Bus 303 Practice Midterm Essay Example for Free

Bus 303 Practice Midterm Essay Characters: Cathy and Dave, a well-to-do professional couple Al, a real estate salesman Cathy and Dave are young, upwardly mobile. They hold good professional jobs in downtown Chicago. One day Cathy received an invitation in-the mail, from a resort called Green Acres (GA), which was located near the mountains, about eight hour driving time from Chicago. The mailer invited the young couple to spend two nights free of cost and receive $50 for expenses. All they had to do in return was to listen to a presentation, see a video, and take a tour of the resort for about two hours. Additional conditions included 24 hour advance booking of the room with 24 hour notice for cancellations, and a valid credit card. Green Acres would charge $50 for a no show. Although the letter made no mention of it, Cathy and Dave knew the invitation was a real estate promotion and guessed that the two hours with the GA staff meant a hard sell for a condo or a timeshare near a lake or a golf course, something they did not want. They decided to take the offer anyway in order to get a free vacation and arranged to visit GA the following weekend. Cathy and Dave had a good time at GA. The only sour point of the trip was the last exchange they had with Al the salesman. At the end of the two hour presentation when Cathy and Dave had refused to buy any GA properties, Al looked at Dave in frustration and said, â€Å"If you knew you weren’t going to buy any property here, why did you come? Our company spent $300 to get you down here, and you have taken food off my family’s table. What you did is immoral. Please don’t do it again. At this Dave retorted, â€Å"Your letter was clear, there was nothing in it that said a purchase of real estate was involved, and we had no obligation to buy anything from you. Your invitation was unconditional, I don’t owe you or your family anything and I resent what you just said.† At the end of this uncomfortable exchange, Cathy and Dave left the room with an unpleasant feeling. But the unpleasant feelings did not last long because the drive back through the mountain s was truly spectacular. After a few days Cathy said to Dave, â€Å"You know, Im still mad about what Al said to us at Green Acres. Should we write to his boss, or to the real estate board or to some government agency? You know, if we don’t do something, some poor unsuspecting people might fall for their gimmicks!† Dave replied, â€Å"No, I dont think we should waste any time on this. Most people know, or should know what they are getting into-there are no free lunches! Besides if we complain, Al may lose his job and when we’ll be really taking the food from his family’s table!† What Are the Relevant Facts? 1. Cathy and Dave are educated and well paid. 2. They received an unsolicited invitation from Green Acres (GA). 3. The invitation had nothing in it that said they had to buy anything from GA. 4. They correctly guessed that the GA sales staff would put pressure on them to buy real estate. 5. GA was eight hours driving time from Chicago. 6. Al made a sales presentation. 7. Cathy and Dave listened to the presentation. 8. Al said Cathy and Dave had acted immorally and had deprived him of a chance of earning a sales commission. 9. Dave said that they had fulfilled their obligation, as outlined in the invitation, and had not acted incorrectly. What Are the Ethical Issues? 1. What is the role of inducements in marketing? 2. What is the responsibility of individuals who accept inducements? Do they have any moral obligations to purchase goods if they accept free gifts from merchants? 3. Is giving attractive gifts to potential buyers an ethical practice? Does the size of the gift or the inducement matter? 4. Cathy and Dave were sophisticated, educated city folk and did not feel pressured to buy from GA. Do less educated or less sophisticated consumers feel pressured to buy merchandise under enticements of gifts? Would such business practice be considered ethical? 5. Should Cathy and Dave complain so that other vulnerable people may not be pressured into buying expensive property they do not want? Who Are the Primary Stakeholders? †¢ Cathy and Dave †¢ Al †¢ GA †¢ GA’s stockholders †¢ Other potential consumers, especially the vulnerable ones What Are the Possible Alternatives? 1. Cathy and Dave can forget the incident and do nothing. 2. They can file a complaint with GA. 3. They can file a complaint with the appropriate authorities. 4. They can write to Al. What Are the Ethics of the Alternatives? 1. What is the best course of action for Cathy and Dave from the moral standpoint? What is the best course of action that Cathy and Dave take that will provide the greatest benefit to the greatest number? 2. Do Cathy and Dave and other potential customers have rights not to be pressured or induced into actions they may not wish to take? Do GA and Al have rights to pursue their business and personal interests? Were any rights violated? 3. What is the just thing to do in this case? Which alternative distributes the burdens and responsibilities fairly? If Cathy and Dave act and if GA improves its practices everyone except Al may benefit. Not complaining may mean Al will continue his tactics and undermine GA’s goals assuming that word would get around and additional customers will be offended. What Are the Practical Constraints? None. What Actions Should Be Taken? 1. What should Cathy and Dave do? 2. Which alternative would you choose? 3. Which approach (utilitarian, rights, or justice) makes the most sense to you in this situation? Clearly, no action could mean unsuspecting people may be lured into buying expensive real estate with potentially severe consequences. Complaints could lead to improved practice and better performance but could lead to the loss of Al’s job. 4. What is the right thing to do?

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

The Integration Of Faith And Learning Education Essay

The Integration Of Faith And Learning Education Essay The integration of faith and learning (IFL) has been defined as a multifaceted attempt to distinguish the underlying truth about ones academics and Christianity research study by Garzon, Milacci Boothe (A Phenomenology of the Integration of Faith and Learning). Various IFL models have been developed by scholars, in which some have focused on the interdisciplinary engagement between theology and academics, and others on the intrapersonal and interpersonal elements (Research study by Garzon, Milacci Boothe (A Phenomenology of the Integration od Faith and Learning). Research studies in the past on have been conducted with the instructors rather than the students. But current studies have been on the students perceptions of IFL experiences. Students have referred to IFL in various studies in terms of the methods used by the instructor as opposed to the learning of integration by the student. They described IFL as primarily being a teacher activity and not the result of the students behavior (Lawrence, Burton, Nwosu (Refocusing on the Learning). I ask, as Sherr, Huff, Curran did in their research study, What good are our efforts as Christian professors to provide IFL experiences if student do not sense these efforts? And if our efforts to provide IFL experiences are incongruent with the expectations of IFL experiences of students, how can we identify those differences and adapt to provide Christ-centered education? (Research study by Sherr, Huff, Curran (Salient Indicators of Integration). The results from various studies have shown that overall most students desire more real life examples by applying theories to real life situations. They want the opportunity to integrate their faith into their academics through assignments and practicum (Research study by Hall, Ripley, Garzon and Mangis (The Other Side of the Podium). The research conducted by Hall, Ripley, Garzon Mangis have found that students learn integration though relational attachments with mentors who personally model that integration. Students valued the quality of the attempt made by the instructor to integrate over the mere act of integration itself. Though they took note of attempts that were half-hearted, insincere, done out of duty, forced, or of poor quality ( all, Ripley, Garzon and Mangis (The Other Side of the Podium). The participants in this study identified three areas that they perceived facilitated integrative experiences in the classroom. The first was the teaching method the instructor used to integrate. They identified five traits of the professor as being significant in effective integration: self-revealing (evidence of an active, personal relationship with God that was observable by the students), caring, welcoming, dedicated and open-minded (the professor must demonstrate genuine care for the students). The second area was in curriculum. The students perceived successful integration when time was intentionally allotted for prayer and integrative discussions and assignments. Sherr, Huff, Curran found that some students did not perceive prayer as being an important factor (Sherr, Huff, Curran (Salient Indicators of Integration). The third area was in the overall climate of the institution. The students welcomed the opportunity to express their Christianity openly and simultaneously with their academic learning. They expressed their satisfaction in not having to separate the two ( Hall, Ripley, Garzon and Mangis (The Other Side of the Podium). Garzon, Milacci Boothe (A Phenomenology of the Integration od Faith and Learning) research findings suggested that students perceived that integration can only occur within a caring relationship with a mentor (in this case a professor, administrator, etc). The mentor must be an individual who has spiritual depth and is able to integrate it both inside and outside the classroom. This would require the professor to take the time and effort to get know each student personally, placing a strong emphasis on the process of learning as opposed to solely the concept of learning. Garzon, Milacci Boothe (A Phenomenology of the Integration od Faith and Learning). This proposes the importance of the type of professors that are hired with the intention of implementing IFL in the classroom (Research study by Hall, Ripley, Garzon and Mangis (The Other Side of the Podium). Lawrence, Burton, Nwosus research concluded that students perceive IFL in terms of the content of the teachings through the methods used by the professor. Most gave examples in terms of the professors actions rather than the students activity. The methods utilized active learning approaches that gave the student opportunities to mentally manipulate and develop ideas (role play, inquiry training). The top three categories were teaching methodologies (conversations with the professor during class time), making connections (the teacher playing an active role in connecting faith with the area of study),and the atmosphere of the classroom (positive, safe, feels accepted and supported) Lawrence, Burton, Nwosu (Refocusing on the Learning). The research of Sherr, Huff, Curran (Salient Indicators of Integration) pinpointed two main indicators of IFL: faculty relationships with God and students, and faculty competence. In terms of the professors relationship with God, the students needed to indentify the professor as being a genuine Christian with a passion for their relationship to the Lord. They also looked for accountability and commitment to God. In terms of the professors relationship with the students, they believed the instructor should demonstrate a genuine care for every aspect of the students live- physical, emotional and spiritual. This was connected to the amount of genuine time and interaction in and out fo the classroom the professor spent with the student. These were individuals who were concerned about helping the student personally develop (Sherr, Huff, Curran (Salient Indicators of Integration). When it came to the professors competence and curriculum, the students belied the professor not only had to be competent, but also able to implement IFL experiences within the curriculum. They made references to using scripture as a reference, assisting students in applying Christian faith in different settings and the professor sharing their personal faith experiences. The students also mentioned the classroom environment as an indicator of IFL. They indentified a sense of belonging acceptance and commitment within the classroom is important. Prayer in the class was not necessarily agreed on as being an important factor (Sherr, Huff, Curran (Salient Indicators of Integration). Reports have indicated that there is no significant learning difference when students are taught at a distance as opposed to traditional face-to-face instruction, though attrition can be higher and satisfaction lower (2006). A blended training approach using videoconferencing for distance education. AND (Research study by Spector (Time Demands in Online Instruction) AND Research study by Muilenburg Berg (Student Barriers to Onlin Learning). Though some argue that the interaction through traditional instruction has the potential for greater social presence, research has shown that online communication does permit high levels of interpersonal communication, thus social presence Research study by Beuchot Bullen (Integration and Interpersonality). There are significant differences in the way students perceive their online learning experiences (Research study by Muilenburg Berg (Student Barriers to Onlin Learning). Asynchronous instruction is independently experienced by the student and does not require simultaneous participation of the student and instructor. It gives every student an opportunity to respond to a topic and encourages more in-depth discussions. It is a more inclusive type of interaction. It limitations include feelings of isolation from students, lack of quick feedback, and not enough time for discussions to mature. This method requires more time to manage and provides less social interaction than synchronous instruction.(Research study by Johnson (Synchronous and Asynchronous CMC) 2006, TechTrends. Synchronous instruction requires simultaneous involvement between the professor and the student. This method allows multiple users to communicate in real time. Limitations include getting the students online at the same time and moderating conversations with larger groups. They are more difficult implement than asynchronous, though it generates a greater sense of presence and spontaneity. Chats and chat rooms can be utilized with this method (Research study by Johnson (Synchronous and Asynchronous CMC) 2006, TechTrends. Research study by Johnson showed that students did not report a clear preference for synchronous or asynchronous instruction. 43% of the variance in perceived learning rested in the difference of the students perception of learning. Both forms have advantages and both contribute to student cognitive and affective outcomes. Though the students characteristics seemed to affect asynchronous online behavior and discussion. Perhaps the two methods can be blended in a systematic and object manner in order to maximize the students learning experience (Research study by Johnson (Synchronous and Asynchronous CMC) 2006, TechTrends. There are significant differences in the way students perceive their online experiences during learning (Research study by Muilenburg Berg (Student Barriers to Online Learning). Results by Johnson have shown that there is no evidence of instructional superiority between the two methods- student achievement was equivalent. Although synchronous is often assumes to be more interactive- students perceptions of such may differ. The reported advantages/disadvantages of both methods may reflect the students personal preference due to characteristics. Students who prefer synchronous over asynchronous text-based computer mediated communication (CMC) may be more social than students who express the reverse preference. Students who expressed the perception that they learned the case studies best when using synchronous chat were at a learning disadvantage when using that CMC mode (Research study Johnson (20008- The Relative Learning Benefits of synchronous and asynchronous). Ng Murphy reported that Some students in their study took an active role(ex: initiating discussion topics and responding to prior contributions), and their involvement did not rely on tutors inputs . Ng Murphy (Evaluating Interactivity and Learning) . This suggests that another factor, like personality, was the cause for such participation. Having two-way audio and video, enabling the students and teachers to see as well as hear each other in real time, is better than those in courses where communication is only one-way. This may increase the sense of social presence, increasing course satisfaction. Blending asynchronous and synchronous methods may eliminate attrition and isolation problems that affect distance education learners (2006). A blended training approach using videoconferencing for distance education Research by Muilenburg Berg (Student Barriers to Onlin Learning) found eight barriers to online learning perceived by students: administrative/instructor issues, social interactions, academic skills, technical skills, learner motivation, time and support for studies, cost and access to the internet, and technical problems. The most critical barriers were social interaction, administrative/instructor issues, learner motivation, and time/support for studies. A lack of social interaction was most severe barrier perceived by students. Findings show that social interaction is strongly related to the effectiveness of learning and course enjoyment. (Research study by Muilenburg Berg (Student Barriers to Onlin Learning). Research study by Beuchot Bullen (Integration and Interpersonality) suggests that interactivity comes about through inner process variables, for example, the interpersonal content of messages. This suggests that online interaction participation may be determined by the type and degree of a students socio-affective content in postings, therefore implying that online instruction should focus more on the development of the socio-emotional climate of virtual communities. It has been shown (Rafaeli and Sudweeks, 1998) that overall, interactive messages are likely to contain self-disclosure, indicating that interactivity may bring about a sense of involvement and belonging. This may be done through allotting additional time for interpersonal connections between students and between the student and professor. One suggestion would be to ensure that students make meaningful interpersonal connections before they engage in cognitive tasks (Beuchot Bullen (Integration and Interpersonality). Online teachers invest significantly more time than traditional face-to-face instructors. Online teachers at various universities believe that preparing and teaching online courses places more stress on the instructors. Research study by Spector (Time Demands in Online Instruction). Research by Dennen indicated that instructors balanced their own need to manage time with the affirming students through regular feedback, but the task was very overwhelming Dennen (From Message Posting to Learning). Based on research results, professors teach online courses out of personal interest, mainly for flexibility. Research study by Spector (Time Demands in Online Instruction). Again rises the importance of carefully selecting professors for clear goals of integration.(Research study by Hall, Ripley, Garzon and Mangis (The Other Side of the Podium).   Course designers and instructors in distant education need to have and understanding on how students interact and how groups develop in computer mediated environments. Research study by Beuchot Bullen (Integration and Interpersonality) Online instruction needs to create and interactive environment for students and teachers through CMC. The Challenge is to balance independent study and bringing each student into contact with others, which is essential in forming any community (interaction/interactivity) (Ng Murphy (Evaluating Interactivity and Learning. Dialogue consists of at least two discussants who communicate in repose to each other. Research conducted by Denned noted that composing and posting messages did not necessarily result in dialogue. Students were going through the motions of dialogue but all aspects were not present.Dennen (From Message Posting to Learning). Conversational treatment by the instructor generated higher quality student contributions, Dennen (From Message Posting to Learning). It has been suggested that moments of conversation within the forum need to be carefully designed themselves- the strategies need to support students construction of meaning. It may not just matter how much instructors post messages that affects class dialogue, but also how why compose those messages. Distant learning activities must be relevant and goal based in order to attract student participation. The learning objective and relevance should be made clear to the students. Socialization can also incite participation. The discussion prompts that where effective in prompting student dialogue had clear guidelines and gave each student the opportunity to have a unique response. The instructor feedback also motivated students to participate more. Dennen (From Message Posting to Learning) The Research study by Ng Murphy (Evaluating Interactivity and Learning) results were that the number of genuinely interactive messages were limited. There was little evidence of the students participating collectively and building upon each others contribution. The learners DID NOT use forum to share experience or self-reflections. Though evidence of cognitive skills in their responses was evident, higher-order reasoning skills- such as critical thinking and problem resolution- were not demonstrated. (Ng Murphy (Evaluating Interactivity and Learning) Research Proposal Research Rational Research studies have given us an idea of some of the primary indicators of IFL experiences perceived by students: relational attachments with mentors Ripley, Garzon and Mangis (The Other Side of the Podium).; Teaching methods with an instructor who is self-revealing, caring, welcoming, dedicated and open-minded ( Hall, Ripley, Garzon and Mangis (The Other Side of the Podium); within a caring relationship with a mentor Garzon, Milacci Boothe (A Phenomenology of the Integration od Faith and Learning); faculty relationships with God and students, and faculty competence Sherr, Huff, Curran (Salient Indicators of Integration). Research has also shown that there is no difference in online and traditional face-to -face learning, as well as no significant different between synchronous and asynchronous teaching methods. This study asks the question, What are distant learners perceptions of efficient faculty efforts in facilitating IFL experiences using current and intentionally increased and focused IFL goals, and current and various CMC (Computer Mediated Communication) methods? Purpose of Research The purpose of this study is to assess whether the same indicators of IFL experiences are perceived by distant learners as traditional, face-to-face learners. If not, what are some of the different indicators? Although studies have proven no significant difference in asynchronous and synchronous methods in learning, we will assess whether or not there are differences when it comes to the students perceptions of IFL experiences. Also, we will increase the focus and goals of IFL experiences, making them intentional and known to the students and assess if there is any differences between intentionally increased and focused methods of IFL goals and the current ones the institution uses. The theoretical implication will be to compare the institutions current IFL goals and methods with the same goals but using primarily asynchronous methods, synchronous methods, and finally a balanced blend of both methods. Then increase the IFL goals and compare them to the same increased goals but using primarily asynchronous methods, and then synchronous methods. The results from the current goals will be compared to the intentionally increased and focused goals. Method Participants The study will be conducted over the course of one complete year, consisting of a 4 consecutive semesters (beginning with a summer semester and the following fall, spring, and summer semesters).Undergraduates and graduate students from three Christian higher education institutions will be surveyed. The institutions will be from different regions of the US, as well as from difference Christian denominations. There will be 8 professors used (2 female undergraduate professors, 2 male undergraduate professors, 2 female graduate professors, and 2 male graduate professors) to implement the current IFL goals and the intentionally increased and focused goals. There will be 8 courses (4 undergraduate and 4 graduate) randomly chosen from 4 various schools of study (education, arts and science, business, religion, etc.) each institution offers. Materials A survey questionnaire will be given to each student to fill out during the last week of the course. It will consist of open-end questions. This method of data collection will be identical between each institution and will be collected over the course of four consecutive semesters. Design Qualitative data will be obtained by having each participant fill out a survey questionnaire. The survey will consist of open-ended questions. in order to give each student an opportunity to give their perception and initiate candid responses. Procedures This experiment would be conducted in 2 parts: Study A and Study B. Study A will be conducted in three Christian institutions, assessing eight courses: four (2 undergraduate and 2 graduate) analyzing current IFL goals and various CMC teaching methods and four (2 undergraduate and 2 graduate) analyzing intentionally increased and focused IFL goals and various CMC teaching methods. Study A Analyze the current IFL goals using the institutions current methods in a Summer Semester, 20YY. Study will have no control over this particular instructor (male or female). Analyze the current IFL goals using mainly asynchronous CMC methods in the Fall Semester, 20YY. Study will have control over instructor (male or female who will be trained in Asynchronous methods). Analyze the current IFL goals using mainly synchronous methods in the Spring Semester, 20ZZ. Study will have control over the instructor (male or female who will be trained in Synchronous methods). Analyze the current IFL goals using a balanced combination of asynchronous and synchronous methods in the Summer Semester, 20ZZ. Study will have control over the instructor (male or female who was trained in a balanced combination of asynchronous and synchronous methods). Study B Analyze intentionally increased and focused IFL goals using current methods simultaneously in study As Summer Semester, 20YY. Study will have no control over this particular instructor (male or female). Analyze intentionally increased and focused IFL goals using mainly asynchronous methods simultaneously in study As Fall Semester, 20YY. Study will have control over instructor (male or female who will be trained in increased IFL goals through asynchronous methods). Analyze intentionally increased and focused IFL goals using mainly synchronous methods simultaneously in study As Spring Semester, 20ZZ, Study will have control over instructor (male or female who will be trained in increased IFL goals through synchronous methods). Analyze intentionally increased and focused IFL goals through a balanced blend of asynchronous and synchronous methods simultaneously in study As Summer Semester, 20ZZ. Study will have control over instructor (male or female who was trained in increasing IFL experiences through a balanced combination of synchronous methods. Data Analysis There will be three researchers. Two of the three researchers will: STUDY A: Analyze and Compare current IFL methods against each group that received treatment; Analyze and Compare each treated group against each other; Come together and resolve any discrepancies. STUDY B: Analyze and Compare Increased, focused and intentional IFL goals through current methods against each group that received treatment of increased, focused and intentional IFL goals; Analyze and Compare each Increased, focused and intentional IFL goals that received treatment ; Come together and resolve any discrepancies. The third blind research will analyze and compare each group as mentioned above; join with the other two to resolve any further discrepancies along the way.

Friendship in Of Mice and Men and The Absolutely True Diary of a Part T

Friendship in Of Mice and Men by John Steinbeck, The Absolutely True Diary of a Part Time Indian by Sherman Alexie and the essay on Friendship by Ralph Waldo Emerson. Friendship is always a sweet responsibility, never an opportunity. -- (Khalil Gibran) Once I was looking on the internet that what is friends and what is friendship, since I never know what it was or never really read a definition of them. I was looking all over the place and I found this: â€Å"No one knows the exact definition of "Friendship"; however, they do have their own way to tell if they have a friend or not.† From the bedtime stories parents read to their children when they are going to bed, to the books students ready in high school, or the books adults ready have one most common element friendship. The history in the American literature three were many books, essays and songs about great friendships some are still famous and some are lost, in the era where people become friends by following each other on Instagram or sending request on Facebook. Mostly all persons on earth have a best friend and people say that it is hard to fine because a best friend should be some on whom you can always count on and that a person always count on you too. Sometimes having right person as your friends can help you but at the same time if you are in companionship of a bad person it can hurt you too. Who is the bad person and who is the good person is totally up to the person’s personal choice? Some books and movies have a stereotypical character o... ...micemen/ Mignon, Charles W., and H. Rose. CliffsNotes on Emerson's Essays. 01 May 2014 literature/e/emersons-essays/ralph-waldo-emerson-biography Van Kirk, Susan. CliffsNotes on Of Mice and Men. 01 May 2014 literature/o/of-mice-and-men/of-mice-and-men-at-a-glance References Steinbeck, J (1937) Of Mice and Men. Covici Friede. Alexie, S (September 2007) The Absolutely True Diary of a Part-Time Indian Emerson, R Friendship. Spark Notes Editors. (2007). Spark Note on Of Mice and Men. Retrieved April 30, 2014, from http://www.sparknotes.com/lit/micemen/ Mignon, Charles W., and H. Rose. CliffsNotes on Emerson's Essays. 01 May 2014 literature/e/emersons-essays/ralph-waldo-emerson-biography Van Kirk, Susan. CliffsNotes on Of Mice and Men. 01 May 2014 literature/o/of-mice-and-men/of-mice-and-men-at-a-glance

Hurricane Andrew Essay -- essays research papers

Hurricanes are notoriously capricious. Andrew was a compact system. A little larger system, or one making landfall just a few nautical miles further to the north, would have been catastrophic for heavily populated, highly commercialized and no less vulnerable areas to the north. That area includes downtown Miami, Miami Beach, Key Biscayne and Fort Lauderdale. Andrew also left the highly vulnerable New Orleans region relatively unscathed.   Ã‚  Ã‚  Ã‚  Ã‚  Andrew moved nearly due westward when over land and crossed the extreme southern portion of the Florida peninsula in about four hours. Although the hurricane weakened about one category on the during the transit over land, and the pressure rose to about 950 mb, Andrew was still a major hurricane when its eyewall passed over the extreme southwestern Florida coast. The first of two cycles of modest intensification commenced when the eye reached the Gulf of Mexico. Also, the hurricane continued to move at a relatively fast pace while its track gradually turned toward the west-northwest. When Andrew reached the north-central Gulf of Mexico, the high pressure system to its northeast weakened and a strong mid-latitude trough approached the area from the northwest. Steering currents began to change. Andrew turned toward the northwest and its forward speed decreased to about 8 kt. The hurricane struck a sparsely populated section of the south-central Louisiana on the 26th. The landfall location is about 20 n mi west-so...

Ethics Case Study Essay

What are Ethics? Ethics are standards of behavior, developed as a result of ones concept of right and wrong (Judson & Harrison, 2010). Code of ethics is a list of principles that is intended to influence the actions of healthcare professionals within an organization. Ethical principles help guide the decision-making process among healthcare workers in complicated situations. This paper will review the assigned case study and provide the reader with the ethical issues and principles. The case study discussed an 8-month old child brought into the emergency department by the mother due to inconsolable crying. It was reported that the child had been crying since picked up at daycare. The nurse found that the child’s vital signs were slightly elevated and child’s thighs were edematous and warm to the touch. The assessment was then presented to the physician; concern was expressed that the child may have been injured. An x-ray was ordered revealing fractures to both femurs. The mother was informed of the findings by the physician; after speaking with the mother the physician decided it was not child abuse and therefore, the situation did not need to be reported. The ethical issues involved in this case study are the nurse and physician’s responsibility to report suspected child abuse. Social services should have been consulted to assist in the process of reporting the situation to Child Protective Services (CPS). Hardy and Armitage state â€Å"In all actions concerning children, the best interests of the child shall be a primary consideration† (2002, p. 109). In this case, the primary consideration was not in the best interest of the child. According to Judson and Harrison the physician has the responsibility to understand the tools for diagnosing and treating abuse and to be familiar with the available resources (2010). The nurse in this case has the ethical duty to report the physician for not having concern regarding a possible child abuse situation. Healthcare professionals have an ethical duty to expose unethical behaviors by other members of the healthcare field. Nursing has progressed over the years into a respected and honorable profession. Insaf Altun (2008) states it best by saying, â€Å"The six key roles  of nursing are: advocacy, promotion of a safe environment, research, participation in shaping health policy, in patient and health systems management, and education† (p. 839). The two key roles the nurse in this case study failed to provide for the patient are advocacy and promotion of a safe environment. If the nurse was using good moral and professional judgment, he or she would have reported the situation immediately for the wellbeing of the child. Three of the ethical principles that are discussed are nonmaleficence, veracity and paternalism. The first ethical principle is nonmaleficence, meaning first do no harm (Judson and Harrison, 2010). A question asked by Milton and Cody (2001 p.290) was, â€Å"How can one keep from doing harm if one acts without knowing what the reality of the lived experience is for the person?† In the case study, neither the nurse nor physician knows what happened to the child or the reality of the circumstances that led to fractured femurs. The healthcare professional would have upheld this ethical principle and the law by reporting the suspected abuse, thus doing no harm and protecting the child from further harm. Another ethical principle that the physician could have used is veracity. The ethical principle of veracity deals with the truth or truth telling (Henry, 2005). The physician in this case should have discussed the health status of the child with the mother, made a â€Å"good† moral judgment, and told the mother the incident must be reported to CPS; the case could have been presented to the mother in a non-accusatory manner. The physician should have been reported for assuming the incident did not need to be reported. Furthermore, the nurse should have stepped forward and reported the incident, thereby promoting veracity on behalf of the case at hand. The third ethical principle in the case study to discuss is paternalism. At times paternalism viewed as a negative act because it allows healthcare providers to make decisions for the patients (Andre & Velasquez, 2008).The facts in the case study provide for an excellent example of when paternalism should be exercised. The nurse or physician in the case study could have used paternalism as a guiding ethical principle to do what was right for the  safety of the child by reporting the incident. The child has no voice to defend or protect him or herself and in this situation, the nurse should have advocated for the patient. The three ethical principles mentioned above are valued in my personal and professional life as a mother, nurse and student. The first ethical principle discussed nonmaleficence has been a personal value of mine throughout my life, used in all aspects of my life. The second ethical principle, veracity is maintained most of the time. I believe there are situations where telling the person the whole truth would not have an optimal outcome. The third ethical principle mentioned paternalism is used at times in my professional life as a nurse and in my personal life as a mother. The outcome of the case study could have been optimal for all involved the child, mother, nurse, and physician if the situation occurred at a children’s hospital. The likelihood of staff in a children’s hospital to have the proper training on signs of, diagnosing, treating, and reporting suspected abuse is much higher than an adult focused medical center. The American Association of Heart Failure Nurses incorporates ethical principles in the respect of advancement of care, education, and research to promote the best outcomes for patients’ with heart failure. One ethical principle used by the association is beneficence; this principle is used by providing evidence-based research, treatment, and education for patients and healthcare professionals. Using evidence-based medicine the association is giving optimal care for this patient population. Using ethical principles healthcare professionals can ensure they are making good moral decisions regarding care and treatment of patients. The ethical principles do not have to be a value of the healthcare professional, but must be upheld for the best interest of the patient. References Altun, I. (2008). Innovation in behavior patterns that characterize nurses. Nursing Ethics, 15(6), 838-840. Retrieved April 20, 2009, from EBSCOHost database. Andre C. & Velasquez M. (2008). For your own good. Retrieved April 21, 2009, fromhttp://www.scu.edu/ethics/publications/iie/v4n2/owngood.htmlHardy, M., & Armitage, G. (2002). The child’s right to consent to x-ray and imaging investigations: issues of restraint and immobilization from a multidisciplinary perspective. Journal of Child Health Care, 6(2), 107-119. Retrieved April 20, 2009, from SAGE database. Henry, L. (2005). Disclosure of medical errors: Ethical considerations for the development of a facility policy and organizational culture change. Policy, Politics, & Nursing Practice, 6(2), 127-134. Retrieved April 21, 2009, from SAGE database. Judson, K. & Harrison, C. (2010). Law and ethics for medical careers (5th ed). New York,NY: McGraw-Hill. Milton, C. & Cody, W. (2001). The ethics of bearing witness in healthcare: A beginning exploration. Nursing Science Quarterly, 14(4), 288-296. Retrieved April 20, 2009 from SAGE database.

Wikipedia: the Basis of Unreliable Sources

Over the years that Wikipedia has been running, there has been a continuous debate on whether it is a reliable resource or not and no one has been able to come up with a conclusion. However, there are many people who think that they have come up with a compromise, which includes Wikipedia being a good source for basic information but not for the sole information of any given topic. Especially for college students, Wikipedia can get tricky in the sense where it is easy to just read through and agree with; however, it may be misleading and have incorrect information.People including college English instructor Jenny Meister, Marquette University Law School student blogger Jessica Slavin, and Internet executive Mark Moran can all agree that Wikipedia is helpful in college research but only for basic knowledge, not for sole information. Throughout college, students must do a lot of book and computer research in order to do papers, class work and home work. The computer research is what sc ares professors the most because they know how easy it is for the students to click the first link that comes up on Google-Wikipedia.These students seem to get all of their information from the website, which can be edited by anonymous sources at any time. This means that the information is not always correct which can be frustrating to professors. â€Å"As someone who's been there and done the grading thing, I can tell you that when a professor flips to your Works Cited page and glances over your list of sources, a page full of Wikipedia entries is going to spark a frenzy of eye-rolling and red pen† (Meister).College students not only have to worry about having the wrong information in their school work, but also need to consider Wikipedia in their future careers. They must ask themselves how they should use it and the proper way to use it without hurting themselves. For example, law student Jessica Slavin had stated she was only going to use Wikipedia for background informa tion. â€Å"I will admit that I sometimes read a Wikipedia entry if I want background information about a topic. I do not think, though, that I would cite an entry as proof of anything in court† (Slavin).Wikipedia can be useful at times for background information about a topic, however, it can not be trusted because you are never sure who is writing it, if it is from a reliable source, or if the information is completely accurate. In Mark Moran’s blog about how Wikipedia is not reliable he makes one important point on why it should not be trusted for sole information. â€Å"In March 2009, Irish student Shane Fitzgerald, who was conducting research on the internet and globalization of information, posted a fake quotation on the Wikipedia article about recently deceased French composer Maurice Jarre.Due to the fact that the quote was not attributed to a reliable source, it was removed several times by editors, but Fitzgerald continued re-posting it until it was allowed to remain† (Moran). Moran is making the point that it is the contributor with the strongest agenda who â€Å"wins† and it is not always the one with the most correct information, which makes the site less reliable. For many years, Wikipedia has been known as an unreliable source and to be unused in school, although, it can be useful at times for basic information.Throughout college, students use Wikipedia as a main source which is giving them the wrong information in papers, class work, and school work, which is extremely frustrating for the professors to see. The students need to consider their future and if they would use Wikipedia as a primary source when they are at their future careers. College students should be allowed to use Wikipedia, but only as a basic information source in order to get background knowledge on a topic.