10 minutes. The supernatant is then transferred to 80 µl isopropanol, followed by microfuging it again for 10 minutes. The pellet can then be used for DNA extraction.

DNA extraction using Qiagen minikit Spin Columns In order to extract the DNA from the solution, the DNA supernatant were added to the spin columns. The spin columns were then centrifuged for 1 minute at maximum speed, and the flow-through was discarded. Afterwards, 750 µl of the PE Buffer was added to the spin columns, which were centrifuged again for 1 minute at maximum speed. The flow-through was drained, and then the spin column was spun again at maximum speed for 1 minute to remove any residual wash. Following this, 60 µl of EB buffer was added to the column to elute the DNA. The solution was incubated at room temperature for 1 minute before it was centrifuged at maximum speed for 1 minute. The flow through is the final DNA solution, which was stored in 4 degrees Celsius for future use.

Ethanol Precipitation of DNA The final supernatant from the Alkaline Lysis miniprep can be treated chemically to extract the DNA. 400 µl of the supernatant was mixed with 1/10 its volume of NaAC (3M) pH 5.2. Then, twice the volume of 95% ethanol is added, and the solution was mixed. The solution was chilled in -20 degrees Celsius for 45 minutes. Subsequently, the solution was microfuged at maximum speed for 15 minutes at 4 degrees Celsius. The liquid was then removed, and replaced by an equal volume of 70% ethanol. The ethanol was then removed by pipette, and the pellet was left to dry for 2 hours. Finally, the pellet was dissolved in 20-50 µl of TE buffer with RNase A.

Phenol Chloroform and Ethanol Precipitation Another method of DNA Precipation is using Phenol Chloroform and Ethanol. First, the Phenol Chloroform bottle was shook, then 1.5 ml was transferred to each tube. This tube was centrifuged for 1 minute at maximum speed and room temperature. The bottom layer of the centrifuged solution was transferred to a fresh tube. The aqueous and organic phases were vortexed, then centrifuged for 2 minutes at 4 degrees Celsius at maximum speed. An equal volume of the upper aqueous layer was pippeted and transferred to each plasmid sample. Twice the volume of 100% ethanol was added to the solution, which was then incubated for 5 minutes at room temperature. The samples were then centrifuged for 5 minutes at 4 degrees Celsius and maximum speed. The supernatant was carefully removed by pippetting it twice. 1ml 70% Ethanol was added to each sample. The samples were again centrifuged, this time for 2 minutes at maximum speed and 4 degrees Celsius. The supernatant was removed, and the DNA pellet was mixed with 50µl TE + RNase A in each sample. The samples were stored and frozen in -20 degrees Celsius.

Restriction Digestion A restriction digestion was performed on the purified DNA to test if the correct size plasmids were present, meaning the correct DNA was isolated without too much contamination. First, a digestion mix was made. The digestion mix is made up of 15 µl Water, 2 µl 10 X Enzyme buffer, 2 µl plasmid DNA, and finally 1 µl total volume of the appropriate enzyme, or .5 µl of two appropriate enzymes if a double digestion was necessary. Each Plasmid requires a different enzyme and buffer. PB[Act-RFP]DS requires HindIII, BamHI, and NEB buffer 2. PB[SV40-neo] requires AscI and NEB buffer 4. PB[PGK-neo] requires EcoRI and NEB EcoRI buffer. Act-PBase requires HindIII, XbaI and NEB buffer 2. CMV-PBase requires HindIII, EcoRI, and NEB buffer 2. After the digestion mixes were made, they were mixed, then incubated for 1 hour at 37 degrees Celsius. Another mix was made for all the plasmids without the enzymes, called the uncut mix. This was used as a means of comparison when the gel electrophoresis was performed.

Agarose Gel Electrophoresis The first step in performing agarose gel electrophoresis is to prepare the gel. The gel was made by mixing 130 ml TAE buffer with 1.3g agarose. This solution was microwaved for 1-2 minutes until the agarose was fully dissolved. 9 µl Ethidium Bromide was added to allow the DNA to fluoresce under Ultra Violet light. Next, the gel was poured into a gel box, and a comb was inserted to form the wells. The gel was washed in TAE buffer. The gel was left to sit for 20-30 minutes for the gel to solidify and the wells to form. 2 µl loading dye was added to each DNA sample. The samples were loaded into the wells, along with the DNA ladder. 100 volts was applied to the gel for 45 minutes, and the results were recorded.

Objectives:
Construction of Positive Control Vector: The first step of this study is to construct the vector that will deliver the gene of interest. Before the SIP-30 is used, a test vector must be constructed to determine if PiggyBac can in fact transpose effectively into cells of the central nervous system as hypothesized. The pSV-(-Galactosidase plasmid contains the Lac Z gene, which dyes blue in the presence of the X-gal substrate and serves as a very sensitive marker for the expression of the transfected gene. The Lac Z gene will be isolated from the pSV-(-Galactosidase plasmid and inserted into the PiggyBac backbone in order to make the complete test vector. Plasmid DNA will be isolated by preparation from transformed E. coli cells. The isolated DNA will be subjected to restriction endonuclease digestion and the digested DNA will be separated by agarose gel electrophoresis. The desired band of DNA, which carries the Lac Z gene, will be isolated from the gel and will be used for insertion into the PiggyBac backbone vector. Parallel to the Lac Z gene isolation, the PiggyBac vector DNA will also be prepared and linearized with the appropriate restriction endonucleases. These two pieces of DNA, the DNA insert and the PiggyBac backbone, will be joined together using DNA ligase to make the final test vector.

Results:
Preparation of Competent Cells: In order to perform the transformation of cells, it is necessary that the cells to be transformed are made competent. This was done with treatment with the Inoue Buffer. This buffer consists of 800 ml Water, 10.88 g MnCl2 tetrahydride, 2.20 g CaCl2 dihydride, 18.65 g KCl, and 20 ml PIPES solution. The PIPES solution was prepared by dissolving 15.1 g PIPES in 80 ml Water, then adjusting the pH with 5M KOH, then adding 20 ml Water. Water was added to the Inoue Buffer until the total volume was 1 liter. This buffer was sterilized through a .45 µm Nalgene Filter. The filtered solution was divided and stored at -20 degrees Celsius for future use. The cells that were going to be made competent were plated at 37 degrees Celsius for 16-20 hours. The following day, I picked a colony from the plate and transferred it to 25 ml LB medium. This was incubated at 37 degrees Celsius for 6-8 hours with vigorous shaking. This was then divided into three flasks: the first was filled with 10ml, the second was filled with 4ml, and flask 3 was filled with 2ml. These flasks were incubated overnight at room temperature with moderate shaking. The OD600 was measured to determine when the growth was sufficient. Once the OD600 reached 0.55, the culture was transferred to an ice water bath for 10 minutes. The cells were then centrifuged at 3900 rpm at 4 degrees Celsius for 10 minutes. The media supernatant was discarded, and the cells were resuspended in 20 ml of the Inoue Buffer. 1.5 ml DMSO was added, and then the solution was stored in ice for 10 minutes. The solution was then divided into aliquots of 50-100 µl. these aliquots were snap frozen in liquid nitrogen, and then stored in -80 degrees Celsius until they were ready for use. A test transformation was performed to calculate the transformation efficiency of the competent cells. This was performed by transforming the DNA at concentrations varying ten-fold from .1 pg/µl to .1 ng/µl. In addition, commercial B10-Beta commercial competent cells were used as a positive control, and cells with no DNA were used as a negative control. By counting the colonies formed from the transformation, the transformation efficiency can be calculated. The DNA used for the transformation was the plasmid PSV/CMV. The colonies were counted and the results were as follows:

|Plate / Number of Colonies |Transformation Efficiency |
|Commercial.1 pg/µl – 6 colonies |3 x 107 cfu/µg |
|1 pg/µl – 6 colonies |3 x 106 cfu/µg |
|.01 ng/µl – 16 colonies |8 x 105 cfu/µg |
|.1 ng/µl – 84 colonies |4.2 x 105 cfu/µg |

Results of Transformation 7/30/10: The average of the transformation efficiencies was calculated as 5.6 x 106 cfu/µg.

Transformation efficiency was calculated by dividing the number of colonies by the amount of DNA used for the transformation. By taking the average of all the transformation efficiencies, the transformation efficiency was calculated as 5.6 x 106 colony forming units per microgram of DNA.

Transformation of PiggyBac Plasmids: Once the transformation efficiency was known, the competent cells were used to transform the PiggyBac vectors for amplification. At first, since the concentration of the PiggyBac vectors was unknown, they were serially diluted to concentrations of 1:10 and 1:100 before the transformation. However, when the transformation was performed, none of the plates showed any growth, implying that the concentration was very low. The DNA concentrations of the undiluted samples were about .1 pg/µl. The transformations were redone using 4 µl DNA, resulting in colonies 1 colony on one plate, and 2 colonies on another plate. The transformation was then redone using 6 µl DNA, resulting in 1 colony on one plate, 4 colonies on another plate, and 57 colonies on a third plate, while the rest still had no growth. For the plasmids that still produced no colonies, the transformation was redone while plating 400 µl of the culture instead of 200 µl. This resulted in a few colonies on each plate, and as a result, all the plates had a few colonies and were ready for plasmid minipreparation.

Plasmid Minipreparation of PiggyBac: Once colonies were isolated for all of the PiggyBac plasmids, it was time to isolate the DNA for amplification. The first method used was the alkaline lysis in compound with spin column extraction. Following this, the nanodrop was used to test the concentration of the plasmids, which ranged from 13.3 ng/µl to 27.6 ng/µl. this yield was not nearly sufficient to perform a restriction digest, so the miniprep was redone. The nanodrop showed that the miniprep yielded similar results the second time. In order to achieve a better yield, I attempted the boiling lysis method for the miniprep. Unfortunately, this method yielded no pellet after the isopropanol was added. The alkaline lysis was then reattempted, however, ethanol precipitation was used instead of the spin columns this time. When the concentration was measured using the nanodrop, at first glance it appeared to have a very strong yield. The concentrations ranged from 135.5 ng/µl to 708.1 ng/µl .
Restriction Digestion and Gel Electrophoresis: The next step was to perform a restriction digestion to determine if the correct size bands would show up in the gel electrophoresis. The gel electrophoresis was a success, since the positive control, the DNA ladders, ran correctly. However, there were no bands in any of the lanes with the PiggyBac plasmids.

[pic]
Results of Gel Electrophoresis 11/18/10: The bands in the marker lanes show that the gel was successful. However, the lack of bands in the Cut and Uncut plasmid lanes suggest a lack of sufficient DNA.

Curiously, there was some minor fluorescence in the wells of a few of the PiggyBac lanes. These results implied that there might not be enough DNA in the PiggyBac samples, so I went back to the nanodrop to see if it could provide any insight. The graph of the UV absorption from the nanodrop shows only a slight hump in the 260-280nm range, when there should be a large peak if there is a large quantity of DNA.

[pic]
Results of Nanodrop 12/10/10: The lack of a peak at around 260 nm suggest that the UV absorption was due to contaminants, and not the desired plasmid DNA.

Plasmid Minipreparation by Alkaline Lysis Method For improved results, the minipreparation was performed using freshly made reagents. This was done with the intention to maximize the yields. First, the overnight cultures containing the desired plasmids were centrifuged to remove the broth supernatant. The cell pellet of each sample was resuspended in 100µl of resuspension Buffer I. Buffer I was prepared with 50mM Glucose, 25mM Tris-Cl (pH 8.0), and 10mM EDTA (pH 8.0). The combined solution was autoclaved for 15 minutes at 15psi. Next, 200µl of Alkaline lysis Buffer II was added to each sample. Buffer II was prepared with 20µl 10M NaOH, 100µl 10% SDS and 880µl H2O. The final concentration was .2M NaOH and 1% SDS. Once Buffer II was added, the samples were gently inverted to mix the solution without damaging the DNA. Next, 150µl Neutralization Buffer III was added to neutralize the samples. Buffer III contains 60ml 5M Potassium Acetate, 11.5ml Glacial Acetic Acid, and 28.5 ml H2O. The tubes were gently inverted to mix, and then incubate on ice for 5 minutes.
Second Plasmid Preparation: Once it was determined that there was not sufficient DNA in the samples, the plasmid minipreparation was attempted again to achieve a better yield. This time, a new method was selected to improve the results. We performed the Alkaline Lysis, this time using fresh reagents. In addition, a new method of DNA precipitation was needed, since the previous methods failed to give sufficient yields. This time, we used the phenol chloroform and ethanol precipitation. The results this time were much better than previously achieved. The DNA concentrations improved by over ten-fold for most of the plasmids. The concentrations as measured by the nanodrop were the following:
| |260/280 |Concentration |
|PB1 |2.04 |1.24µg/µl |
|PB2 |2.04 |1.64µg/µl |
|PB3 |1.91 |0.71µg/µl |
|PB4 |2.02 |1.46µg/µl |
|PB5 |1.95 |0.39µg/µl |

Results of Nanodrop 2/8/11: These nanodrop results suggest a large amount of DNA present. The DNA appears to be sufficient for a gel electrophoresis.

Second Restriction Digestion and Gel Electophoresis: First, all the DNA samples were diluted to .4µg/µl. These were then mixed with the proper buffers and Restriction Endonucleases. In addition, uncut control samples were prepared with DNA and buffer, but no enzymes. The samples were run in the Gel electrophoresis, the results were as follows:
[pic]
Results of Gel Electrophoresis 2/25/11: The bands in the marker lane show the gel was again successful. However, the lack of bands in any other lane suggest that once again there is not enough plasmid. DNA is present in the samples.

DNA appeared in the ladder lane, but not in any of the sample cut or uncut lanes. The restriction digest and gel electrophoresis were attempted again, this time using diluted enzymes, and undiluted DNA. The results were as follows:

[pic]
Results of Gel Electrophoresis 3/4/11: The bands in the marker lane show the gel was again successful. However, the lack of bands in any other lane suggest that once again there is not enough plasmid. DNA is present in the samples.

Once again, there was DNA in the Ladder lanes, but not in any of the sample lanes. This implied that there was not enough DNA in the samples. To test if the DNA was lost, the remains of the sample were tested using the nanodrop. The results were approximately the same, indicating the DNA loss was not the issue.

[pic]
Results of Nanodrop 3/22/11: There is a strong peak at 260 nm, suggesting that there is a lot of DNA in the sample. However, the gel electrophoresis contradicts these results.

Discussion: The gel electrophoresis was successful once again, however, the correct bands of our desired DNA have yet to be isolated. The nanodrop indicates that the concentration of DNA is sufficient, yet the results of the gel electrophoresis contradict this, indicating that there is not sufficient DNA in our samples. One possible explanation is that the DNA in our samples is a contaminated mixture of nucleic acids, which would give good results in the UV fluorescence test of the nanodrop, but poor results in a gel electrophoresis. Furthermore, all the DNA in the samples was used up, so new DNA must be isolated, and the minipreparations must be redone. Once the DNA is isolated from the miniprepations, it must again be tested for the proper fragments using restriction digestion and gel electrophoresis. Once the correct bands are successfully isolated, I can then proceed to perform a maxipreparation of the plasmids, to isolate a larger amount of the plasmids. This will then be used to construct the complete plasmid vector with the PiggyBac backbone, lac Z gene, and the oligonucleotide linkers. Once this vector construction is complete, it will be tested in mice through Intrathecal injections, spinal cord extraction, and x-gal staining. After it is shown that the PiggyBac transposon can successfully transpose itself into mice nervous system cells, the lac Z gene will be replaced with SIP 30 and RNAi, to test if this transposon can be used to treat neuropathic pain in mice. SIP 30 will overexpress neuropathic pain, while RNAi should block the expression of neuropathic pain. After the PiggyBac vector shows success in treating neuropathic pain when intrathecally injected into mice spinal cords, it will be tested in mice brains.

Vector Construction and Future Implications: When the test vector construction is complete, it will be administered to mouse spinal cord by intrathecal injection. After allowing various time for gene expression, the spinal cord will be dissected. Successful PiggyBac transposition and subsequent Lac Z gene expression will be tested with X-gal staining; if the transposition is successful, the spinal cord should turn blue. Once PiggyBac is shown to successfully transpose into cells of the central nervous system, two new vectors will be designed to alter the expression of neuropathic pain gene in mice. The first vector will consist of the PiggyBac backbone with the SIP-30 gene as the insert. When this is given to mice, it should result in increased neuropathic pain. The second vector will consist of the PiggyBac backbone and antisense SIP-30 as the insert. This should inhibit the expression of SIP-30 gene, and therefore reduce neuropathic pain. This research has many important implications and applications to medicine. If transposition occurs successfully in the spinal cord and alters the expression of neuropathic pain, not only will this be important to gene therapy for neuropathic pain, but more generally, it will also present a new method of stable, long-term alteration of gene expression in cells of the central nervous system, which is currently very difficult. This can be used to treat a wide variety of neurological disorders that are currently difficult to treat.

References: Ding, S., Wu, X. Li, G., Han, M., Zhaung, Y., and Xu, T. 2005. Efficient Transposition of the piggyBac (PB) Transposon in Mammalian Cells and Mice. Cell 122: 473-483.

Yu-Qiu Zhang, Ning Guo Guangdun Peng, Mei Han, Jeremy Raincrow, Chi- hua Chiu, Lique M. Coolen, Robert J. Wenthold, Zhi-Qi Zhao, Naihe Jing, and Lei Yu. "Role of SIP30 in the Development and Maintenance of Peripheral Nerve Injury-Induced Neuropathic Pain." Pain 146.1-2 (2009): 130-140.…...

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...Christina M. Owens Writing Assignment October 29th, 2012 Capital Punishment by Lethal Injection Capital Punishment is defined as the execution of a convicted criminal by the State as punishment for crimes known as capital crimes or capital offences. Capital Punishment is given when the crime is considered so vast and so horrible that it is over the realm of being forgiven or pardoned. Capital punishment in the United States is officially certified by 38 of the 50 states; the minimum age at time of crime to be subject to the death penalty is 18. Throughout history, statistics have proven that Capital Punishment furthermore known as the death penalty to be a working prevention of major crimes. When the death penalty is carried out, it illustrates to the society that committing a capital crime has deadly consequences, and it seems reasonable that a person is less probable to commit a given act if it results in the persons enduring instant and definite punishment. However, there has been some controversy on rather or not Capital punishment should be used as a way of penalizing criminals. Over the past two decades, there has been a colossal increase in violent crimes. As most Americans come to an understanding, death is the only fitting sentence for these crimes. Even in ancient times' capital punishment was not something that came as a surprise. Even the Bible states, "Who so sheddeth man's blood, by man shall his blood be shed: for in the image of God made he man"......

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Capital Punishment

...Capital punishment, or the death penalty, is when a person is put to death by the state as a result of a crime. Crimes that can result in the death penalty are known as capital offences. The death penalty is a long debated topic across the world. The word capital comes from the Latin word capitalis, which translates to “of the head.” The word refers to the ancient time when beheading was used to as capital punishment for the crimes. Punishment by death is usually reserved for murder, treason, spying, or military justice. Only the mentally competent adults could receive the capital punishment. The process of capital punishment today is very costly. It takes millions of dollars and only a small amount of people are actually executed. The judicial system is not a hundred percent accurate either, therefore innocent lives could be taken. The increase in cost as well as inconsistent executions results in a flawed capital punishment system which should be abolished. The death penalty began in the ancient times when murder, treason, or killing the ruler of an enemy. In the western culture, death penalty laws were established as early as the Eighteenth Century. Britain had heavy influence on America’s use of the death penalty. Each colony’s laws regarding the death penalty differed. The abolitionist movement started during the colonial times. The northeast was soon influenced by the abolitionist movement in the early to mid Nineteenth Century. In 1846, Michigan was the first state in...

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Capital Punishment

...Capital Punishment Is No Resolution For Juvenile Offenders Juvenile Delinquents Should Not Have To Worry About Capital Punishment Tkemia S. Gibson 1000 Huntington Mews. Clementon, New Jersey 08021 Juvenile Delinquency Capital Punishment 2 Introduction/Abstract….. page 3 Judicial System…… page 5 Juvenile versus Adults…… page 6 Rehabilitation……. page 8 Conclusion……. page 9 Reference page… page 11 Capital Punishment 3 Introduction/Abstract In today’s society our judicial system has been trying to use rehabilitation, treatment services, and reformation to aide our youth in place of Capital Punishment. Capital Punishment was done away with on March 1, 2005. When dealing with medical research and therapist teams the process has become more opened to helping juveniles to overcome any negative behaviors in the legal system. Capital Punishment 4 Capital Punishment over the years has always been one of the most controversial topics that stay in communication with our legal system. In committing murder as a child, it is hard not to become judgmental in thinking that a child knows right from wrong. When thinking of a teenage child committing murder at times you may think do they really realize what they have just done and do they even know the consequences. My position is juveniles who commit the act of murder should not have to suffer the resolution of capital punishment because they are still children and their brains are......

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Capital Punishment

...get rid of capital punishment? I believe that it should be kept because it benefits the U.S. Here is my point of view, if you take someone’s life what gives you the right to enjoy the gift of living? I believe that if there is strong evidence, and you are not mentally insane and authorities know for sure that a person has committed murder, there should be a penalty involved. The Bible says that if a person should murder, they should deserve to lose their life also. I believe in the death penalty because it eliminates criminals who commit horrific murders, it gives closure to many who have been affected by those criminals, and it also provides more space in jail and prisons. Today, I strongly believe that our consequences are a joke and that is why killing sprees are getting worse and more violent. It’s some people out here that enjoys killing people like serial killers, which they need to be stopped. A person’s religious matter might prevent them from supporting capital punishment. This is why some judges sentence murderers or serial killers to life in prison or a even lesser punishment with parole. Just giving them a life sentence is overcrowding prisons. I don’t think we should reward murderers by keeping them alive and feeding them. While the family of the victim forever mourns. Another thing that I think is dumb is to let them out for good behavior. They can kill again when they get out. I have seen it happen before. People that are against capital punishment are......

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Capital Punishment

...Capital Punishment What is Capital Punishment? Capital Punishment which is also known as the Death Penalty. Death Penalty is the infliction of the death penalty as punishment for certain crimes according toDictionary.com. In the following essay will show why Capital Punishment is implemented to our society. Capital Punishment in any form is acceptable according to the following to the following articles will help justify this concept. To some people Capital Punishment would be considered an act of cruel and unusual punishment; while for others it is a system that should be considered needed in all the States in order to keep the country more safe for the people. Many states within the U.S. have performed executions of convicts since the early 1600’s. Views on capital Punishment vary with people in different ways; there are various organizations within the country that have different opinions on the subject, and organizations such as Religions, Political, or Humanitarian have diverse perspectives on the Death Penalty. “The death penalty is also most commonly argued to be a violation of the right to life or of the "sanctity of life." Many national constitutions and international treaties guarantee the right to life. the right to life demands that a life only be taken in exceptional circumstances, such as in self-defense or as an act of war, and therefore that it violates the right to life of a criminal if she or he is executed, since this is purely murder by the State”......

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Capital Punishment

...Level 6 Name Teacher Benefits of Capital Punishment In order to understand this topic is important to define ´´Capital punishment´´, which is also called death penalty, according to the Encyclopedia Britannica is ´´the execution of an offender sentenced to death after conviction by a court of law of a criminal offense´´ published on Apr. 25, 2008. This type of punishment has been used for many years, in almost all societies and religions. In most countries (Saudi Arabia, Africa, Somalia, and United States) this count of method it is reserved from those who commit murder, treason, but there are other countries where it is applied for those who commit sexual felonies, such as rape, incest, pedophilia, adultery. On the other hand, crimes as drug traffic are considered capital punishment as well. In China, human trafficking, and other corruption cases lead to the death penalty. In this context, there are many people who are against capital punishment, as said by the Abolitionists, death penalty or capital punishment is not only is an act of violence but also a crime, it is considered an inhumane act tolerating the killing of another human being. In fact, it is intricate that the government allows this type of punishment since is taking away the life of a human being as a solution to fight against crime and violence. They argue that violence should never be the answer for the problems that the society is going through. Although, the society that we are living now is full......

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Capital Punishment

...Murder is wrong. Since childhood we have been taught this indisputable truth. Ask yourself, then, what is capital punishment? In its simplest form, capital punishment is defined as one person taking the life of another. Coincidentally, that is the definition of murder. There are 36 states with the death penalty, and they must change. These states need to abolish it on the grounds that it carries a dangerous risk of punishing the innocent, is unethical and barbaric, and is an ineffective deterrent of crime versus the alternative of life in prison without parole. Capital punishment is the most ­irreparable crime governments perpetrate without consequence, and it must be abolished. “We’re only ­human, we all make mistakes,” is a commonly used phrase, but it is tried and true. Humans, as a species, are famous for their mistakes. However, in the case of the death penalty, error becomes too dangerous a risk. The innocent lives that have been taken with the approval of our own government should be enough to abolish capital punishment. According to Amnesty International, “The death penalty legitimizes an irreversible act of violence by the state and will inevitably claim innocent victims.” If there is any chance that error is possible (which ­there always is), the drastic measure of capital ­punishment should not be taken. Also, it is too final, meaning it does not allow opportunity for th accused to be proven innocent, a violation of the Fifth Amendment which guarantees due......

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Capital Punishment

...history capital punishment has been opposed on many premises. In discussion forums across the world many individuals often cite deterrence of crime as a viable defense of capital punishment. However, comprehensive studies, including the 1994 FBI Uniform crime Report, indicate that capital punishment does not serve as a deterrent to crime. According to the American Civil Liberties Union, the death penalty not only does not deter crime- among states that have either abolished or instituted the death penalty crime and murder rates have remained unchanged. Additionally, Eric Pooley of Time magazine, in his research, reports that no proof exists to substantiate claims that capital punishment discourages crime by anyone other than the criminals whom are executed. Glenn Lammi, of the Washington Legal Foundation is quoted as saying that there are no convincing studies [connecting] the death penalty and the crime rate. Whitehead 3 In the absence of persuasive studies linking capital punishment and crime rates, who better to turn to than the individuals who walk the thin blue line- law enforcement officials may be better equipped to address this subject. Time magazine reports that 67% of polled police chiefs also did not believe that the death penalty deters [crime such as] homicide. According to a 1994 Government Accounting Office report (GAO) substantial evidence indicates that courts have been unfair in death sentencing. The 1990 GAO report, summarizing numerous capital punishment......

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Capital Punishment

...The human rights activists are against this type of punishment I believe in second chances. The Disadvantages of Death Penalty * Though there are scientific methods available to investigate the crime, nothing is guaranteed. You cannot remove the chances of punishing innocents completely. * The cost involved on the death penalty prosecution is greater than the expenses occurred in the life imprisonment of the accused. The appeals against such capital punishments take too long to decide, and often it takes years to decide the fate of the death penalty. All these things make the death penalty an expensive option for the governments who spend millions of the dollars of the taxpayer money on death penalty prosecutions. * It is reported that some of the jury members are not completely impartial as they decide the penalty on racial or religious basis. * Some of the accused are mentally ill, and it is ethically wrong to put mentally ill patients to the death. * In most cases people who can afford to hire the expensive lawyers often survived from such kind of capital punishment. People who are poor, and cannot afford to get a quality legal assistance becomes the victim of this penalty. * Some of the experts believe that life prison is a more effective punishment to control crimes as compared to the death penalty. The countries where the death penalty is banned have less capital crime rate as compared to those countries where the death penalty is practiced. ...

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Capital Punishment

...Capital Punishment  Many distinctive doctrines in criminal law originated in efforts to restrict the number of capital crimes and executions. For instance, in the late 18th century, when all murder in the United States was punishable by death, Pennsylvania pioneered in dividing murder into two categories. The state enacted laws that authorized punishment of first-degree murder by death, while second-degree murder was punishable by imprisonment only. Elsewhere, penal codes uniformly required death for certain serious crimes. In these jurisdictions, discretionary powers to commute death sentences gradually expanded. (A commutation substitutes a lesser penalty for a more severe one—for example, replacing execution with a life sentence.) Today in many nations, including Turkey and Japan, the death penalty remains legal but the number of executions has declined over time.  Although many jurisdictions limited imposition of the death penalty, no government had formally abolished capital punishment until Michigan did so in 1846. Within 20 years Venezuela (1863) and Portugal (1867) had formally eliminated the practice as well. By the beginning of the 20th century the death sentence had been abolished in a handful of nations, such as Colombia, Costa Rica, Ecuador, Norway, and The Netherlands. Although not formally eliminated, it had fallen into disuse in many others, including Brazil, Cape Verde, Iceland, Monaco, and Panama. The defeat of the Axis powers provided a foundation......

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