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Insulin and Erythropoietin Production

Insulin and Erythropoietin Production. Insulin is a protein (polypeptide) discovered in 1921 by Banting with the pancreas being the site of its production. It is made up of 51 amino acids, divided into 2 chains; A and B, bonded by disulfide linkages. Chain A is made up of 21 amino acids with an intra-disulphide linkage, while chain B is made up of 30 amino acids (4). Why Insulin? Insulin is important in glucose metabolism, and is being used for the treatment of Diabetes mellitus; a metabolic disorder of glucose in the body. Initially, Insulin from animals was used to treat this disorder however nowadays synthesized human Insulin is being used, this is because; it is fast absorbed by the body, it has less allergic reactions, it contains less impurities, and it produces good results (3). Recombinant process of producing Insulin Synthetic Insulin was first produced in 1983 through genetic Engineering, which involve extraction of the human DNA (1), once extracted, the gene for Insulin is isolated, and enzymes are used to cut it. The gene is then cut using enzymes and put into the plasmid of a vector, where in most cases E. coli plasmid is used. Since Insulin contains two chains, two pieces of DNA are extracted, and the genes for the two chains are linked to β galactosidase enzyme of the bacteria. The plasmids formed are then inserted into a host cell E. coli and sealed using another enzyme called ligase. And the host on replicating produces the enzymes each containing one of the two chains each. Production is followed by extracting and purifying the chains which are mixed in a reaction to reconstitute the disulphide bridges (1). ESCHERICHIA COLI AS RECOMBINANT INSULIN HOST Entero-bacillus, gram-negative E. coli is about 1 – 2μm, it can survive in the presence/absence of oxygen, and it also grows in an optimum pH and temperature of 7.0 and 37oC respectively. It utilizes glucose as its major carbon source and can also use other carbon sources like pyruvate, glycerol, acetate, and other sugars. K-12 and B strains are mostly used in the laboratory (20) Reasons for choosing E. coli Genetic Engineering technologies were developed using E. coli as a role organism, and so, the genetics of E. coli are well known among other microorganisms, as such it’s the most used organism for the production of different proteins (14). Moreover E. coli has a well known safety and production abilities, stable plasmid, controllable promoter, cheaper and easily cultured (6), E. coli also has fast growth rate, it’s easy to handle, and has well known fermentation skills and the ability to produce high protein content (14). That is why most of the proteins licensed recently by FDA and EMEA, were produced in E. coli (5). With these, and the fact that Insulin is a simple polypeptide (protein) which does not require glycosylation for its bioactivity and stability, E. coli carrying the plasmids for production of insulin will be used as the host for the production of Insulin Strain and plasmids: BL21 strain containing the pMYW-A and pMYW-B plasmids and temperature repressor λ-c1857, will be used for insulin production (21). Growth strategy The various growth strategies that will be used to grow E. coli in order to make it happy and produce the desired product (11) include: Medium: E. coli needs nutrients like carbon, nitrogen and others; thus a carbon source; glycerol will be provided since it’s cheaper and more soluble than glucose (12), a source of nitrogen in the form of ammonium sulphate will also be provided. However such nutrients in large quantities can inhibit the growth of E. coli, as such a defined medium that contain optimum concentrations 20gl-1 glycerol and 2gl-1 ammonium sulphate will be used (11). The medium will also consist of the following; 3gl-1 KH2PO4, 1gl-1 MgSO4.7H2O, 0.8gl-1 citrate, and 6gl-1 K2HPO4 (23). Some trace elements will also be added to the medium. (23) Process and culture-strategies: E. coli will be grown submerged in a sterile controlled stirred tank reactor, and fed-batch will be used as the growth strategy so as to avoid accumulation of acetate which can be inhibits its growth, and reduce the production of the insulin (18). The growth strategy will be divided into two; initially batch mode will be used to initiate growth, after which the fed-batch exponential feeding will be used to produce the insulin (21). After adapting the medium and feeding method, oxygen transfer rates (OTRs) had to be increased through a suitable bioreactor design and over-head pressure (16). Large scale reactors usually reach high ORTs using air and normal aeration pressure, and so the oxygen partial pressure (pO2) will be increased by adding pure oxygen to the air-stream entering the reactor, thus increasing its oxygen transfer rates (16) DO will be maintained at 40% of air saturation and aeration rate at 1vvm. Foaming arising due to large number of cells and high aeration-rates will be solved by use of impellers for stirring simultaneously at 300rpm and the use of antifoam (ucolub N115) (16, 21). The process temperature and pH will be maintained at 30oC and 6.8 respectively so as to avoid partial proteolysis of the insulin protein. Bioreactor Design: Bioreactor vessel is usually cylindrical and made up of stainless steel. It is composed of impeller for stirring, Air sparger is placed at the bottom of the vessel for introduction of air, it has some inlets for introduction of acid/alkali for pH control and also for introduction of antifoams, nutrients and inoculum; It is also has pH, DO and temperature probes for sensing (22), Microbial activity during fermentation usually produces heat, so the bioreactor design must allow for removal of heat, and this can be achieved by cooling with jackets and coils (16) Bioreactors must also be designed in a way that it can withstand high temperature and pressure and to allow cleaning-up and sterilizing (22). Growth analysis Temperature, pH, DO, foam, partial oxygen and carbon dioxide pressures, will be analysed on-line, other parameters like biomass, will be analysed by using optical density (OD600) and dry cell weight (offline). Cell viability will be analysed by using flow cytometry, the concentrations of substrates and metabolites by enzymatic methods while insulin will be analysed using electrophoresis methods like SDS-PAGE, and ELISA, while its purity will be determined by HPLC (8). Limitations/Problems There are several problems that may arise during processing and can limit the use of this organism for Insulin production, these are; Poor secretion because of the structure of its membrane (and tough cell wall), small amount of foldases, chaperones and increased concentrations of proteases, leading to low productivity (7). Solutions to this problem include all measures taken to increase quality of secretion and production such as: Use of secretion systems like the system of α-haemolysin (7) co-expression after co-cloning of foldases and chaperones (13) Improving the rates of gene-expression and using proteases deficient mutants like BL21 (18). use of E. coli mutants that are deficient of cell-wall (12) Limited post translational-modifications; including disulfide-linkage formation, which is important for the insulin stability and biological activity (9). Solutions to this problem include; Production of insulin with altered amino acid sequences through genetic engineering (9) Using E. coli mutants to enhance the formation of disulfide linkages e.g. Origami (15) iii. Exporting proteins into the periplasm which has disulphide bonding mechanisms (19). Codon biases; due to large quantities of exact transfer-RNAs found in E. coli, the codons in the human-genes are often different from those that are found in this organism. This results in inefficient expression of some of these rare codons by the organism resulting in an unexpected protein synthesis termination or wrong incorporation of the amino acids (12). This problem can be solved by replacing codons that are rare in the desired gene by codons that are often found in the E. coli and by co-expressing the rare transfer-RNAs (15). Acetate is usually formed as a by-product, and is inhibitory to growth of the cells (20). Solution is by using a fed-batch feeding method and by limiting DO level (11). Another problem is that large proteins are often obtained in an insoluble form (5); forming aggregates called inclusion bodies; IBs (20). This can be solved by adjustment of temperature, increasing the strength of the promoter, adjusting the number of plasmids, concentrations of the inducer, and the composition of the media (9). Erythropoietin EPO EPO is a glycoprotein that is produced in the renal cortex of the kidney (10, 11). It has also being shown to be present in the brain, spleen, liver and the lungs (7, 17). It is made up of 165 amino acids of about 18kDa (25), with a number of carbohydrates linked to the polypeptide through O and N glycosidic-bonds giving the glycoprotein a total weight of 34kDa.Two disulphide linkages hold the molecule together (15) and the carbohydrates are responsible for the stability of the glycoprotein in-vivo,and increasing its half-life in the body (24). Why EPO? EPO functions to regulate the amount of red blood cells (RBC) in the blood by controlling the proliferation and differentiation of its immature cells to mature cells (1, 2, 22,). It is also involved in the growth and formation of blood vessels, and healing of wounds (6), it functions in the brain is not clear, but studies showed the glycoprotein to have some protective effects (18). Because of these functions EPO has being used in the treatment of anaemia caused by kidney failure and other causes (25). Recombinant production of EPO Despite its importance, EPO in body is found in very small amounts and mostly in the urine (4), as such there is the requirement to produce EPO in large amounts, this leads to the work of isolating the glycoprotein from the urine (12, 21), and was used to identify it’s amino acid sequences, and synthesis of its DNA (9, 12), furthermore the human erythropoietin genes were cloned by Lin et al. (17), and consequently recombinant human EPO (rhuEPO) was produced in 1985 using CHO cells (14, 16). Chinese -Hamster- Ovary (CHO-Cells) as rhuEPO host: These are epithelial cells derived from the ovary of Chinese hamster (a mammal). They grow well in culture and looks like cobble stones. The cells usually attach to a surface available but can be grown in suspension (20). CHO cells are grown best at 37oC and at pH 7.4; they are cultured in a suitable complex medium which can support their growth for many generations (20). CHO cell lines are now available from cell culture collections like the American type culture collection; ATCC. Moreover human EPO expression plasmids are now also commercially available, and are usually used for production of EPO using the CHO cells (27). Reasons for choosing CHO-cells Karthik et al. (13) showed that CHO-cells are being used extensively in the industries for the production of many proteins, because they have demonstrated, to possess some qualities like: They can modify biological products post-translationally; Proteins produce in CHO-cells have high glycosylation quality making them compatible and stable (13) Safety of the product; Studies in 1989 have shown that most viruses do not multiply in CHO-cells (13) Ability to adapt easily and be grown in suspension (13). Products can now be purified to contain less contaminant (13). CHO cells have being used for a long time; as such much data has being accumulated for regulatory reasons (13). They are easy to manipulate genetically (13). The isolation of cells deficient in Dihydrofolate-reductase enzymes leads to stable clones’ selection and genes amplification to increase production (13). With all these, and the fact that EPO is a glycoprotein that requires glycosylation for its stability and activity, recombinant CHO cells are chosen to produce EPO. Cell lines and plasmids: Cell lines which have the capability of glycosylating proteins (Pro-5), harboring the pGEX-HET-puro expression plasmid, will be used to produce the recombinant human erythropoietin (27). Growth strategy Medium: Complex culture medium will be provided with; Glucose as a source of carbon and energy, Amino acids as source of nitrogen, Salts will be included to make the solution isotonic Vitamins and hormones will be added as co-factors Serum is usually added to the culture medium to enhance the growth of the cell (20), but has the following disadvantages: It chemicals are not defined and can cause cell growth inconsistency between batches (20) It is very expensive (20) The serum may contain proteins which can be difficult to separate and purify from the proteins secreted by the cells during downstream processing (20) It increases foaming and can be a source of contamination by viruses. (20) Therefore a serum-free (SF) media (16) will be used for the growth of the E. coli. Process and culture-strategies: The cells will be grown adherent on micro-carriers in a sterile controlled packed bed reactor, and perfusion method of production where some amounts of the medium is removed and replaced by fresh one and the cells are grown slowly will be used (28); because it was found to improve the glycosylation of the proteins more than fed-batch where there is fast growth of cells, (8). Before, many processes were run in a simple batch method, but nowadays, Perfusion or fed-batch methods are mostly employed and higher products are now realized (22). The production will be carried out in two stages; the growth stage and the production stage. Normally stirring will be kept at 100 to 150 rpm, foaming will be avoided by adding Pluronic F68 (16).Temperature will be maintained at 37oC initially during growth and then reduced to 33oC during production, as was shown to increase the overall protein production, while maintaining the quality of the glycoprotein (3, 26). pH will be kept at 7.1 initially and then reduced to 6.8 (8, 26), by passing CO2 gas to the culture or by addition of concentrated sodium-bicarbonate solution in low quantities, because CO2 is also toxic to the cells and can also affect the production of EPO (20). In order to avoid the depletion of oxygen, the oxygen transfer rates (OTRs) will be increased above its utilization rate, with a constant supply of pure oxygen and air, while DO will be maintained at 20-50% of air saturation (20). Bioreactor Design: Since the cells are big and fragile, the design of the bioreactor has to be considered. Mammalian cell culture bioreactors are designed with bottoms that are round and are usually made up of glass/stainless steel (20). The impellers are usually marine or pitched blade types fitted at the end of mechanical drives shafts so that both vertical and horizontal mixing are allowed at low stirring-rates (20). Temperature is controlled through coiled pipes or open ended fermenter jacket (20). pH, DO and temperature probes are used for sensing and have both air inlet and outlet for respiration. Growth Analysis Temperature, pH and DO will be monitored on-line, because cells are immobilized, biomass formed cannot be measured directly therefore it will be monitored by measuring rate of glucose consumed daily and the rate of lactate produced (28) Cell viability by flow cytometry, Glucose, glutamine, and lactate concentrations will be analysed using multi-parameter Bio-analytical system (26); while ammonia formed as waste product of amino acid metabolism, will be analysed by colorimetric assay and by the use of detection-kit (26). EPO formed will be analysed using HPLC to determine its purity and its quality by Isoelectric focusing, SDS, and Bradford assay (26). The activity of EPO will be analysed by bioassay and by the use of protein assay-kit (27) Limitations/Problems. There are many limitations associated with CHO cells culture processes and they include; They are fragile and highly sensitive to shear stress caused by agitation and bubble because the cells are large and have only cell membrane (20). This is usually solved using a suitable bioreactor-design and use of Pluronic F68 (20). They need a complex medium including serum which can cause problems in the downstream processing and is expensive (20). Solution to this is by using serum- free media (24, 25). Low yield of proteins have been produced from these cells, the productivity using the microbes being higher than the use of these cells. They also have slow growth rates (13). The problem of low productivity and slow growth rates can be solved through selecting cell lines that are better and optimizing cultural-strategies. Ammonia and lactate are generated during growth and can inhibit growth and also affect glycosylation (8). Solution is by optimizing the strategies of feeding and by monitoring (8). Glycosylation differences may arise from the EPO produced in the CHO-cells and the human EPO as seen in the way the two are sialylated terminally, as a result that the CHO-cells are not able to express an enzyme called alpha-2,6, sialyltransferase (27). Solution is by the use of CHO-cells harboring alpha-2, 6, sialyltransferase-cDNA expression-cassettes (27). REFERENCES: 1. Alcamo, I., DNA Technology; the Awesome-Skill. Farming-dale. New York: Academic Press. (2001). 2. Banting – Grolier Electronic publishing www.littletree.com.au/dna.htm accessed on 30/12/2010 3. Carbs information, www.carb-information.com/insulin-synthetic.htm accessed on 30/12/ 2010. 4. Charce, R.E., and Frank, B.H., Research, Production and Safety of Biosynthetic Human Insulin. (1993). www.littletree.com.au/dna.htm accessed on 30/12/2010. 5. Ferrer-Miralles N. Domingo-Espín, J. Corchero, J.L. Vázquez, E. and Villaverde, A. Microb. fact. for recombinant pharmaceuticals, Microbial factories , 8:17, 2009. 6. Fox, S. Improved processes and new capacity for pipeline to commercial production. Biopharmaceutical contract manufacturing, Volume 1 (report). High Tech Business Decisions: San Jose, CA. 2005 7. Genschev, I., Dietrich, G., Goebel, W.,The E. coli alpha-hemolysin secretion system and its use in vaccine development. Trends Microbiol. 10: 39-45. 2002 8. Hewitt C.J., Nebe-von Caron G., Axelsson B., McFarlane C.M, Nienow A.W Studies related to the scale-up of high-cell-density E. coli fed-batch fermentations using multi-parameter flow cytometry: effect of a changing microenvironment with respect to glucose and dissolved oxygen concentration. Biotech. Bioeng. 70: 381-390. 2000 9. Hite P.F, Barnes A.M.J.P.E. Exhuberance over Exubera. Clinical Diabetes 24: 110-114. 2006. 10. Jana, S., Deb, J.K. Strategies for efficient production of heterologous proteins in Escherichia coli. Appl. Microbiol. Biotech. 67: 289-29. 2005. 11. Joseph S., and Raphael F., growing E. coli to high- cell density-A historical perspective on method development Biotech. Advances 23: 345-357 2005. 12. Korz D.J, Rinas U., Hellmuth K, Sanders E.A, Deckwer W.D. Simple fed-batch technique for high cell density cultivation of E. coli. J Biotechnology, 39: 56-65. 1995. 13. Kujau, M.J., Hoischen, C., Riesenberg, D., Gumpert, J. Expression and secretion of functional mini-antibodies McPC603scFvDhlx in cell-wall-less L-form strains of Proteus mirabilis and E. coli: a comparison of the synthesis capacities of L-form strains with E. coli producer strain. Appl. Microbiol. Biotech. 49: 51-58. 1998. 14. Lund, P.A. Microbial molecular chaperones. Advanc. Microbiol. Physiol. 44: 93-140. 2001 15. Makrides S.C. Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol. Rev. 60: 512-5388. 1996. 16. Meyer, H.P. Brass, J. Jungo, C. Klein, J. Wenger, J. and Mommer, R. an emerging Star for Therapeutic and Catalytic Protein Production. Bioprocess International. 2008. 17. Nacelle, G. J. V. and Coppel, R. L. Reshaping Life; Key Issues in Genetic Engineering, Novo-Nordisk Promotional Brochure. Melbourne: Melbourne University Press. 1989. 18. Schmidt, F.R. Recombinant expression systems in pharmaceutical industry. Appl. Microbiol. Biotech. 65:363-37. 2004. 19. Wacker M., Linton D., Hitchen P.G., Nita-Lazar M., Haslam, S.M., North, S.J., Panico M., Morris H.R., Dell A., Wren, B.W., Aeb, M. N-linked glycosylation in Campylobacter jejuni and its functional transfer into E. coli. Science 298:1790-1793. 2002. 20. Demain, L. A., and Vaishnav, P. Production of recombinant proteins by microbes and higher organisms. Biotech.Advan. 27: 297-306. 2009. 21. Schmidt, M., Raman Babu, K., Khanna, N., Marten, S., Rinas, U., Temperature- induced production of recombinant human insulin in high cell density culture of recombinant Escherichia Coli. Journal of Biotech. 68:71-83. 1999. 22. Ratledge, C. and Kristiansen, B. Basic biotechnology. Cambridge: Cambridge university press. 2001. 23. Tabandeh, F., Shojaosadati, S.A., Zomorodipour, A., Khodabandeh, M., Sanati, M.H., Yakhchali, B. Heat induced production of human growth hormone by high cell density cultivation of recombinant E. coli. Biotech. Letters. 26: 245-250. 2004. Insulin and Erythropoietin Production
Humans learn how to behave by copying the examples they see, humanity is a species that learns social behavior by the example of . This system works well enough when a child’s main observation of human behavior are humans they interact with in real life. However, the amount of time children spend consuming mass media and, by extent, the violence present in over sixty percent of all media not intended of audiences under the age of thirteen is generally thought to teach the wrong lessons. The various degrees of violence depicted in multiple different types of media affects behavior, but the extent of those effects are widely disputed. Violence in media is one of the many factors that may potentially cause violent behavior, but it has a far less direct correlation with criminal behavior, and the strength of the correlation between violent behavior and violence in media is questionable. One of the main arguments for the correlation between criminal behavior and violence in media is video games, specifically first person shooter games, which are often linked to glamorization of mass shootings. However, it would not be nearly as influential because the media generally shows pain, blood, and other negative consequence to these actions, even in video games.(Annual Reviews). In addition, for the vast majority of people the reconnection with the real world allow them to rationalize the compulsiveness of these electronic, violent actions that occur only in fiction. For instance, people know that pain is real, and introducing someone to the pain and anguish of the victim who is injured and the long term consequences that jail and being forced to flee from society has on the perpetrator would greatly discourage any passing thoughts of committing murder. When a well publicized act of violence occurs, humans attempt to rationalize such behavior. The most frequently used claims are that the inflictor’s actions were influenced by drugs, a mental illness, or the way they were raised. One such question as to the manner in which a criminal was raised is their exposure to violence as a child or adolescent. Violent video games are commonly accused of causing or encouraging violent behavior that escalates to criminal behavior later in life. The National Rifle Association frequently claims that criminal behavior, especially gun violence, being caused by drugs, mental illness, or exposure to violence in video games. The U.S. Supreme Court stated “Psychological studies designed to show a connection between exposure to violent video games and harmful effects on children do not prove that such exposure causes minors to act aggressively. Any demonstrated effects are both small and indistinguishable from effects produced by other media.” (AP NEWS). in response to the state of California attempting to ban the sale of violent video games to children in 2012. The U.S. Supreme Court acknowledged that witnessing violent crimes and behavior in media may cause children to imitate the actions they see, but likewise acknowledged that the connection between long term behavior and exposure to violence in various media forms is not certain or strongly proven. The mere fact that California was willing to blame violent video games for the actions and behavior of children is a prime example of a desperate attempt to rationalize behavior and failing to examine every possible cause or admit that numerous causes are responsible for the affirmation, encouragement, and continuation of criminal intentions that could lead to criminal actions. One of the main reasons studies that analyze the correlation between violent behavior and exposure to violence in media are questionable is because the majority of studies usually only record and examine the behavior of prepubescent children for a few hours immediately after showing them a movie that does or does not contain violent or criminal actions. While studies such as these are useful for analyzing the immediate effects exposure to violence in media have on children, they do not gather data or properly examine the long term effects violence in media has on children. One study conducted in 2004 had a sample group consisting of 1,254 students in seventh or eighth grade and 500 parents and was focusing on what video games kids were playing, the duration and frequency of play, and the possible relationship with violent or aggressive behavior.(Massachusetts General Hospital). The study was supervised by Dr. Cheryl Olson and the researchers were a team of Mass General research professionals. The study found some correlations between the amount of violent games played and the intensity and frequency of self-reported physical altercations and antagonistic behavior when subjects spent a longer sum of time playing violent games. However, this relationship only occurred in a few children , all of which had previously exhibited high levels of stress and aggressive traits. (Massachusetts General Hospital). The results of this particular study indicate that increased use of violent video games is the result of combative tendencies and stress being channeled into violent games, instead of violent games causing the aggressive tendencies and stress. Studies that examine the immediate results of children being shown physical violence in media have generally indicated a more direct relationship between combative behavior and seeing violence in media, and imply a relationship between aggressive behavior and the possibility of future criminal behavior. A study conducted in 1963 by Bandura, Ross, and Ross attempted to replicate the effects on violent media on children by showing preschoolers a clip of an adult physically assaulting an inflatable dummy or showing a clip that contained no violence.(Bandura, Ross,
This assignment is for a doctoral level course and should be written at a scholarly/academic level. The writer should use technical vocabulary specific to the field of higher education. The focus will be on identifying and comparing the academic missions for five (5) different types of postsecondary institutions: Public; Private Not-for-Profit; Denominational; For-Profit; and Corporate. paper specifics will be included in an attached document.

formulating strategy and strategic management

formulating strategy and strategic management. Help me study for my Business class. I’m stuck and don’t understand.

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http://www.csun.edu/~hfmgt001/formulation.docPick any corporation you are familiar with and identify one of the competitive tactics it uses from this week’s reading “Formulating Strategy.” Analyze the competitive tactic it uses. Consider its internal and external environment using any of the strategic management tools in the course (SWOT, PEST, 5 Forces). Synthesize (develop a new idea) for a competitive tactic it should attempt.

Prepare a two page (double-spaced) essay. The paper should be 12-point font, Times New Roman, be at least 500 words, and include a final source list.
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formulating strategy and strategic management

Project Statement

cheap assignment writing service Project Statement. Paper details Submit a brief Project Statement (850-1000 words) in which you detail at least two key points of your proposed exhibit. Your proposal, including your initial statement and thesis • The artist you have chosen to present. • Three key images • What your exhibit intends to say about this artist. The statement must include an initial thesis – This Project Statement is important, because it will serve as the basis of what will be your Curatorial EssayProject Statement

Al Yamamah University Lethal and Nonlethal Force Discussion

Al Yamamah University Lethal and Nonlethal Force Discussion.

Project GuidelinesTopic: One real Problem as you seen around youThe purpose of this project is to develop an understanding of basic marketing research process.Total length of the project should be 13-15 pages of text (Time New Roman references and any appendix material), single-spaced (12-point font) Title (16-point font bold), heading (14-point font bold all capital) and sub heading (12-point font small letter). Be sure to include a cover page that includes the name of university, project title and names of all group members. Use subheadings to divide the sections. Use colored A4 pages to divide the sections.Cover PageExecutive SummaryTable of Contents1. Introduction2. Literature ReviewTalk about all previous relevant work done in this topic.3. Conceptual framework and hypotheses development.Develop the concept of the variables you will use and draw the hypotheses using the concept.4. MethodologyDemographic characteristic, Survey instrument, sample and procedure 5. Data AnalysisApplication of statistical technique.6. Results7. Discussions and Conclusion8. Practical ImplicationUses of your research in real world9. Limitation of the study 10. References11. AppendixThe Demographic characteristic should include: the geographic location of the country; a brief history; the economic situation, currency and current exchange rate and living conditions; cultural background such as demographics, language(s), major religions, consumer lifestyles, food and dress, business etiquette and important norms and traditions; political system; legal system; involvement in multinational market groups, barriers or encouragements to trade.Reference Style:Be sure to cite all sources used in compiling information, whether paraphrasing or directly quoting those sources. If you are using direct quotes, include page numbers in the citation. Examples of bibliographic references:For Journal Papers:Sitkin, S. B., & Weingart, L. R. (1995). Determinants of risky decision-making behavior: A test of the mediating role of risk perceptions and propensity. Academy of Management Journal, Vol. 3 No. 2, pp. 1573-1592.For Websites:World Trade Organization (2012). Statistics database, International Trade and Tariffs Data. Retrieved July 31, 2016, fromhttp://www.wto.org/english/res_e/statis_e/statis_e.htmFor Books:Carson, D., Gilmore, D., Perry, C., & Gronhaug, K. (2001). Qualitative Marketing Research. Great Britain: SageThe most critical issue with respect to citation of sources is that the citation given in the text of the paper must match alphabetically a source in the reference list. Double-check and reconcile your reference list to make sure every citation is included!Reference Quantity and Quality:Your paper will be judged in part on the basis of your reference materials, both that you have used adequate sources to gain multiple perspectives and that these are respected, credible sources. It is expected that most of these are secondary sources, although primary sources are encouraged as well. A bibliography with fewer than 10 sources is likely to be judged as less than adequate. Wikipedia may be used to help you identify credible sources, but do not rely on it as a reference. Multiple citations to Wikipedia will hurt your grade.Tables:All tables, figures, graphs, and exhibits included within the paper should be numbered (i.e., Figure 1) and given a descriptive heading. At the bottom of each, the word `Source’ with full bibliographic citation should be included, as shown below.Due Date:The project submission is due on 07/12/2020.Three (02) students in each group.You are required to submit the electronic copy of the project through LMS.Criteria for Project EvaluationCompleteness Is all the information specified in the outline for the project included? Are the topics chosen in the situation analysis adequate/reasonable? Do reference citations reflect thorough use of available information sources? Are appendixes used effectively to supplement the text of the paper?Format Has the specified structure for the document been followed? Is information presented in a neat, logical, well-organized manner? Have all external information sources been credited through reference citations? Has the specified reference style been followed? Do citations in text and reference list match?Creativity and Logic Do the strategy recommendations contain imaginative ideas? Do they reflect a logical thought process? Are they reasonable and consistent with basic marketing concepts and principles? Is the paper’s overall presentation (using such elements as cover, diagrams, maps, drafts of promotional materials) inviting, interesting, effective? Has the group developed an overall plan that is practical, usable? Could the company actually implement it?Writing Style Is the paper free of spelling, grammatical and typographical errors? Is the style of expression clear, concise and appropriate for its audience? Has the paper been edited so that it reads as one document (rather than as segments contributed by different authors)? Does it flow well?
Al Yamamah University Lethal and Nonlethal Force Discussion

Ralph Bunche and James Meredith Essay

Table of Contents Introduction Ralph Bunche (1904 to 1971) James Meredith (1933) Conclusion Works Cited Introduction This is a short essay focusing on some of the prominent African-Americans in history. It highlights brief achievements and contributions of Ralph Bunche and James Meredith. Contributions of these individuals are fundamental for people who seek to understand African-American literature and history (Flynn 1). Ralph Bunche (1904 to 1971) Bunche had a difficult childhood. He did odd jobs to supplement the family finances. However, Bunche demonstrated his academic prowess by being a valedictorian in his graduating classes, and he excelled as an athlete in different sports. He became an academic, adviser, political scientist, and a diplomat with the UN and in the administration of John F. Kennedy. Bunche had some of the most outstanding achievements in the African-American history. He was the first black to receive the Nobel Peace Prize (1950) because of his mediation efforts between Arabs and Jews in Palestine. Bunche did not actively engage in civil rights movements. However, he criticized racial segregation by maintaining that racial segregation had no scientific basis. He claimed that democracy and segregation were not “compatible and urged blacks to maintain their struggle and accept responsibilities that come with freedom” (Nobel Media 1). He made such claims through personal speeches and published works. Bunche was a member of the Black Cabinet, and Roosevelt’s administration consulted him on several occasions. Bunche rejected the position of Assistant Secretary of State from President Truman because of segregation in Washington, D.C. He was a part of the Civil Rights Movements organized by Martin Luther King, Jr. in 1965. He also supported other black movements such as the Urban League and NAACP (National Association for the Advancement of Colored People). James Meredith (1933) Meredith is a writer, an activist, and a political adviser, who actively participated in the American Civil Rights Movement. He was the first black to attend the University of Mississippi in 1962 during racial segregation (Eagles 9). Meredith shaped American politics and the fight against racism. Meredith saw an inaugural address of President John Kennedy as an opportunity to demand his constitutional rights and applied to the University of Mississippi. This was an attempt to pressure the government to enforce equality for all races. Meredith started his campaign against racism (the March Against Fear) in 1966. This marked the beginning of civil rights movement against racism in America. Meredith attempted to join Congress, but withdrew his candidature on all occasions. He accused whites as the greatest enemies of blacks. Conclusion Meredith and Bunche were among the first African-Americans who pioneered the fight against racial segregation and had excellent achievements in education. As a result, their works shaped African-American history. Get your 100% original paper on any topic done in as little as 3 hours Learn More Works Cited Eagles, Charles. The Price of Defiance: James Meredith and the Integration of Ole Miss, Chapel Hill, NC: University of North Carolina Press, 2009. Print. Flynn, James. Negroes of Achievement in Modern America, New York: Dodd, 1970. Print. Nobel Media. n.d. Ralph Bunche: Biography. n.d.