Sand Pebbles Essay The Movie Sand Pebbles Focus Was On The Flag. The F

Sand Pebbles essay The movie Sand Pebbles focus was on the flag. The flag was not only a symbol of a nation, but of nationalism, militarism and imperialism. During the 1800's and early 1900's there was a severe foreign influence in China. During this time period the European nations wanted to trade with the Chinese, but the Chinese had no interest in the European products for at the time China was self sufficient. The European nations in order to keep from losing all of the money to the Chinese decide that they would start an Opium trade them. Against the wishes of the China, the European nations continued to sell the Opium to the Chinese. Finally war erupted because of this. The European nations were victorious in a series of Opium Wars against China. From these Opium Wars many treaties in the favor of the west were signed which gave those European nations greater access to China. After the European nations, got their peace of China so did the United States. Soon after the United St ates defeated the Spanish in the Spanish American War, the United States received the Philippines from the Spanish. With that the United States declared the Open Door Policy, which all of the European nations accepted. Now in China it was not only the European nations, but also the Untied States. The Chinese flags were a symbol of nationalism, of their nationalism. That they were their own nation. It was a reassurance of their own nationalism. It proved to the Chinese that even though there were many countries slowly taken over China, that they were still their own nation, and they weren't going to let anymore foreign influence into China. The flags also symbolized militarism, because where ever there were two different flags in the same area conflicts arose. When these conflicts arose they often resulted in the militaries getting involved. These militaries were the foreign militaries in China and the Chinese nationalist military. All of these stemmed back the imperialism that was b rought to China. The flags also symbolized the imperialism of the foreign nations in China. Every non Chinese flag was a symbol of the imperialism in China. This imperialism stemmed back to the Opium Wars were when the treaties were made countries like Britain able to setup colonies in China. All of the flags in the movie symbolized either nationalism, militarism or imperialism in China, which was all caused by China loss of all of the Opium Wars against the European nations.

top events of 1968 essays

top events of 1968 essays After reading through newspaper articles for the year 1968, I realized that the year was quite an eventful one. Politically, socially and economically speaking, the country endured a great deal of influential circumstances. Although the studying of vast articles from the New York Times succeeded in painting a clear, factual picture of that turbulent year, I was still eager to discover how incidents affected people growing up in that era. This fueled my motivation to begin the interviewing process, and to choose participants. In the end, I decided to interview my father, Mr. John Arthur Bartle, and a friend of my mothers, Mrs. Linda Pacelli. Although both came from completely different backgrounds, and both have differing views, their stories and descriptions were equally fascinating. Since I grew up with both my parents, I assumed that I knew a great deal about my father, John Bartle. I could not have been more wrong! I had heard stories about his being in the United States Airforce, but I never knew the governing factors surrounding them. It turns out that in 1968, my father, age twenty-two, was stationed in Spain. Apparently, he had enlisted in the Airforce because he was about to be drafted, and he claimed, There was no way in hell I was going to Vietnam. He said he had even considered running to Canada. Much to my surprise, my father revealed that he had been part of the counterculture during that time, and also vehemently opposed the war. I could not quite picture my father that way, for today he fits the description of a hard-working, clean-cut, rigid, white-collared father of three. My father was interesting to interview since he was overseas for 1968, and learned of all American events second hand. My interview with Linda Pacelli showed a sharp contrast with that of my fathers. Linda, nineteen years old at the time, was attending St. Lawrence University during the year of 1968. She...

Question Essay Example | Topics and Well Written Essays - 500 words - 9

Question - Essay Example These defined liabilities are the results of the past events that will be turning into future economic outflows from the company, these liabilities successfully meet the standard definition of obligation but their measurement and computation is often observed to be a debatable issue. As the above-discussed liabilities are future expenses for the company, their future value computation may depend upon numerous factors. These liabilities are very sensitive to the rates of interest of the country and other external factors such as government policies, inflation rates, time value of money, and the probable date of maturity. Their date of maturity may change and solely depends upon the clauses and covenants placed upon them in the contracts. The lease commitments are the future payments of the leased item. Only the current liability under the lease agreement contains a true value of the lease payment for the year, the non-current liabilities hold an estimated figure to be paid in the future. The purchase obligation makes an organization to bound into a commitment of purchase of an item in the future date. The market value of the item in the future cannot be defined in the present period; an estimated value is considered in this case as well. In the case of marketable securities, the rates and maturity periods, and markets for the item are estimated based on assumptions. The derivatives also fall into the same category. The nature of these obligations makes it difficult for the company to reflect and present the real and accurate value of these items. Hence, there is a possibility that the liabilities been shown by the companies may differ from their actual worth. Ernst & Young LLP is the audit firm which performs external audit of Apple Inc. and PriceWaterhouseCoopers performs external audit of Dell Inc. The auditors of both

Speech Essay Example | Topics and Well Written Essays - 1000 words

Speech - Essay Example The freedom is necessary, but we do not like the freedom, which cause unnecessary change in the society. â€Å"Hate speech is against the law, communication that classifies a person or a group on the basis of color, disability, ethnicity, gender, nationality, race, religion, sexual orientation, or other characteristic is regarded as hate speech.† (http://en.wikipedia.org/wiki/Hate_speech) Hate speech occur in all of countries but in different ways. There are hate speeches against religion, homosexuality, and racialism. The speech should be respectful of other people’s religion. When necessary, criticism should be polite. Hate speech cause a lot of problem between people, countries, and religion. If anyone espouses Christianity, Islam religion or any other religion, we should respect his/her opinion. If someone chooses to be a Muslim, someone will tell him that all Muslim kill other people or Muslims are barbarian and terrorist. That is wrong since not all Muslim kill ot her people, but the question is (Why the people kill others?). There are various causes of hate speech. For example, â€Å"Following the end of world war II, 24 German leaders were brought before the international Military Tribunal at Nuremberg.† (https://www.google.com/search?q=what+is+hate+speech%3Fpdf&ie=utf-8&oe=utf8&aq=t&rls=org.mozilla:en-US:official&client=firefox-a). Two of the leaders were linked to incitement and distribution of the Nazi propaganda. This is kind of Hate of speech and it caused deaths of many people in the world. In the same way, the racialism was huge problem in United State. The Black people lived miserable lives due to hate speech. They were lived with people who hated them and they did many kind of the callousness. In addition, they were not getting their rights, in other words they were enslaved. They had to demonstrate against the government to get their freedom. â€Å"Pornography is material designed to arouse and has no legal or consistent d efinition.† http://www.ffeusa.org/html/statements/statements_pornography.html Pornography most of time is dangerous for the societies. The commercials do not care about pornography and its effects. They care about their money and the ways to get more money. Sometimes, they show pictures and films have contempt for women. Moreover, men learn violence and force women to do sex as shows depict. â€Å"What is obscenity? It is not synonymous with pornography, as most pornography is not legally obscene. Most pornography is protected by the First Amendment.† (Freedom of speech and press, Henry Cohen consequently, people should have freedom of speech and opinion, but their opinion should be reasonable. â€Å"Freedom of speech is the political right to communicate one's opinions and ideas via speech.† (http://en.wikipedia.org/wiki/Freedom_of_speech). People’s opinion is significant for the government and for the other people. We cannot develop our country if we do not listen to people's opinion without interference. They can say idea through any media whether by write, TV, or radio. Most of great countries have freedom speech. The government and people listen for their voice, what they need, what they criticize and reasons for their criticism. If any person or government said this is our country we should not criticize them since, they too are citizens. The country belongs to all people and, not an individual’s property. Everyone has the right to say his opinion and idea to develop his country and

Tell how stepping outside of your comfort zone taken you on an Essay

Tell how stepping outside of your comfort zone taken you on an unexpected path to change your life. Also tell about a childhood event that you feel has most sh - Essay Example Many of the men and women involved in these construction efforts are community volunteers. Several years ago, my long-time acquaintance, "Thomas" suggested that we volunteer for a specific project, which involved a rapid construction of a two-bedroom dwelling. My initial response to his request was quite negative, offering an argument that such an activity is only suited for skilled laborers. After an extended period of disagreement, I finally relented and allowed Thomas to sign us up as contributors to the project as one-day only volunteers. Thomas and I showed up for "duty" on the final day of construction, when the kitchen flooring, cabinetry, and other finishing touches were being accomplished. We were each assigned a partner and given multiple tasks to perform. Throughout the course of this extremely arduous day, I learned valuable skills such as how to install countertops and how to lay ceramic tile. I realized the importance of do-it-yourself knowledge as it provides a broader set of skills which can be applied to everyday living. However, my learning consisted of much more than just a fundamental knowledge of home improvement. On that day, the future owner of the home came to inspect the property with her two small children. Armed with cookies, tears, and excessive gratitude, this woman made an effort to embrace the entire volunteer staff and express that the home would take her life from destitution to an opportunity for hope and a future for her children. I realized instantly that my minimal volunteer efforts had made a tremendous impact in the life of a hardened family. In one day, I learned the long-term value of compassion and community relations. This ties in with an event which I experienced as a child that fundamentally changed my viewpoint about caring. My long-time friend, "Jessica", had invited me over for our usual playtime. On this particular

Human Carbonic Anhydrase II

Human Carbonic Anhydrase II Human carbonic anhydrase II is one of the fastest studied enzymes known with a variety of roles in reaction catalysis. Its primary function is to catalyze the reversible hydration reaction of carbon dioxide. In addition to carbon dioxide hydration, it is also capable of other latent skills, such as catalyzing esterase activity. The ability of human carbonic anhydrase II to function as a catalyst derives from key residues in and around the active site that play crucial roles in the mechanism. Substitutions to two of those particular key amino acids were performed via Quick-change site directed mutagenesis: H64A and V142D, to investigate the particular role they have in the catalytic active site. Various kinetic experiments and structural analyses were performed on wild-type carbonic anhydrase and the mutants to discern and compare their activity to each other and to literature, including Michaelis-Menten parameters for PNPA hydrolysis, CO2 hydration, and inferring function molecular m odelling. Though the same trends can be seen as the literature, individual values were found to be much lower owing to errors in measurement and equipment. Trends were found to coincide with the mutants known roles in the active site: His64 is the proton shuttle that facilitates proton transfer during the rate limiting step and Val142 participates in the hydrophobic pocket to bind and recruit substrates to interact with the active site. Mutations to both of these sites show that enzyme efficiency and activity strongly decreases. Introduction Human carbonic anhydrase II (hCAII) is a zinc metalloenzyme that catalyzes the following reversible reaction: . The enzyme commonly functions to help shuttle carbon dioxide in red blood cells to rid the body of metabolic waste, and catalyzes the hydrolysis of many aromatic esters [1, 2]. Structurally, a zinc ion is located in the active site, coordinated to 3 histidine residues (H94, H96, H119) and usually a hydroxide ion or water molecule [2]. The mechanism of hCAII proceeds through two major steps: 1) the conversion of carbon dioxide to bicarbonate, and 2) the regeneration of Zn-OH by proton transfer. The active hydroxide that is bound to zinc nucleophilically attacks a nearby carbon dioxide molecule, resulting in a bicarbonate ion binding to zinc [3]. The zinc-oxygen bond breaks to subsequently release a bicarbonate ion, which is replaced with water [3]. The Znà ¢Ã¢â€š ¬Ã¢â‚¬â„¢OH bond is regenerated by a proton transfer to the external buffer, which is facilitated by the His64 residue [3]. The proton transfer step is the rate limiting step of the reaction [3]. The diazole side chain on the histidine residue is what gives it the ability to be a proton acceptor and donor. Mutations in that position (His64) usually result in decreased enzyme activity due to a lack of proton transfer; however the reaction does proceed to a lesser degree without an active His residue, possibly due to its extensive water network in the activ e site forming secondary proton wires [4]. Carbonic anhydrase catalyzes one of the most rapid reactions; it is one of the fastest enzymes studied [1]. Its reaction speed is due, in part, by the amphiphilic nature of the active site [1]. The hydrophobic side is used to bind carbon dioxide, while the hydrophilic patch functions to optimally orient the carbon dioxide molecule for the reaction [1]. The hydrophobic wall forms a well-defined pocket near the zinc-hydroxide and is composed of the following amino acids: Val142, Val121, Leu197 and Trp208. The hydrophilic patch consists of Thr198 and Glu106, which form a hydrogen bond network with the Znà ¢Ã¢â€š ¬Ã¢â‚¬â„¢OH to stabilize and orient it for nucleophilic attack on CO2 [2]. Therefore, any modifications to the hydrophobic pocket would change its structure, and consequently, its catalytic efficiency [1]. In this study, the importance and role of His64 and Val142 to the structure and mechanism of hCAII are determined through site-mutagenesis and subsequent characterization of the new mutants, H64A (His64 Æ’Â   Ala) and V142D (Val142 Æ’Â   Asp) via kinetic and structural analysis. The changes that arise from the substitutions may prove to be applicable to drug synthesis because hCAII is known to be involved in a variety of diseases, for example, Marble brain disease, where mutations in the hCAII gene leads to a deficiency in the enzyme which is an autosomal recessive disease [5]. Studies in hCAII mutations can be used to design folding modulators to suppress misfolding which frequently occurs due to hCAII destabilization [5]. Another major disease involved with hCAII gene is osteopetrosis. The hCAII genes inactivation decreases osteoclast function in bone, and knowledge of hCAII mutations that inactivate the enzyme may lead to better understanding of bone remodelling [6]. Some carbonic anhydrase diseases use inhibitors (CAI) to suppress the hCAII as a therapeutic treatment. Inhibitors prevent hCAII activity by inhibiting either of the reaction steps: the conversion of CO2 which involves V142 in the hydrophobic pocket, or the rate limiting step, proton transfer, in which His64 is crucial. Experimental Procedure Site directed mutagenesis via the PCR-based Quick-change method was performed on hCAII as cited in Woolley (2011) for 10 ng and 20 ng wild-type plasmids (hCA2pET24b from Novagen) [7]. Table shows the sequence of the primers used in the PCR reactions. Products of PCR mutagenesis reactions were run on 0.7% agrose gels to determine size. The gels were run at 150 V in 1X TAE buffer. Red safe dye from Intron Biotechnology was used in the agrose gel instead of ethidium bromide for safety reasons [7]. The standard molecular weight ruler used was a 1 kB DNA ladder from Fermentas. Table : Primer sequences used in mutagenesis of hCAII in the forward and reverse direction for mutants H64A and V142D Mutant Direction Sequence MW (Da) %GC TM ( °C) H64A Forward GGATCCTCAACAATGGTgcTGCTTTCAACGTGGAG 10778 51 67 Reverse CTCCACGTTGAAAGCAgcACCATTGTTGAGGATCC 10709 V142D Forward CTGATGGACTGGCCGaTCTAGGTATTTTTTTG 9868 44 62 Reverse CAAAAAAATACCTAGAtCGGCCAGTCCATCAG 9779 The enzyme, DpnI, was then used to digest methylated DNA (the parent template DNA). The DNA vector that contained the mutation was transformed into supercompetent E.coli turbo cells from New England Biolabs by heat shock [7]. LB-agar plates were prepared to grow the transformed cells containing mutant genes (i.e. H64A and V142D hCAII gene) [7]. Both were injected with Kanamycin to ensure that the culture that grows will have the desired mutation [7]. A miniprep culture was set-up from the LB-agar plate into LB medium to grow one colony for DNA analysis [7]. Restriction enzyme mapping was prepared and XhoI and BglII were chosen, they were used under buffer 3 for optimal efficiency. Plasmid putification was performed using the QIAprep Spin Miniprep Kit, and then the chosen restriction enzymes were carried out and were run on 1% agrose gel [7]. A sample of the purified DNA was sent to an external company (ACGT) for commercial sequencing (Sanger dideoxy type) to verify if the mutagenesis occurred correctly. The sequence was analyzed using the program BioEdit. To determine the level of confidence of the sequencing results, the purified DNA was quantified using UV/Vis absorption via a spectrometer [7]. The concentration was calculated using Ecà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1(260) as 50 ÃŽÂ ¼g/mL. Purified plasmid DNA was transformed into E.coli BL2(DE3) cells to initiate protein expression by heat shock, similar to the transformation into turbo cells [7]. The cells were cultured and a single colony was grown. Once sufficiently grown, ITPG and ZnSO4 were added to induce protein expression [7]. SDS-PAGE was used to confirm protein expression and was analyzed against an unstained protein molecular weight marker by Fermentas. The protein and ladder was stained with coomassie blue [7]. Affinity chromatography was used to purify the mutant hCAII proteins [7]. The matrix used was agrose linked to p-(aminomethyl)benzenesulfonamide, exploiting the tight binding that occurs between hCAII and sulphonamides. Once purified, the protein was dialyzed using a 6000-8000 Da dialysis membrane to replace the elution buffer with protein buffer and removes the matrix from the protein [7]. SDS-PAGE is again used to confirm the protein is still present after purification and to check its approximate molecular weight. It was run for two different amounts of protein, 2 ÃŽÂ ¼g and 10 ÃŽÂ ¼g, and also ran 10ÃŽÂ ¼L of wash fractions from affinity chromatography [7]. Protein concentration was determined by UV absorption at 280 nm in a final concentration of 6M guanidine hydrochloride. From the calculated concentrations, purity of the protein could be assessed via SDS-PAGE. To characterize this purified hCAII protein, a variety of analyses were done. Two types of mass spectrometry (MS) were performed: electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) [7]. The MS analysis was used to confirm the presence of the mutation in hCAII with intact and digested protein. Protein samples (H64A and V142D hCAII) were not diluted for either of the MS analyses as cited in Woolley (2010). Samples of 10 ÃŽÂ ¼L of stock protein concentrations (37.6 ÃŽÂ ¼M H64A and 3.2 ÃŽÂ ¼M V142D hCAII) were used for analysis of the molecular weight of the intact protein by ESI-MS. Both mutants were then digested by Trypsin Gold (MS grade) from Promega and the resulting fragments were evaluated by ESI-MS as well [7]. A 50 ÃŽÂ ¼L sample was used for each mutant, 40 ÃŽÂ ¼L of the mutant at stock concentration and 10 ÃŽÂ ¼L of the Trypsin Gold. A couple ÃŽÂ ¼L of the digested mutants were saved for MALDI-MS and the rest was used for ESI-MS. Once the molecular weights for each of the digested fragments were determined by ESI-MS, the products were run through a protein database to confirm the identity of the protein and mutations [7]. The 1 ÃŽÂ ¼L of the tryptically digested mutants prepared for ESI-MS, subsequently underwent MALDI-MS. The 1 ÃŽÂ ¼L samples were mixed with a matrix consisting of 1 ÃŽÂ ¼L ÃŽÂ ±-cyano-4-hydroxycinnamic acid (CHCA) and 1 ÃŽÂ ¼L of 0.1% trifluoroacetic acid (TFA) [7]. The entire mixture was pipette onto a MALDI well and was inserted into the mass spectrometer and a MALDI-MS spectra was obtained. Michaelis-Menten kinetics was used to determine the KM and kcat of the p-nitrophenyl (PNPA) hydrolysis reaction [7]. The ionized product from the hydrolysis, p-nitrophenol (PNP) produces a bright yellow colour that was used to follow the rate of the reaction via the Perkin Elmer Lambda UV/Vis spectrophotometer [7]. Various sample concentrations of PNPA were set up to have a final enzyme concentration of 0.2 ÃŽÂ ¼M in protein buffer [7]. The initial rate measurements of each PNPA concentration were taken for wild-type enzyme, H64A mutant, V142D mutant, and a blank with no additional enzyme added (refer to data tables in Enzyme Kinetics I [7]). PNP has a molar absorption coefficient (ÃŽÂ µ) of 1.73ÃÆ'-104 Mà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1cmà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1. This was used to calculate Michaelis-Menten values: Vmax, KM, kcat, and kcat/KM [7]. The ability of hCAII mutants (H64A and V142D) and wild-type hCAII to catalyze the hydration of CO2 was measured. The pH of the solution was measured to track the progress of the reaction because the reaction generates protons. Enzyme solutions were prepared according to table 2 in [7]. The buffer used in the table was 50 mM TRIS buffer (pH 7.8). Additional enzyme samples were prepared for 25 nM of wild-type hCAII and 100 nM of H64A mutant in a final concentration of 22.5 and 29.92 mM imidazole buffer (pH 7.8) respectively to determine chemical rescue of mutant H64A. The pH of the CO2 hydration assay was measured using a pH probe and pH meter at 5 second increments for a total of 90 seconds starting at the beginning of the reaction [7]. The slope of the initial changes in the first 2 points was considered to be the V0 for each enzyme concentration. From the initial velocity, a kcat value can be calculated for each enzyme using the assumption that [S] >> KM, the Michaelis-Menten equati on simplifies to kcat=V0/[E]. The third kinetics experiment used fluorescence to determine the binding constant of dansyl amide (DNSA) and acetazolamide (AZ) (from Sigma-Aldrich) to H64A and wild-type hCAII was performed using the Perkin Elmer Fluorometer [7]. Stocks of 1 mM and 200 ÃŽÂ ¼M of DNSA were prepared from a 21.6 mM DNSA stock by dilution with DMSO. Enzyme stocks were diluted to 0.25 ÃŽÂ ¼M with TRIS buffer to make a 10 mL solution. A 1 mL sample of H64A from stock made was titrated with DNSA in small increments [7]. The fluorometer emissions were taken at 470 nm. AZ titration in competition with DNSA was not able to be completed. The last characterization experiment done was molecularly modelling the hCAII wild-type enzyme, as well as the mutants H64A and V142D. The molecular model of hCAII analyzed was derived by x-ray crystallography and found in the Protein Data Bank (PDB) repository. The wild-type and H64A hCAII structures examined had a PDB code of 1CA2 and 1MOO respectively. At present, no crystal structure has been found for V142D hCAII. The Swiss PDB Viewer program was used to visualize the protein structures. Secondary structures of the proteins were able to be observed. Residues around the metal active site and the Ramachandran plot were explored. Homology between hCAII and other carbonic anhydrase isozymes, hCAIV (PDB code 1ZNC) and hCAI (PBD code H1CB), were also studied by performing an iterative magic fit on the ÃŽÂ ±-carbons and structure alignment for each pair. The root mean square (RMS) between hCAII and the other isozymes were also analyzed to determine conserved and deviated regions in the structures. The binding of cobalt in the hCAII active site was also investigated (PDB code 3KOI). The structural inhibition of hCAII by AZ was also gleaned by structural analysis (PDB code 3HS4). Its mode of inhibition and binding sites were shown through the crystal structure. Lastly, the Swiss PDB Viewer program was used as a tool to theoretically synthesize mutations and compare it to the actual structure as determined by other scientists, for example, by aligning the virtual and crystallized mutations to determine deviations in structure by performing RMS. Results Site-directed mutagenesis PCR. Products from the PCR mutagenesis reactions were examined using 0.7% agrose gel electrophoresis. Two samples of differing amounts of template DNA (10 ng and 20 ng) were used for each mutant (Figure ). Bands were only observed for samples containing 20 ng of the hCA2pET24b DNA template plasmid (Figure ). The size of the bands observed coincides with the size of the plasmid used, 6018 bp. Heat shock transformation and isolation of plasmid. Several colonies were observed after plasmid transformation for both mutants, and 1 colony from each mutant was chosen for restriction enzyme digest with BglII and XhoI. Figure : Electrophoretic run on 0.7% agrose gel of DNA of hCAII mutants from PCR mutagenesis reactions. Lane 1 is the GeneRuler ladder by Fermentas and lanes 10-13 are the following: V142D (10 ng), V142D (20 ng), H64A (10 ng), and H64A (20 ng). As suggested from the gel, the mutants in the 20 ng plasmid was more successful than the 10 ng plasmids in determining relative molecular weights. Both mutants in the 20 ng plasmid show a band at approximately the 6000 base pair mark, which coincides with the number of base pairs in the hCA2pET24b plasmid that was used (6018 base pairs). Quantification of pure plasmid DNA. A 1/20th dilution was carried out on the purified DNA with elution buffer (EB; 0.1 M Tris, 0.4 M KSCN, pH 7). The absorption of the diluted DNA at 260 nm and 280 nm was taken by a UV/Vis spectrophotometer and the relative DNA purity was determined (Table ). The assumption that Ecà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1 260 = 50 ÃŽÂ ¼g/mL for DNA was applied in the calculation of concentrated and diluted concentrations of purified DNA (Table ). Table : Relative DNA purity for mutants V142D and H64A determined by UV/Vis spectrophotometer absorbance at 260 and 280 nm. Calculated concentrations of mutants from absorbance data, where Ecà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1 260 = 50 ÃŽÂ ¼g/mL. Mutant Wavelength, ÃŽÂ » Absorbance Units Relative DNA Purity (A260/A280) Concentrated (ÃŽÂ ¼g/mL) Diluted (ÃŽÂ ¼g/mL) V142D 260 nm 0.3117 1.7852 311.70 15.59 280 nm 0.1746 H64A 260 nm 0.2653 1.7581 265.30 13.27 280 nm 0.1509 Enzyme restriction digest. Purified plasmid DNA of mutants were digested with XhoI and BglII, separately and together in a single and double digest for both mutants. The digested and undigested samples were run on 1% agrose gel, and 2 bands were observed around the 6000 and 7000 bp marker for all 8 samples (Figure , Figure ). The expected length of the bands in the double digest should be 892 bp and 5126 bp (Figure ). Figure : Electrophoresis performed in 1% agrose gel of digested V142D hCAII in lane 1-4. The (1 kB) GeneRuler DNA ladder is shown in lane 5. Lane 1-4 contain the following: V142D plasmid, V142D + XhoI, V142D + BglII + XhoI, and V142D BglII. Double bands are shown at the 6000 and 7000 bp marker for all 4 V142D samples. Figure : Electrophoresis performed in 1% agrose gel of digested H64A hCAII in lane 1-4. The (1 kB) GeneRuler DNA ladder is shown in lane 5. Lane 1-4 contain the following: H64A plasmid, H64A + XhoI, H64A + BglII + XhoI, and H64A BglII. Double bands are shown at the 6000 and75000 bp marker for all 4 H64A samples. Figure : Restriction enzyme cut sites and position of hCAII gene (5072-5854) on the hCAI2pET24b plasmid DNA Sequencing. The mutations for both V142D and H64A in the hCAII gene were successful according to the sequenced DNA result obtained from ACGT. Other mutations in the DNA sequence were observed in both mutants, but since the aligned protein sequence was the same, mutations were likely to be silent mutations due to amino acid redundancies. When sequenced in the forward direction by T7 polymerase, a protein mutation was found (K153N) other than the desired mutation of V142D; however, when sequenced in the reverse direction by T7 polymerase terminator (T7TER), K153N was not observed. Plasmid DNA transformation into E.Coli BL21(DE3) cells. Following transformation into BL21(DE3) cells, colonies were observed for both hCAII mutants (V142D and H64A). A random colony was chosen to be cultured and then was induced to express protein with 270 ÃŽÂ ¼M IPTG and 0.1 mM ZnSO4. SDS-PAGE for protein expression. Protein expression was tested with SDS-PAGE. The expected molecular weight of V142D hCAII is approximately 29.2 kDa and the expected molecular weight of H64A hCAII is approximately 29.1 kDa. SDS-PAGE bands are observed between the ladder markers 25.0 kDa and 35.0 kDa for both mutant proteins (Figure , Figure ). Figure : SDS-PAGE loaded with V142D hCAII proteins to examine protein expression. Samples were loaded in different volumes of protein to ensure gel visualization. Lane 15 contains the Fermentas protein molecular ladder and lane 1-4 contain the following: 1 ÃŽÂ ¼L à ¢Ã¢â€š ¬Ã¢â‚¬â„¢IPTG, 4 ÃŽÂ ¼L à ¢Ã¢â€š ¬Ã¢â‚¬â„¢IPTG, 1 ÃŽÂ ¼L+IPTG, 4 ÃŽÂ ¼L +IPTG. All 4 samples had some form of protein expression between 25.0 to 35.0 kDa. Figure : SDS-PAGE loaded with H64A hCAII protein to examine protein expression. One sample was loaded with 4 ÃŽÂ ¼L of H64A protein and +IPTG in lane 10. Lane 6 contains the Fermentas protein molecular ladder. The one H64A sample loaded showed an expression between 25.0 and 35.0 kDa. Calculation of pure protein concentration and extinction coefficient. Following affinity purification and dialysis, pure protein concentration was calculated from UV absorption measurements at 280 nm and the known extinction coefficient of hCAII as 50070 Mà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1cmà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1 (Table ). The final concentration of the samples of V142D and H64A hCAII were 3.2 ÃŽÂ ¼M and 37.6 ÃŽÂ ¼M respectively. Table : UV absorption measurements at 280 nm of purified protein and the resulting final concentration Mutant Average A280 Protein concentration (ÃŽÂ ¼M) V142D 0.01583 3.2 H64A 0.1884 37.6 SDS-PAGE to assay purity and check approximate molecular weight. Several samples were loaded into the SDS-PAGE for each mutant protein: lysate and wash fractions (collected from affinity chromatography), 2 ÃŽÂ ¼g protein, and 10 ÃŽÂ ¼g protein. For H64A, a visible band was only observed for the 10 ÃŽÂ ¼g sample (Figure ). The band was located between the 35 kDa and 25 kDa markers on the ladder. For V142D, none of the 4 samples resulted in a band on the gel (Figure ). Figure : SDS-PAGE shown for H64A mutant protein. Lane 1 contains the Fermentas protein molecular weight marker. Lane 11-14 contains H64A samples of the following (in order): lysate, wash fraction, 2 ÃŽÂ ¼g protein, and 10 ÃŽÂ ¼g protein. Only the 10 ÃŽÂ ¼g protein had (faint) observable bands located between the 25 and 35 kDa markers. Figure : SDS-PAGE shown for V142D mutant protein. Lane 4 contains the Fermentas protein molecular weight marker. Lane 12-15 contains V142D samples of the following (in order): lysate, wash fraction, 2 ÃŽÂ ¼g protein, and 10 ÃŽÂ ¼g protein. No observable bands are seen for any of the samples. Mass spectrometry. ESI-MS was not successful in analyzing the molecular weight of intact and digested protein of both mutants. A MALDI spectrum was able to be generated for the digested proteins; however, without the digested ESI spectrum to compare to, the peaks from the MALDI spectrum can only be speculatively assigned. Kinetics: Hydrolysis of PNPA. Using the molar absorption coefficient of PNP (1.73ÃÆ'-104 Mà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1cmà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1), the rate of each reaction was determined. The predicted rate was calculated using the Michaelis-Menten kinetics: . The plot of predicted rates and actual initial rates vs. PNPA concentration can be seen in Figure , Figure , Figure for wild-type, H64A, and V142D hCAII respectively. The Vmax and KM values for each enzyme were calculated by minimizing the square difference between the predicted and actual reaction rates, and the kcat was calculated using the equation: (Table ). Table : Calculated Michaelis-Menten parameters for wild-type, H64A, and V142D hCAII catalyzing the hydrolysis of PNPA. Wild-type hCAII H64A hCAII V142D hCAII Vmax (ÃŽÂ ¼M/sec) 1.202 0.812 0.218 KM (mM) 1.280 1.957 8.362 kcat (sà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1) 5.141 ÃÆ'- 10à ¢Ã¢â€š ¬Ã¢â‚¬â„¢3 2.159 ÃÆ'- 10à ¢Ã¢â€š ¬Ã¢â‚¬â„¢2 6.825 ÃÆ'- 10à ¢Ã¢â€š ¬Ã¢â‚¬â„¢2 kcat/KM (Mà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1sà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1) 4.02 11.032 8.162 Figure : Michaelis-Menten plot of initial rate vs. concentration of PNPA added for wild-type hCAII enzyme. Figure : Michaelis-Menten plot of initial rate vs. concentration of PNPA added for H64A hCAII enzyme. Figure : Michaelis-Menten plot of initial rate vs. concentration of PNPA added for V142D hCAII enzyme. Kinetics: CO2 hydration. Initial velocity (V0) values were calculated by measuring the progression of the reaction (via concentration of protons) with time (Table , Table , and Table ). kcat values were then calculated using the same equation as in the hydration of PNPA and averaged for the individual enzymes (wildtype, H64A, and V142D hCAII) in a particular buffer (i.e. TRIS or imidazole). Table : Initial velocity (V0) and kcat values calculated for the hydration of CO2 by wild-type hCAII in TRIS buffer and imidazole buffer. Wild-type concentration (nM) V0 for WT+TRIS (M/s) V0 for WT+Imidazole (M/s) 0 1.3E-08 6.05778E-08 1.5 1.1E-08 N/A 2.5 1.1E-08 5.63E-08 5 2.1E-08 5.16E-08 12.5 5.9E-08 5.63E-08 Average kcat (sà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1) 5.3 ±1.62 12.44 ±9.19 Table : Initial velocity (V0) and kcat values calculated for the hydration of CO2 by H64A hCAII in TRIS buffer and imidazole buffer. H64A concentration (nM) V0 for H64A+TRIS (M/s) V0 for H64A+Imidazole (M/s) 12.5 1.4E-08 6.57E-08 25 1.4E-08 5.8E-08 50 1.7E-08 7.53E-08 Average kcat (sà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1) 0.67 ±0.39 3.03 ±1.97 Table : Initial velocity (V0) and kcat values calculated for the hydration of CO2 by V142D hCAII in TRIS buffer. V142D concentration (nM) V0 for V142D+TRIS (M/s) 12.5 6.2E-09 25 5.4E-09 50 5.5E-09 Average kcat (sà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1) 0.27 ±0.19 Fluorescence detection of ligand binding. DNSA was titrated with H64A hCAII to determine its affinity for the enzyme. The dissociation constant, KD, for DNSA was determined to be 0.086 ÃŽÂ ¼M when protein concentration was 0.25 ÃŽÂ ¼M. Competitive titration of H64A-DNSA hCAII with AZ was attempted, but was not successful as DNSA binding was too tight, making it difficult to be displaced by AZ. Molecular modeling. Literature models of wild-type (PDB code 1CA2) and H64A (PDB code 1MOO) hCAII were analyzed. There is no available structure of V142D hCAII at present. The secondary structure of wild-type is composed of 18 ÃŽÂ ²-sheets (77 residues) and 10 ÃŽÂ ±-helices (42 residues), with the majority of the ÃŽÂ ±-helices falling in the domain of right-handed helices, while very few show left-handed helical properties according to the Ramachadran plot. It also seems that the active site is solely composed of ÃŽÂ ²-sheets, and no ÃŽÂ ±-helices (Figure ). Analyzing PDB structure 3HS4 (AZ bound hCAII), the mechanism as to how AZ inhibits hCAII function can be seen. AZ has 3 binding sites, 2 are novel binding sites and the other provides a mechanism of inhibition. AZ binds the zinc directly at the active site, displacing crucial ligands needed for catalysis. There were some discrepancies found between the crystal structure of H64A [1MOO] as cited on PDB and virtually mutated H64A from wild-type hCAII, resulting in a RMSD (root mean square deviation) of 0.29 Ã… (Figure ). Since no literature structure of V142D is available, no comparison between virtual and crystal structures could be made. Figure : Secondary structure of wild-type hCAII overlain with ribbon to visualize the higher arrangement. Figure : RMSD between H64A hCAII virtually mutated and literature crystal structure. Blues denote the same or similar residues, while reds and oranges indicate completely different amino acids. Discussion Agrose gel results were only visible for samples that contained 20 ng of the plasmid template DNA, rather than the 10 ng plasmid. This may be a result of more amplification during PCR with the 20 ng plasmid, and so would intensely be more visible. Though the 20 ng samples showed bands at the appropriate 6000 bp mark, there was also a faint band that can be seen near the end of the gel. This may be due to non-specific primer annealing. Quantification of DNA purity was done by exploiting the peak absorbances of protein and DNA. DNA maximally absorbs at 260 nm, while protein dominantly absorbs at 280 nm. The purity ratio reports the relative amount of DNA compared to protein present in the sample. The purity of both mutants were approximately 1.8, which is regarded as a relatively pure sample; however, a purity ratio of more than 2.0 would have been ideal. The restriction enzyme digest showed 2 bands (7000, 6000 bp) for all samples, which may have been a sign of poor mixing/ pipetting since the volumes of restriction enzyme were extremely small amounts. If this is the case, only some of the DNA was nicked and some were not, which would result in 2 bands. It was expected that the plasmid sample would have a high band (supercoiled), each of the singly digested samples would have a slightly lower band (nicked), and the doubly digested would show 2 bands that indicated the fragment size of 892 and 5126 bp. Sequencing results showed that a protein mutation occurred when the sample was sequenced in the forward direction by the T7 polymerase. A lysine at position 153 had mutated to glutamine (K153N). However, this mutation was not observed when the T7 polymerase terminator was used to sequence the sample in the reverse direction. A mutation that occurs in one sequencing direction and not the other is usually attributed to sequencing errors, which may be the reason in this case. The SDS-PAGE bands for protein expression coincided with the expected molecular weight for both mutants, which could suggest that the correct proteins were expressed; however, there is a possibility that the proteins expressed could be of similar weight, but completely different. Interestingly, the V142D samples that did not include the protein inducer, IPTG, had a more intense band than the faint ones found for the samples that did include IPTG. This may just be a result of mislabelling. The SDS-PAGE performed to assess purity after the purification process. Mutant V142D had low protein expression as evidenced by its concentration of 3.2 ÃŽÂ ¼M. The V142D mutant should have very low protein expression according to Fierke et al. (1991) because valine at position 142 is uniquely required for maximal expression in E.Coli. It is suggested that by altering position 142, protein stability decreases [2]. Therefore, the protein that was expressed in the previous SDS-PAGE gel may not be V142D hCAII at all. The sample may have been small fragmented contaminant proteins that would have completely run off the gel altogether. However, the low concentration of V142D after purification may also be a major factor in the lack of gel bands observed as well. Unlike V142D, H64A hCAII concentration should not have affected its lack of bands because it was calculated to have had a reasonable concentration of 37.6 ÃŽÂ ¼M. There were some problems loading the samples into the wells; t his could be an explanation as to no observable gel bands. ESI-MS is dependent on concentration because it affects the size of primary droplets [8]. The unsuccessful determination of molecular weight of V142D hCAII may be attributed to its low concentration. The H64 hCAII mutant was also not able to be successfully analyzed with ESI-MS. A possible reason for the failure was that it was not kept on ice while it was not being used. The enzyme may have become inactive and degraded into smaller fragments. This would explain the ESI-MS output obtained for H64A. No definite molecular mass was determined, but the spectrometer did detect a lot of small protein fragments in the sample, all under 1000 amu. The kinetic values obtained from PNPA hydrolysis do not follow similar trends found in literature [2]. The kcat/KM for wild-type hCAII (2500 ±200 Mà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1sà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1) was found to be significantly larger than V142D hCAII (3 ±0.3 Mà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1sà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1) in literature, more than 800ÃÆ'- larger [2]. Experimental calculations yielded kcat/KM for V142D (8.16 Mà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1sà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1) to be about 2ÃÆ'- larger than wild-type (4.02 Mà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1sà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1), which did not follow literature patterns. The literature trends make more biolog

Aliens Built the Pyramids Essay -- essays research papers

  Ã‚  Ã‚  Ã‚  Ã‚  Aliens built the Egyptian pyramids. Recent research supports the theory that the Pyramids were built long before humans inhabited the area now known as Egypt. It is also nearly impossible for the Egyptians to have lifted and moved the limestone brick used to build these massive structures. Only a more advanced form of life could have constructed such an enormous undertaking, while using advanced mathematics and geography that were not yet known to ancient peoples.   Ã‚  Ã‚  Ã‚  Ã‚  Archeologists suggest that the large stones used in building the pyramids were transported by rolling them over logs or a wet, slippery, clay surface. These methods may have been effective in moving the blocks close to the building site, but do not explain how the massive bricks, weighing as much as a Ford F250 truck, were lifted on top of each other.   Ã‚  Ã‚  Ã‚  Ã‚  When the Great Pyramids at Giza were built, the Egyptians had not invented the wheel yet, but the limestone blocks that they grudgingly transported, in an effort to build pyramids, weighed about 2 tons each. If all of the stone from the pyramids was cut into one foot, square blocks, it would extend two thirds of the way around the earth. No human life forms could have possibly erected these structures using that much limestone, because they did not have the technology to work in such scale. Only aliens, with more advanced mechanical and mental abilities, could have designed and ...

The War over How Content Providers

The other opponents are the large electrification companies and the Internet Service Providers who will be referred to as Sips. These companies have a new business plan that if allowed to be implemented by the FCC will change the way Content Service Providers Caps and the end users, which are anyone using the internet today, will have their data routed and how they must pay for it. What will happen if the large Corporations win this â€Å"War† and how will it affect the status quo that we've all become used to as the Internet standard? Net Neutrality: The War over How Content Providers and Users Will Pay for Speed andRoutes of internet traffic. What is the real problem being debated? The new laws governing internet traffic if passed will give the large Sips the right to dictate how internet traffic is routed, and how they will charge the content service providers (Caps) and Internet end users (Sis's). Net neutrality has been a core principle of the Internet since its inception . According to (Vogue, 2014) Internet service should be very similar to telephone service. As an analogy, the phone company cannot make the connection poorer if they do not like the person you are ailing.The Sips and the large telecommunication companies don't like this concept and are working very hard to change it. Advocates of this policy are licking their wounds because the FCC recently reported they would likely leave Broadband services deregulated. Many activists for this movement had hoped that the Obama administration would not allow Internet Service Providers (Sips) to charge individuals by the amount of bandwidth they consume (Hudson, 2010). â€Å"Does this threaten freedom and openness on the Internet as net neutrality activist's claim? How would et neutrality impact future investments in broadband?Here are two opposing views on the issue† (Hudson, 2010). It seems as if the favoritism game has begun. A group of Internet service providers, mainly Commas has already begun to raise, and overcharge certain websites for their service. Yet other service providers who are a subsidiaries of theirs, like Hull who provide the same types of service have not had their fees increased (Cry, 2014) This is a growing concern of AN supporters. What is net neutrality? Law professor Www (2003) coined the term â€Å"net neutrality'. Lawrence (Lessee, 2001, p. 68175) can trace the idea of Internet neutrality back to the open access movement that was lead.The debate on AN centers on the potential consequences of network owners exercising control over the data traffic in their networks. The meaning of â€Å"control† can mean anything from blocking certain types of traffic (Www, 2007), to termination fees (Lee & Www, 2009), to offering preferred services to customers willing to pay a fee for it (Hahn & Wallet, 2006). To date, there is not a generally accepted definition of AN. Consumer rights groups have among others; put a strict definition f AN forth. The i nternet has developed at a tremendous rate of speed. It provides users with a platform for information, entertainment, and communication.The role of content providers has shifted to an essential gatekeeper position on the information superhighway. â€Å"Therefore, the public and politicians alike are concerned about how Internet service providers (Sips) are going to noontime access and usage of the networks in the future. The discussion on the future of the Internet is known as the net neutrality (AN) debate† (Kramer & Warrior, 2013, p. 1). Definition 1 Strict Net Neutrality. Net neutrality prohibits Internet service providers from speeding up, slowing down or blocking Internet traffic based on its source, ownership or destination.As mentioned above, the Sips are already planning to implement these prohibitions in their networks. This will endanger the â€Å"openness† of the Internet that has been the standard since its inception. (KerrГmere & Warrior, 2013). Defin ition 2 How AN Applies To Service Providers. â€Å"Net neutrality usually means that broadband service providers charge consumers only once for Internet access, do not favor one content provider over another, and do to charge content providers for sending information over broadband lines to end users. (Hahn and Wallet, 2006) The Pros and Cons The Cons: Sips can block any derogatory statements said about their company. They can block peer-to peer technologies, even those used by software developers used to enhance and grow technology. They can also block certain â€Å"Blobs† because of deals they have made with other higher paying â€Å"Blobs†. Just to name a few, and this list is growing by the day. If the Sips continue with the new business model they have landed they will not only change the face of the internet as we know it, they will lose all trust in the public and customer relations will suffer greatly.No one will trust or buy service from a company who wants t o undermine an institution standard that's been in place since its inception. Most customers will lose trust in them and the customer relations departments of the Sips will be working overtime to try to regain the publics trust and convince them to come back and be a loyal customer once again. I myself hope that it sparks a whole new line of smaller ISP startups who will epithelial on the publics distrust and resentment of the large Sips for betraying the publics trust.If I had the capital, I would start an ISP that would NOT use the new business model they so desperately want to implement. I am sure any company who could do this would grab a huge share of the market. The pros, which in my opinion are few and far in between, and are practically meaningless in my opinion. Congress claims its Constitutional authority to set interstate communications policy, the Constitution's protections, and court precedent, as well as encourage private investment and innovation Just proves Congress' bipartisan Internet policy.It fails to keep a competitive free market, which is not hampered by government regulation. Congress also claims it respects the rule of law, and it also encourages public and Private Corporation's to get the fastest broadband to all Americans under the National Broadband Plan. Smoke and mirrors I say. Legislation, Regulation, and Constitutional Rights Since 2005, the Federal Communications Commission (FCC) has been working towards a set of principles that will ensure the open and interconnected character of he Internet, a restriction to try to avoid the biased term AN.The FCC is seeking to maintain the current status quo and has followed the presented views in this section. There exist several examples of Sips that have blocked voice over IP (Poop) traffic, which is in competition to their regular telephone service. The most prominent example is that of Madison River Communications, which was subject to an investigation by the FCC in 2005 for exactly suc h practice. The case was settled under the old common carrier powers of the FCC, which applied at that point in time o DSL service (c. F. FCC, 2011).Traffic management techniques may be used by the ISP to avoid or limit traffic that, in their view, generates nothing but higher costs. Here, the most prominent example is that of Commas, the largest cable company in the US, which was subject to scrutiny by the FCC in 2008 because it had restricted the flow of peer-to-peer (APP) traffic. The FCC issued a cease or desist order against Commas in 2008, which was overturned by the US Court of Appeals in 2010, because it was found that the FCC has failed to tie its assertion of regulatory authority to an actual law enacted by Congress (McCullough, 2010).In its final Report & Order from December 2010, the FCC adopted the following AN framework. Definition 3 FCC. â€Å"A person engaged in the provision of fixed broadband Internet access service, insofar as such person is so engaged, shall 1 . Transparency â€Å"C†¦ ] publicly disclose accurate information regarding the network management practices, performance, and commercial terms [†¦ ]. â€Å"(FCC, 2010, Section 54) 2. No Blocking not block lawful content, applications, services, or non-harmful devices, subject to reasonable network management. (FCC, 2010, Section 63) 3.No Unreasonable Discrimination not unreasonably discriminate in transmitting lawful network traffic over a consumer's broadband Internet access service. † (FCC, 2010, Section 68) The FCC acknowledges the usefulness of reasonable network management, but also says that pay for priority arrangements will raise significant reasons for concern (FCC, 2010, Section 76). They also said that transparency and competition are the main remedies to ensure AN. It is also important to note that wireless network services are not subject to the restrictions of network management.The main reason for this is the competition between wireless network oper ators. Because the effect of competition is still unclear, it is going to be interesting to see whether the PC's AN ruling, which took effect on November 20, 2011, is going to lead to changes of the fixed and wireless networks in the US. The New FCC Rulings In January 2014, the DC Court of Appeals agreed with Verizon and said that the FCC cannot stop Internet service providers from blocking or discriminating against websites or any other Internet traffic unless the Internet is reclassified as a public utility.However, the court also said the FCC does have some authority to implement net neutrality rules as long as it promotes broadband deployment across the country. On May 1 5, the FCC voted to move forward with their proposed rules for net neutrality, the principle that all Internet traffic should be treated equally. The proposal, which is now open for public comment for four months, would dramatically change the Internet. The new rules would allow Internet service providers (Sips) like Verizon or AT&T to charge websites like Backbone and Twitter for faster service.This as a whole range of consequences for all avid Internet users. (Miranda. 2014) The Public Fear The public AN debate it is related to the fear that Sips may be in the position to limit the freedom of speech. Sips could block access to politically controversial (but legal) information, or shut down websites of unwanted organizations, Ex. The websites of labor associations to prevent an assembly of workers (Austin, 2005) Evidence of such practices is not necessarily true, because it will almost certainly cause a loss of reputation for the Sips.It seems obvious that such limitations of freedom of speech would be addressable by constitutional law of the respective country. However, people are aware that there are remarkable differences in the legal basis for preserving free speech online. The Other Side of the Coin Opponents say that strict AN would be taking a step backwards from the status quo of the Internet. If any network management practices are forbidden it could lead to congestion problems at peak times, which could only be counteracted by over provisioning of a networks capacity.In any case, Sips' revenues would be reduced because business models that rely on managed services, like PIPIT, could not be liable offered anymore. The likely result of this strict interpretation of AN would be that consumer prices for (full) Internet access will increase, or that the rate of investments in network infrastructure is reduced which will reduce the SO (Quality Of Service) we are all accustomed to. They also claim that customers with limited needs for internet access will not have the opportunity to purchase these services if they want to.Vice-president of the European commission Nellie Zeroes who said that â€Å"requiring operators to provide only full internet could kill innovative new offers Even worse, it could mean higher prices for those consumers with more limited needs w ho were ready to accept a cheaper, limited package† (Meyer, 2011). Conclusion In general what all of this means is that the Sips have an agenda to create a new business model. If the laws are changed that currently govern internet traffic, it can, and will change the way Internet access is routed and how the users are charged for it.However, for now, they are gunning for the website owner/operators or Caps (Content Service Providers) as they are also called, who provide content to the end users. Specifically the large Caps who rely on fast data transfer rates to provide customers with the services they offer. For Ex. Nettling which is a company that offers subscribers a service that allows them to instantly stream TV shows and movies would be put out of business if they did not agree to pay for their data to travel in the so-called â€Å"fast lane†.This type of service relies heavily on the fast transmission of data packets across a network in order to provide uninterru pted service. If the Sips and large telecommunication companies get their way they can restrict certain kinds f data/traffic at their discretion and direct it into the â€Å"traffic lanes† of their choosing. They wish to divide the Intervention into predetermined lanes of slow, medium, and fast data transfer speeds. Then charge Caps (Content Service Providers) according to the speed that they want, or essentially need their data to travel at.Step 2. Focus on the accuracy of the assumptions and conclusions. I used the scoring guide and the instructions for the assignment to ensure that I have met the requirements and feel confident with my submission. Step 3. Break the problems into workable parts. I used the discussion posts and suggestions of the other classmates as well as the instructor's comments to tackle each perceived item that was recommended to me that needed revision.I approached this by looking at them as a whole and then breaking them down and working on them one at a time. Step 4. Do not guess or Jump to conclusions. I feel I did not Jump to conclusions by using the many credible sources and references to Justify my conclusions used in my draft. Step 5. Employ meaningful self-dialogue throughout the process, including written or drawn prompts as well as spoken words. I'll be quite honest here I still have not mastered how to accomplish this step.I do not talk out loud to myself but do talk silently to myself while writing to make sure my words sound correct and flow nicely together, so in a sense I guess I do implement this process in that way. Step 6. Briefly describe what it felt like to go through the process. Going through this process is a constant learning experience for me. I'm realizing that as my paper develops I find my steps of using the critical thinking process are beginning to change. I'm not sure if this is a good thing but I have noticed a difference in my processes from the beginning until now.

Moss and Mcadams Accounting Firm

RUNNING HEAD: MOSS AND MCADAMS Assignment #1: Moss and McAdams Accounting Firm Strayer University Bus 517 Bruce Palmer was a good guy looking to make a difference. He was lead to believe that Zeke Olds was going to be available to him throughout the project and that was not the case. He was led astray and betrayed by Ken Crosby, a new guy to M&M. Crosby knew if he made the case to Sands early, that he would get his way. The client was one that M&M was competing to get with two other big 5 accounting firms and since Crosby came from a Big 5 accounting firm, M&M was going to give him whatever he needed to complete the project.Crosby was recruited specifically to manage special projects and this qualified as such. Even though Olds was already slated to work on the Johnsonville audit, Sands was persuaded by Crosby to let Olds work on his team due to the expertise that was essential to the Springfield project. If I was Palmer I would have talked to management about how the process is bein g handled and offer up a suggestion so that no other project manager would be misled by another. Palmer should have stood firm with Crosby from the beginning and maybe there would not have been a major issue along the way.He waited too long to talk with Sands and it proved that Palmers passive way was his demise in dealing with Crosby and ultimately lost him Olds on his project. Once Olds started to do consulting work he realized that auditing was not as fun as consulting work. I do not believe Palmer could have anything to prevent losing Olds. Olds might have had good intentions but he showed a lack of focus when he started showing up 30-60 minutes late. Palmer should have scheduled a meeting with Olds and try to figure out what Olds wanted to do. A mistake on Palmer’s part was never sitting down with Sands and telling her what was going on.There may have been no change but at least he would have stated his frustration and put the responsibility to resolve the issue with San ds. The lack of focus on the project by Olds being stretched in by two teams and conflicting priorities caused Olds to feel stressed. Palmer should have prioritized Olds objectives and set clear deliverables for him to measure up against. Had he told Olds what was expected he could have got ahead of the problem but instead he let Crosby interfere and drive Olds away from his project. Crosby never had any intention of letting Olds work on both projects.He made it clear to Sands when he first started that Olds was critical to the project and he wanted him on his team. Sands went along because Olds is a valuable player in the office and felt that it would be a good match. The problem with this situation is that Olds was being pulled in too many directions and not allowed to focus on projects that suited his talents. This is often the case in matrix type organizations. The disadvantage of a the matrix organization is instead of delegating segments of a project to different unit or creat ing an team, project members report to simultaneously to both functional and project managers.Having resources shared across multiple projects and divisions sounds like an efficient way to divide the energy of individuals across multiple projects on an as needed basis is a disadvantage as seen at M&M. Olds was divided across multiple projects and ended up not being very efficient. Have a strong project focus is a clear advantage of a matrix organization because it provides having a formally designated project manager who is responsible for coordinating and integrating contributions of several units.This helps sustain a holistic approach to problem solving that is most often missing in functional organizations. Although this is an advantage, it was not the case at M&M. The lack of project focus resulted in one project lacking the resource to complete the project. There was no coordination or integration between the two teams and resulted in two project managers fighting over one empl oyee. The dysfunctional conflict between Crosby and Palmer opened up the tension between the two managers. There was a legitimate conflict that arose from conflicting agendas and responsibilities.The Springfield project was clearly a higher priority than the Johnsonville audit and Sands should have told Palmer, that although he is valuable to the audit, he would be more successful for the Springfield project. Palmer could have pulled another resource to take Olds place or hire a contractor to fill in the time when Olds would not be available. Olds was caught in a stressful situation where he was working in an environment in which he was being told to do two different tasks and had two different agendas by two different managers.This all led to infighting over Olds being shared across both projects and the two managers competing over for one resource. Palmer and Crosby were only concerned about their respective projects and Crosby knew what his end game was all along. Palmer was not clever enough to outwit Crosby and therefor he lost out on Olds. M&M management clearly needs to set priorities and objectives for projects. The matter in which Sands handled Olds splitting time between two managers shows the type of organizational culture at M&M.The matrix is one part of the equation but the culture is how that matrix is driven. The type of culture in M&M lends the opportunity for Crosby to manipulate another manager for his personal gain. Sands knew that the Springfield project was high profile and Crosby was hired from a Big 5 firm for projects like these. Sands should have taken the time to hear Palmer’s concerns and make an informed decision. She only heard from Crosby and was told by Olds and with no regard for Palmer, made a decision to move Olds to Crosby’s project to satisfy the organization at the cost of another project.The norm, customs, shared values, and the â€Å"rules of the game† for getting along and getting ahead in the M&M or ganization are clear to see from this project. They need to change how they select and assign project staff to multiple projects. They need to develop a system that appropriately balances the needs of the project with those of the organization. They could send out a survey to poll the employees to see how they would like to be selected for a project in order to get employees that wanted to do certain types of projects. Culture encourages the implementation of projects.

Free Essays on Ancient Egypt

A. Summary: Many different kings and pharaohs governed Egypt. The kings and pharaohs were grouped into thirty-one dynasties. These dynasties were divided into smaller categories, which consisted of an â€Å"Old Kingdom†, â€Å"Middle Kingdom†, and â€Å"New Kingdom†. The basic assumption of Egypt and pyramids came from the Old Kingdom. Ancient Egypt became famous for having the longest river in the world, the Nile. It was the â€Å"most important feature of life in ancient Egypt† (page 10). The country’s fertile fields and food resources such as poultry and livestock relied on the soil of the river. The Nile was where the abundant amount of fish and nourishment came from. This body of water attracted many outsiders. Most of which were from Asia. Egyptians reacted to the Asian outsiders with disrespect. Although the Asians were not accepted at first, once they settled into Egypt, they could obtain jobs and marry Egyptians. The exchange of foreign g oods and services among countries was controlled entirely by the Pharaohs. After the Nile River, the main source of food was fish. The quantity of fish was so high, Ancient Egypt was then known as â€Å"a land of abundance† (page 60). Along with fish, agriculture became popular at this time period. The agriculture relied greatly on the Nile River as a source of fertility for the land. Another source of food was wild cattle that were hunted. The hunting procedure dropped soon after agriculture came into place. Egypt had the richest of lands; The soil contained stones such as gold, copper, malachite, alabaster, limestone, and granite used in building monuments. Society in Egypt was almost the total opposite of other Middle Eastern countries. Along with men, women’s positions depended upon their fathers and husbands. Women were ranked according to their husbands’ positions, but they were in their own economic status. Women had the right to own or rent property, engage in business, and trade ... Free Essays on Ancient Egypt Free Essays on Ancient Egypt Ancient Egyptian Agriculture There are many valid points to be made in Ancient Egyptian agriculture. Irrigation, ploughing and planting, harvesting, and of course, crops. These will be some of the subtopics I will be touching upon in this essay of ancient Egyptian agriculture. Irrigation When the Nile is overflowing, it floods the Delta and the lands called Libyan and Arabian, for a distance of a journey of two days from both banks in places, and sometimes, sometimes less. I could not learn anything about its nature, neither from the priests nor from anyone else. I was curious to learn why the Nile is flooding for a hundred days from the summer solstice; and when this time is passed, sinks again, and the river is low during the whole winter until the summer solstice again. -Herodotus, Histories 2,19 Above, is a quote from a man recovered from an article of writing back in the ancient Egyptian times. Irrigation is a form of re, there are two crops, one crop is getting all the water, and it’s flooding. With irrigation, the farmer will re-route the water towards the other crop, as well as sharing the water with the crop that was being flooded. So now, both crops are getting enough water and they are not flooding nor suffering from drought. Natural river irrigation shaped the early landscape of ancient Egypt. Drainage was not required for the Valley to become liveable. With the natural flooding and draining of the floodplain, the annual flood allowed a single crop-season over two-thirds of the alluvial ground. Once the main canals, many of them natural, were in place, they just had to be scoured yearly to prevent their clogging up. The levees had to be raised, and smaller ditches had to be re-excavated. Organized by the regional authorities, every Egyptian had to move about thirty cubic metres of soil in about ten days every ... Free Essays on Ancient Egypt A. Summary: Many different kings and pharaohs governed Egypt. The kings and pharaohs were grouped into thirty-one dynasties. These dynasties were divided into smaller categories, which consisted of an â€Å"Old Kingdom†, â€Å"Middle Kingdom†, and â€Å"New Kingdom†. The basic assumption of Egypt and pyramids came from the Old Kingdom. Ancient Egypt became famous for having the longest river in the world, the Nile. It was the â€Å"most important feature of life in ancient Egypt† (page 10). The country’s fertile fields and food resources such as poultry and livestock relied on the soil of the river. The Nile was where the abundant amount of fish and nourishment came from. This body of water attracted many outsiders. Most of which were from Asia. Egyptians reacted to the Asian outsiders with disrespect. Although the Asians were not accepted at first, once they settled into Egypt, they could obtain jobs and marry Egyptians. The exchange of foreign g oods and services among countries was controlled entirely by the Pharaohs. After the Nile River, the main source of food was fish. The quantity of fish was so high, Ancient Egypt was then known as â€Å"a land of abundance† (page 60). Along with fish, agriculture became popular at this time period. The agriculture relied greatly on the Nile River as a source of fertility for the land. Another source of food was wild cattle that were hunted. The hunting procedure dropped soon after agriculture came into place. Egypt had the richest of lands; The soil contained stones such as gold, copper, malachite, alabaster, limestone, and granite used in building monuments. Society in Egypt was almost the total opposite of other Middle Eastern countries. Along with men, women’s positions depended upon their fathers and husbands. Women were ranked according to their husbands’ positions, but they were in their own economic status. Women had the right to own or rent property, engage in business, and trade ... Free Essays on Ancient Egypt Ancient Egypt Between 3100 and 332 B.C was the rise and climax of one of the richest and oldest ancient civilizations. It’s lifeline was the Nile river in the Nile valley. Here, Egyptian dynasties ruled from the first cataract of the Nile to the Mediterranean Sea. At the it’s height it ruled an empire that reached from Syria in the east to Nubia in the south. In this report I will be covering the Archaic Period, the Old Kingdom, the Middle Kingdom the New Kingdom and The Late Period or 3100-332 B.C. Archaic Period: 3100 B.C to 2750 B.C There long history began with there first King who began the first Egyptian dynasty. In 3100 B.C Pharaoh Menes united upper and lower Egypt. Making Egypt’s first empire. In doing so, he made the Egyptian double crown. It was made by putting the red crown of Lower Egypt on top of the white crown of upper Egypt. Menes ruled from the ancient city of Thinis near Abydos. Under his reign the first hieroglyphic writing was made. He is also credited with making his empire interdependent. Old Kingdom: 2750 B.C to 2181 B.C / First Intermediate Period: 2182-2260 Little is known about Menes successors until the reign of Zoser at the end of the 3rd dynasty. His capital was located at Memphis on the Nile’s west bank. He built the world’s first pyramid and the first building of that size to be entirely made of stone. Even though it was a pyramid it wasn’t a true pyramid, but a step pyramid. After the reign of the last king of the Sixth dynasty (the last dynasty in the old kingdom.) Pepi II in 2181 B.C, there was a period of crisis and social upheaval known as the First Intermediate Period. The reasons leading up to this dark time, was a series of low floods and the result was famine during the Sixth dynasty. This undermined the stability of Egypt and provoked rebellion. What followed put Egypt in rapid decline. With no central power the provinces b...

MSAT Compare and contrast three old testament kings Essay

MSAT Compare and contrast three old testament kings - Essay Example The first king of the united kingdom of Israel was Saul. Saul was born in 1079 BC, at Gibeah in Judah (History of Israel Kings, n.p). He was a farmer from the tribe of Benjamin, and was not expecting to be king of Israel because he was a Benjamite, the least tribe of Israel, and his family was the least of all the families of the tribe of Benjamin (1Samuel 9:20). Saul was a tall handsome young man (1Samuel 9:2). He was appointed the king of Israel when the Israelites demanded for a human whom they could physically approach and relate to (1Samuel 8:5, JKV). Saul reigned in the period between 1050-1010. David on the other hand was a heard boy from the tribe of Judah the Judah; David was born at Bethlehem in Judah. David was born in the year 1040, when Saul was the king of the united kingdom of Israel (History of Israel Kings, n.p). Just like Saul, David was not expecting to be the king of Israel because he was a little boy, and Saul was still in power as the king of Israel; David was t he youngest of the eight sons of Jesse and he was handsome (1Samuel 16:11-12). King David ruled Israel between 1010-1002. Un like King Saul and King David, King Solomon was raised in a royal environment and he was a prince; King Solomon was the son of King David (1 Kings 2:12). King Solomon therefore was destined to be king of Israel unlike Saul and David. But just like King Saul and King David, King Solomon was from Judah, he was born at Jerusalem in Judah. Solomon reined Israel between the years 970 -931 BC (History of Israel Kings, n.p). Having analyzed the historical background and the beginnings of kings Saul, King David and King Solomon, let us now look at how these three kings of the united monarchy of Israel were loyal and faithful to God. When Saul was appointed the king of Israel, he was loyal to God and God had destined him to liberate the Israelites from their enemies, the philistines (1Samuel 9:15,

Edward Estlin Cummings Essay Example | Topics and Well Written Essays - 750 words

Edward Estlin Cummings - Essay Example four lean hounds crouched low and smiling the merry deer ran before. †¦my heart fell dead before (Cummings lines 1-5/ 31-35). The last line varies at the end of the poem from â€Å"the merry deer ran before† to â€Å"my heart fell dead before.† Even with this slight variation, the repeated lines have a magic effect of overturning the theme of the poem from tragic to that of vitality and life. Two most evident details in this repeated verse is the use of colors and numbers for the purpose of description. The persona is describing the green garb of his lover and the golden color of the horse as well as silver dawn. These details chronicle the beautiful atmosphere of the lovers at the time. There are also numbers, which are repeated throughout the poem. In this specific stanza, the persona talks of â€Å"four lean hounds† of deer (Cummings, line 4). The numbers and the colors are a combination of beauty and terror, especially when the carrier resembles the war carriers that take soldiers to war (Bloom 22). At some point in the end when the lines are repeated, the poem draws the beauty of the described place and the lover by engulfing them in an atmosphere of death. The poem describes from the start a chronological event where it first describes the chase and the death of the persona and the collapse of his lover. These images are alluded to but when the repeated lines appear the poem takes the form of vitality and life. As the two lines unite the poem, the lovers are unified in elation. The colors green is the color of life and connects the lovers who are partakers in the succession of life and fatality. B: Edward Estlin Cummings. Xaipe/Seventy-one poems The poem selected from this collection is titled â€Å"I thank You God for most this amazing.† The poem is like praise lyric made whole by playing with words, grammar, punctuation and syntax (Bloom 28). For example, there is lack of spacing and punctuation, which make the speaker, speak without breathing; thus showing the intensity of his overwhelming joy and appreciation. With only four stanzas, the first stanza captures the spirit of the rest of the piece, and it goes thus: i thank You God for most this amazing day:for the leaping greenly spirits of trees and a blue true dream of sky;and for everything which is natural which is infinite which is yes (Cummings, lines 1-4) In this stanza, the speaker describes the day as amazing with the â€Å"spirit of trees† and a sky of â€Å"blue true dream† with â€Å"everything† ordinary and inestimable and â€Å"yes.† By writing continuously without spacing, regardless of the presence of punctuation, the poet renders the speaker breathless. Reading the poem kills it and gives it a new life afterwards when it is reborn as the speaker reunites and communicates with Mother Nature. Like a small child, the speaker describes the new life in terms of his newly founded cognizance because he is rebor n. The first stanza is precedence to what has already happened, and the speaker is joyous of the renewed sun, life, love and wings; including the earth itself. The praise is merely a subjective experience because the speaker renders everything new because of his personal renewal. With rebirth or a new perspective in life, things become new and one is able to picture things from a different point of view. The persona establishes a personal deity, â€Å"