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Cell Culture and Protein Detection

Overview: This practical is split into three main parts, each part allows us to develop certain techniques. The first part of the practical is focused on the techniques needed to successfully carry out cell cultures. The second part is using an analytical technique known as ELISA this is used to measure the amount of proteins secreted by the cells that were cultured in part one. Lastly, part three focuses on western blotting, this is a technique used to measure proteins too but it differs from ELISA in that it measures proteins that are present inside the cell. The fundamental objective of this practical is to look at how Bacterial lipopolysaccharide (LPS) alters the growth and the expression of the smooth muscle cell α-actin (SMA) in vascular smooth muscle cells (vSMC) while also detecting and quantifying cell signalling molecules (JNK’s) and cytokine secretion (TNF-α) in vascular cells. Section 1 – Subculture Objective: The aim of this part of the practical is to subculture the bovine aortic vascular smooth muscle cells and to count the cells and check for viability by counting the cells in suspension on a haemocytometer using a microscope. The cells are also treated with a bacterial antigen (LPS) to look at its effect on viability, differentiation state and the activation of intracellular signalling and secretion of proteins. Immunocytochemistry is also carried out in this part of the practical. Method: Trypsin is used to remove the cells from the bottom of the flask. When the cells have dissociated, medium which contains a trypsin inhibitor is added. The medium stops excessive trypsin action from damaging the cells. The cell suspension is first diluted using trypthan blue to allow viable cells which remain white to be distinguished from non- viable cells which are blue. This method allows us to get a total cell count of cells/mL and the percentage of viable cells. Our total cell count was found to be 7.45×105 cells/mL The non-viable cells were found to be 1.5×104 cells/mL This means that the culture contains more 98% viable cells which indicates that it is a healthy culture. 3 different 6 well plates were then set up using different densities of seed cells. 3×105cells/ml seeding density was used in one plate. This density was used as a lot of protein needs to be present for the assessment of JNK and α-actin protein cytokine production. 1×105 cells/ml seeding density was used in another 6 well plate. These plates are used to assess the effect of LPS on growth and viability of the cells. This density is used to give a clear picture of the cells to make counting easier. 5×103 cells/ml seeding density was used in the last plate. This plate is used for α-actin expression by immunocytochemistry. The low density will give us a clearer picture. The three different sets of plates are all treated with varying concentrations of LPS. Each plate has two wells which are used as controls, containing no LPS, two wells containing 1µg of LPS and two wells containing 10µg of LPS. Immunocytochemistry is carried out on the plate with a seeding density of 5×103cells/ml. This technique is used to determine if a particular protein or antigen is present. An unlabelled primary antibody is used to bind to the antigen desired antigen. The presence of contractile protein α-actin can determine the differentiation state of vSMC. Immunocytochemistry is used as a qualitative method of determining the presence of a protein, it is not quantitative. Results: Calculations for different seeding densities: Initial concentration = 7.45×105 cells/mL Formula = Plate 1, seeding density 1.5×105 cells/ml Plate 2, seeding density 0.5×105 cells/ml Plate 2, seeding density 2.5×103 cells/ml Table 1: Cell growth for each group Group number Cell growth with 0µg of LPS Ratio of cell growth with 1µg of LPS Ratio of cell growth with 10µg of LPS 1 1 0.75 3.5 2 1 0.66 0.24 3 1 0.662 0.309 4 1 0.952 0.214 5 1 6.477 3.809 6 1 0.822 0.422 7 1 0.333 0 8 1 0.786 0 9 1 0.625 0.542 10 1 0.838 0.108 11 1 3.929 1.286 12 1 0.706 0.018 13 1 0.28 0.666 Immunochemistry results: Figure 1: Cell growth in the control (0µg/ml LPS) Figure 1: Cell growth in the control (1µg/ml LPS) Figure 3: Cell growth with 10µg/ml of LPS Discussion: The first part of this section was to carry out a cell count and determine the viability of the suspension. Our sample had 7.45×105 cells/ml and it contained more than 98% viable cells. This meant it was a healthy cell suspension and it was suitable to run tests on for the practical. A seeding density of 1×105 cells/ml seeding density was used to assess the effect of LPS on growth and viability of the cells. This density was used to give a clear picture of the cells to make counting easier. The results of our experiment correlated with the majority of the class. However there were some unexpected results in some of the groups but this could be down to plates being labelled incorrectly or mistakes while counting when using the haemocytometer. Immunocytochemistry is used to allow us to analyse the results visually. We found that as the amount of LPS increased the number of cells decreased, this is illustrated in figures 1, 2 and 3. LPS is an endotoxin and it inhibits the growth of α-actin. LPS also damages the structure of α-actin, high levels of LPS stop the correct formation of the filaments and so affects the function of the cell which is to facilitate cell contraction and migration. This result was expected as LPS is a major mediator to septic shock and is known to directly affect vascular smooth muscle cells. Question: What could you do to improve this experiment? To improve this experiment I would use a wider range of concentrations for LPS. This would give a better understanding of its affects. Repeating the experiment several times and getting an average of your results would also help. Section 2 – ELISA Objective: The aim of this section of the practical is to use Enzyme-Linked Immunosorbent Assay (ELISA) to detect production of the cytokine TNFα from the cells activated with LPS. Detection is based on a colour change. Firstly a standard curve must be generated so that absorbance values can be converted into concentrations of TNF-α. Once the curve is completed we can determine the unknown concentrations of TNF-α in our samples. Method: Firstly the antibody is immobilised onto the surface of the plate. The plate is then washed to remove any excess antibody, antigen is then added and it is allowed to bind to the antibody. A secondary antibody is then added, this antibody is labelled with an enzyme. The enzymes substrate is then added, this causes a colour change. The amount of coloured product formed is determined spectrophotometrically. The amount of coloured product is proportional to the amount of enzyme present and also to the concentration of the antigen. Results: Table. 2 Absorbance values of samples at 450nm. Sample Absorbance at 450nm Concentration of TNF-α from equation in fig. 4 Control (0µg/ml LPS) 0.0923 9.9 pg/ml Sample with 1µg/ml LPS 0.0981 12.8 pg/ml Sample with 10µg/ml LPS 0.1228 25.15 pg/ml Figure 4. Plot of standard curve of absorbance versus concentration. Discussion: The aim of this experiment was to quantify the amount of TNF-α present in our samples. To do this a set of known standards were used and their absorbance values read. This data produced a straight line with an R2 value of 0.99 indicating that a straight line was an excellent fit for absorbance versus concentration, and so the equation of the line could be used to determine unknown concentrations of TNF-α based on their absorbance values. Our results showed that TNF-α was present in its highest concentration of 25.15pg/ml in the sample with the highest concentration of LPS and it was found in its lowest concentration of 9.9 pg/ml in the sample containing no LPS. This result was expected as cytokines such as TNF-α are produced in large quantities to respond to endotoxins such as LPS. Question: What could you do to gain more information from this experiment? To gain more information from this experiment you could test for other cytokines which are also activated by LPS, correlating these results would make your data more meaningful. Section 3 – Western Blotting Objective: The aim of this section of the practical is to prepare cell lysates from the vascular smooth muscle cells which were activated by LPS previously. SDS PAGE and western blotting will then be used to detect the activation of the intracellular protein JNK. Western analysis quantifies the amount of protein present in the cell. To do this cell lysis must be carried out. In this practical we used a method which generates whole cell lysates. To do this lysis buffer is added to the cells followed by sonication. Method: Cell lysis is carried out first. The next step is SDS polyacrylamide gel electrophoresis. Western blotting involves transferring the protein bands from an acrylamide gel to a more stable and immobilising medium such as nitrocellulose paper so that analytical procedures such as detection with antibodies can be carried out. We carried out western blotting using iBlot dry blotting system. After blotting probing is carried out to determine the presence of phosphorylated JNK protein. Results: Figure 5. Ponceau S Staining Figure 6. α-actin Figure 7. pJNK Discussion: To see if our transfer was a success before probing, the blot was stained with Ponceau S stain. The proteins can be seen as red bands with this stain. The result of this stain can be seen in figure 5. red bands are present which indicates our transfer was a success and that there are proteins present. The western blot analysis showed that α-actin was present in all the samples as a strong band around 42kDa was observed which is expected for α-actin. The results for pJNK did not work out as it was washed incorrectly, because of this no bands were observed, however bands would be expected in the samples containing LPS. Why did you run the sample on the gel before blotting? The sample was run on gel first as proteins are separated by molecular weight. This allows us to distinguish α-actin from other proteins. It is then moved to the nitrocellulose paper so that analytical procedures such as detection with antibodies can be carried out Why measure the phosphorylated form of JNK? The phosphorylated form of JNK is a signal a cell sends out when it is stressed. Therefore pJNK should be present in the samples with LPS. If it is present then it confirms the fact that the cell is stressed as a result of the presence of the endotoxin.
Enthalpy of formation.

Given the following heats of combustion.CH3OH(l) + 3/2 O2(g) → CO2(g) + 2 H2O(l)ΔH°rxn = -726.4 kJC(graphite) + O2(g) → CO2(g)ΔH°rxn = -393.5 kJH2(g) + 1/2 O2(g) → H2O(l)ΔH°rxn = -285.8 kJCalculate the enthalpy of formation of methanol (CH3OH) from its elements.C(graphite) + 2 H2(g) + 1/2 O2(g) → CH3OH(l)
Enthalpy of formation

3 history question

3 history question.

President Lyndon B. Johnson was able to get a civil rights bill through Congress thatA. addressed voting rights in the South.B. established full citizenship for African Americans.C. established quotas for the admission of black students to universities.D. prohibited discrimination in public accommodations.Why did President Nixon authorize the illegal wiretapping of his own officials and members of the press?A. He wanted to discover who was leaking classified information to North Vietnam.B. He sought to contain leaks about the secret bombing of Cambodia in 1969.C. He had received a tip that a Soviet spy was in his inner circle.D. He wanted to gather information with which he could blackmail them.Popular television programs of the 1950s were known forA. moral absolutes and happy endings.B. fair depictions of African Americans.C. realistic depictions of family life.D. appealing primarily to children.
3 history question

SDSU Law Enforcement Agency Higher Standards Reading Response Discussion

write my term paper SDSU Law Enforcement Agency Higher Standards Reading Response Discussion.

I’m working on a writing discussion question and need support to help me understand better.

Write a 100-word response to each student in first person as if you were writing it to the students. Talk about how you agree with their ideas and add your own thoughts. Make sure it’s respectful. Student1: It seems hard to argue that law enforcement officers and those in the criminal justice field should not be held to a higher standard, even outside of the hours of work. While I’m sure we are all aware that this is not always the case, one would hope that those attracted to this field of work should be interested in upholding strong ethical values. Personally, I would feel far more comfortable in society with people that have strong moral values being in these positions of power. Having an understanding and some amount of practice of the principles of tolerance, understanding, and standing up against evil both inside and outside of work seem like they should be standard operating characteristics of what criminal justice representatives should be (Banks, 2020).However, our society is not perfect and many people in positions of power in the criminal justice field lack these traits. So, when they are found in situations in which they may face punishment for violating the law, I also think they should be punished equally to or harsher than an average citizen, who may have less access to training on the law, ethics, de-escalation, and crisis intervention, all of which can address the sensitivities of dealing with the public and morality.Banks, C. (2020). Criminal justice ethics (5th ed.). Thousand Oaks, CA: Sage Publishing. ISBN-13: 9781544353593Student 2: Although attorneys, police officers, and judges are all a part of the criminal justice system, they are also part of everyday civilian life. However, due to their professions, they are often faced with making many decisions that involve ethical issues (Banks, 2020). These decisions also must be made without bias or the inclusion of personal morals and values. However, having the title of a professional within the criminal justice system also comes with being held at a higher ethical standard. While these professionals also have a personal life, they also have a separate code of ethics to uphold. For instance, police officers are to behave in a manner that does not discredit themselves or the agency in which they are employed (Banks, 2020). According to the International Association of Chiefs of Police (IACP, n.d), part of the law enforcement code of ethics is to keep their personal life impeccable as an example to all. Because of the nature of their professions, they should be held at a higher standard of ethics. These professions deal with highly sensitive and confidential topics regularly, which also requires a different ethical standard than regular citizens. Having a life outside of work is also important. Those who hold positions such as these also face a vast amount of stress, but this does not mean that they should not uphold their ethical responsibilities outside of work. If these professionals are found to be in violation of these standards, it is my belief that they should not get a lesser punishment than if they were not. They are fully aware of the standards that they are to abide by and should not be able to use their power as means to receive a lesser punishment.SavannahReferences:Banks, C. (2020). Criminal justice ethics (5th ed.). Thousand Oaks, CA: Sage Publishing. ISBN-13: 9781544353593International Association of Chiefs of Police. (n.d.). Law enforcement code of ethics. https://www.theiacp.org/resources/law-enforcement-code-of-ethics
SDSU Law Enforcement Agency Higher Standards Reading Response Discussion

General Portfolio?

General Portfolio?. I need an explanation for this Business question to help me study.

Option #1: Final Portfolio Project (General)
[Product or service associated with a United States of America (U.S.) for-profit business of interest to you]
You are the International Business Management Adviser to the Chief Executive Officer (CEO) of a successful corporation of your choice. The company desires continued market growth. In addition to financial success, the company’s CEO is interested in maintaining the highest of ethical standards and engaging in sustainable business practices.
Your job is to use the resources provided in the modules from this course, and your independent research, to create a market entry strategy to tap an appropriate foreign country market not currently aligned with this corporation. The project should cover the following points.

Introduce the company including the company’s full legal name. Identify if the company is publicly traded and its market capitalization.
Provide a description of the company.
Define and introduce the industry(ies) in which the company operates.
Provide a description of the industry(ies).

Include a figure showing the analysis of the company’s position given Porter’s (1990) Diamond Analysis. Identify if the company is likely to succeed outside of U.S. given the findings of the analysis.
Describe the company’s competitive strategy(ies) used in the U.S.

Describe the company’s corporate level strategy(ies) used in the U.S.

Identify the company’s domestic customer(s).
Analyze the readiness of the company to engage in international business.
Justify the selection of the company’s target country market you selected using primary and secondary screening techniques. What cross-border opportunities are present, which opportunities are the most viable?
Include a table that contains a CAGE analysis (cultural distance, administrative and political distance, geographic distance, and economic distance), required reading in Module Five, (Ghemawat, 2001) for the target country market.
Analyze the target country markets relative to labor (e.g., costs, work ethic, productivity, availability, etc.), infrastructure, natural resources, regulations and sustainability. Identify synergies between the organization’s culture and societal factors of the selected country for expansion efforts.
Describe the company’s target country customer (is the target country’s customer different from the U.S. customer)—if so explain how.
Define and defend the entry strategy used to enter the target country market.
Define and defend the company’s international competitive strategy(ies) and international corporate level strategy(ies) likely to employed.
Explain if outsourcing is used in the target country market and if you recommend this action.
Identify CSR opportunities, ethical risks and actions to support or mitigate these areas.
Justify follow-on/new opportunities that the company might pursue. Include an appendix with a memo communicating this intended action for distribution throughout the company.

Submission Requirements:

Your paper must be 11-14 pages in length, not including the title and reference pages.
Your paper must cite and reference at least seven outside sources not associated with the textbook, course readings, or course assignments. The CSU-Global Library (Links to an external site.) is a great place to find sources.
Your paper must comply with the requirements defined in the CSU-Global Guide to Writing and APA (Links to an external site.).
Refer to the Portfolio Project Rubric for specific grading criteria.

General Portfolio?

Police organizations are instruments through which policing services are organized and delivered to the public. The primary function of the Essay

Police organizations are instruments through which policing services are organized and delivered to the public. The primary function of the police force is crime prevention. As an aspiring police officer, it is crucial to understand how the organizational structure of the police department and its various practices influence its day-to-day functions. Imagine you have completed your probationary period and are being interviewed for the post of a police officer. Your hiring officer wants to evaluate your understanding of the practical implications of police theory and practices on its functions of crime prevention and keeping order. Respond to the questions provided below. Do your research and choose 1 of the following formats to document your responses to your hiring officer: A written speech of 700-1,050 words A recorded video of 5-7 minutes (if you choose to record a video of yourself, please see the note at the end of this assignment) Scoring Note: I do take notice in the scoring that the video option takes more time and courage to complete. Students choosing the video option tend to score higher for this week’s summative assessment. Write or record your answers to each of the following questions as if you were responding to your hiring officer in an interview: Why are you interested in a career in law enforcement? Does legitimacy in the police force enable effective crime-fighting? Explain how. Is problem-oriented policing an answer to building partnerships with the public? Explain why. Is civilianization* a beneficial practice in police work? Provide reasons to support your answer. What are the positive and negative impacts of bureaucracy on communication within a police department? Which type of mentality makes the best police officer: warrior or guardian? Explain why. Is the code of silence a desired police subculture? Explain why. What impact has been seen with the inclusion of women and minorities in the police force? How is crime control influenced by the contingency theory? How does the environment in which police organizations function influence its operational activities? Explain your reasoning in context with the appropriate policing theory. * Civilianization is a process involving the replacement of fully attested, sworn police officers with ‘civilian’ staff who have either no police powers or limited police powers and who provide either administrative or specialist support to policing. Example News Article. Identify any sources you used to support your responses. Submit your assignment. Note: If you choose to record a video for this assignment, remember: Present yourself professionally, as if you were interviewing for a potential employer. Watch the video on How to Film a Video of Yourself to prepare for recording. You do not have to read the question prompts in your video, but you may. If you read the question prompts, that does not count toward the video time for your responses. You have access to Microsoft Stream®️ as a University of Phoenix student. This tool helps you to convert a video file into a link that you can submit for an assignment. To learn how to use this tool, watch the video on Using Microsoft Stream®️: Recording a Screen or Video and refer to the guide on Using Microsoft Stream to Create a Video for a Discussion or Assignment. Submit the link to your video for your assignment. Resources Using Microsoft Stream: Recording a Screen or Video Using Microsoft Stream to Create a Video for a Discussion or Assignment How to Film a Video of Yourself Center for Writing Excellence Reference and Citation Generator Grammar Assistance

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