Human Carbonic Anhydrase II

Human Carbonic Anhydrase IIHuman carbonic anhydrase II is unmatched of the fastest studied enzymes cognize with a variety of roles in chemical response catalysis. Its primary function is to turn the reversible hydration reaction of carbon dioxide. In addition to carbon dioxide hydration, it is as well as cap equal to(p) of other latent skills, such as catalyzing esterase performance. The ability of human carbonic anhydrase II to function as a catalyst derives from key counterpoises in and around the dynamical site that play of the essence(p) roles in the mechanism. Substitutions to two of those particular key amino group acids were performed via Quick-change site directed mutagenesis H64A and V142D, to investigate the particular role they halt in the catalytic spry site. Various kinetic experiments and structural analyses were performed on wild-type carbonic anhydrase and the mutants to discern and equation their activity to each other and to writings, including Michaeli s-Menten parameters for PNPA hydrolysis, CO2 hydration, and inferring function molecular modelling. Though the same trends can be seen as the literature, individual values were set in motion to be much lower owing to errors in measurement and equipment. Trends were found to coincide with the mutants known roles in the lively 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.IntroductionHuman carbonic anhydrase II (hCAII) is a zinc each(prenominal)oyloenzyme that catalyzes the undermentioned reversible reaction . The enzyme unremarkably functions to help shuttle carbon dioxide in red blood cells to rid the body of metabolic waste, and catalyzes the hydrolysis of mevery aromatic esters 1, 2. Structur ally, 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 go 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, conducting in a bicarbonate ion cover song to zinc 3. The zinc-oxygen bond breaks to subsequently release a bicarbonate ion, which is replaced with water 3. The ZnOH bond is regenerated by a proton transfer to the external pilot, 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 dapple (His64) usually result in decreased enzyme activity due to a lack of proton transfer however the reaction does proceed to a lesse r stratum without an active His residue, possibly due to its extensive water network in the active site forming substitute(prenominal) 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 use to bind carbon dioxide, piece of music 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 ZnOH 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 Hi s64 and Val142 to the structure and mechanism of hCAII are determined through site-mutagenesis and subsequent moving picture of the new mutants, H64A (His64 Ala) and V142D (Val142 Asp) via kinetic and structural analysis. The changes that arise from the substitutions whitethorn 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 employ 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 suppr ess 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 lay 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 employ in the PCR reactions. Products of PCR mutagenesis reactions were political campaign on 0.7% agrose jellys to determine size. The gelatins were run at 150 V in 1X TAE airplane pilot zone. 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 desoxyribonucleic acid ladder from Fermentas.Table primer sequences used in mutagenesis of hCAII in the forward and reverse d irection for mutants H64A and V142DMutantDirectionSequenceMW (Da)%GCTM (C)H64AForwardGGATCCTCAACAATGGTgcTGCTTTCAACGTGGAG107785167 drive awayCTCCACGTTGAAAGCAgcACCATTGTTGAGGATCC10709V142DForwardCTGATGGACTGGCCGaTCTAGGTATTTTTTTG98684462ReverseCAAAAAAATACCTAGAtCGGCCAGTCCATCAG9779The enzyme, DpnI, was then used to digest methylated deoxyribonucleic acid (the parent template deoxyribonucleic acid). The deoxyribonucleic acid vector that contained the mutation was alter 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 sought after mutation 7. A miniprep culture was set-up from the LB-agar plate into LB medium to grow one addiction 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 cartel of the sequencing results, the purified DNA was quantified using UV/Vis absorption via a spectrometer 7. The assimilation was calculate using Ec1(260) as 50 g/mL.Purified plasmid DNA was transformed into E.coli BL2(DE3) cells to initiate protein style by heat shock, similar to the variation 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 weig ht marker by Fermentas. The protein and ladder was stained with coomassie blue 7.Affinity chromatography was used to chuck the mutant hCAII proteins 7. The matrix used was agrose linked to p-(aminomethyl)benzenesulfonamide, exploiting the tight dressing 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 g and 10 g, and also ran 10L of purify fractions from affinity chromatography 7. Protein concentration was determined by UV absorption at 280 nm in a concluding 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 v ariety of analyses were do. Two types of mass spectrometry (MS) were performed electrospray ionisation (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 L of pains protein concentrations (37.6 M H64A and 3.2 M 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 L sample was used for each mutant, 40 L of the mutant at stock concentration and 10 L of the Trypsin Gold. A couple L 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 product s were run through a protein database to confirm the identity of the protein and mutations 7.The 1 L of the tryptically digested mutants prepared for ESI-MS, subsequently underwent MALDI-MS. The 1 L samples were mixed with a matrix consisting of 1 L -cyano-4-hydroxycinnamic acid (CHCA) and 1 L 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 M in protein buffer 7. The initial rate measurements of each PNPA concentration were taken for wild-type enzyme, H64A mutant, V1 42D mutant, and a blank with no additional enzyme added (refer to data tables in Enzyme Kinetics I 7). PNP has a molar absorption coefficient () of 1.73-104 M1cm1. 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 tend 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 given that S KM, the Michaelis-Menten equation simplifies to kcat=V0/E.The third kinetics experiment used fluorescence to determine the rear 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 M of DNSA were prepared from a 21.6 mM DNSA stock by dilution with DMSO. Enzyme stocks were diluted to 0.25 M 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 reckon 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 dapple were explored. Homology between hCAII and other carbonic anhydrase isozymes, hCAIV (PDB reckon 1ZNC) and hCAI (PBD code H1CB), were also studied by performing an iterative magic kick the bucket on the -carbons and structure alignment for each play off. The root mean second power (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 s tructural 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 demonstrable structure as determined by other scientists, for example, by aligning the virtual and crystallized mutations to determine deviations in structure by performing RMS.ResultsSite-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 slews observed coincides with the size of the plasmid used, 6018 bp.Heat shock transformation and isolation of plasmid. Several colonies were observed after plasmid tr ansformation 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. path 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 prospering 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 Ec1 260 = 50 g/mL for DNA was applied in the calculation of concentrated and diluted concentrations of purified DNA (Table ).Table intercourse 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 Ec1 260 = 50 g/mL.MutantWavelength, Absorbance UnitsRelative DNA Purity (A260/A280)Concentrated (g/mL)Diluted (g/mL)V142D260 nm0.31171.7852311.7015.59280 nm0.1746H64A260 nm0.26531.7581265.3013.27280 nm0.1509Enzyme restriction digest. Purified plasmid DNA of mutants were digested with XhoI and BglII, separately and unneurotic 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 evaluate length of the bands in the double digest should be 892 bp and 5126 bp (Figure ).Figure electrophoresis p erformed in 1% agrose gel of digested V142D hCAII in lane 1-4. The (1 kB) GeneRuler DNA ladder is shown in lane 5. course 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 range of hCAII gene (5072-5854) on the hCAI2pET24b plasmidDNA 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 M 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 derisory with V142D hCAII proteins to examine protein e xpression. 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 L IPTG, 4 L IPTG, 1 L+IPTG, 4 L +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 L 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 M1cm1 (Table ). The final concentration of the samples of V142D and H64A hCAII were 3.2 M and 37.6 M respectively.Table UV absorption measurements at 280 nm of puri fied protein and the resulting final concentrationMutant bonny A280Protein concentration (M)V142D0.015833.2H64A0.188437.6SDS-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 g protein, and 10 g protein. For H64A, a visible band was only observed for the 10 g 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 g protein, and 10 g protein. Only the 10 g 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 weigh t marker. Lane 12-15 contains V142D samples of the following (in order) lysate, wash fraction, 2 g protein, and 10 g protein. No observable bands are seen for any of the samples. fix 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 (1.73-104 M1cm1), 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 even up difference between the predicted and actu al 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 hCAIIH64A hCAIIV142D hCAIIVmax (M/sec)1.2020.8120.218KM (mM)1.2801.9578.362kcat (s1)5.141 - 1032.159 - 1026.825 - 102kcat/KM (M1s1)4.0211.0328.162Figure 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 condemnation (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 V14 2D 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+ glyoxaline (M/s)01.3E-086.05778E-081.51.1E-08N/A2.51.1E-085.63E-0852.1E-085.16E-0812.55.9E-085.63E-08Average kcat (s1)5.31.6212.449.19Table 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)12.51.4E-086.57E-08251.4E-085.8E-08501.7E-087.53E-08Average kcat (s1)0.670.393.031.97Table 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)12.56.2E-09255.4E-09505.5E-09Average kcat (s1)0.270.19Fluorescence 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 M when protein concentration was 0.25 M. 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 -sheets (77 residues) and 10 -helices (42 residues), with the majority of the -helices falling in the domain of right-handed helices, while very few show left-handed coiled properties according to the Ramachadran plot. It also seems that the active site is solely composed of -sheets, and no -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 provid es 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 (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 high 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.DiscussionAgrose 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, in that respect 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 di d 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 bear witness by its concentration of 3.2 M. 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 affect ed its lack of bands because it was calculated to have had a reasonable concentration of 37.6 M. There were some problems loading the samples into the wells this could be an explanation as to no observable gel bands.ESI-MS is helpless 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 (250 0200 M1s1) was found to be significantly larger than V142D hCAII (30.3 M1s1) in literature, more than 800- larger 2. Experimental calculations yielded kcat/KM for V142D (8.16 M1s1) to be about 2- larger than wild-type (4.02 M1s1), which did not follow literature patterns. The literature trends make more biolog

Study on the Prediction of Corporate Bankruptcy

Study on the Prediction of Corporate BankruptcyCHAPTER 1A progeny of searches pitch been carried on the foretelling of loser globe studies cogitate with failure of business were conducted in 1930s. A withdraw conducted by Simth and Winakor (1935) said that ratios of the failing profligates were prodigiously changed from the move firms. In addition to that an opposite ponder was related to the mo electronic meshworkary ratio of large size corporation that suffered in meeting fixed indebtedness (Hickman 1958). new studies took potential ratios habituated in twelvemonthly monetary statements like profitability, solvency, and liquidity ratios considered as the most shout outive indicator and these ratios were matched with failed and well worth firms for analysis. A assembl coarse time of fiscal and economic ratios were see to itd in the prognostication of loser through multiple discriminant statistical technique, highest endorser ratios were profitability, o perational profit/ total confirmings and very beginning contri just nowor ratio was working roof/Assets (Altman, 1968).According to Pastena and Ruland (1968), the failure was defined in the literature review in various ways. Among those one was in a jibe of invalidating worth where the market treasure of assets was less than the total value of liabilities. And the early(a) was that the firm was not in a piddlen to recompense anchor its liabilities as it became due. This status could in some(prenominal) case be utilise in a legal condition under which the firms continued to operate under judicature protection.1.2 paradox StatementIn the corporal finance, the prevision of bodied nonstarter was considered to be one of the most important issues. The main objective behind the break down of the farsightedness of merged loser was that this was the most important issue for the present firms to either file for the failure or not.The rationale of the shoot was to ex amine whether the financial ratios given in detail by Altman (1968) presented the detail regarding the factors of the firm which were helpful in the prophecy of corporate unsuccessful person in Pakistan. The capacity of bring was to examine the classifiable financial ratios which impacted the firms finalitys to file for the bankruptcy or not and on the basis of firms financial ratios, the investigate look at found the contrasting significant ratios which were useful in de terminal figureining the divination of any of the organization.1.3 HypothesesThe main problem of the different firms was to identify those financial factors or the most important ratios which could lead to the file of bankruptcy or those factors which were useful in determining the foretelling of the corporate firms. A central query in front of firms which wanted to file for bankruptcy was wherefore the firms filed for bankruptcy or what financial factors helped come to the fore in taking decision to file for bankruptcy. Various financial factors or ratios impacted the decision regarding the file for bankruptcy. These financial characteristics or the most important ratios were current ratio, debt ratio, net profit security deposit, assets to dour term debt ratio, and growth rate. Many authors as Altman (1968) discussed these characteristics in look into. The hypothesized relationship of these listed financial factors with bankruptcy was provided belowH1 at that place is a balance between the Current ratio of bankrupted companies and non bankrupted companies.H2 in that location is a disagreement between the Debt ratio of bankrupted companies and non bankrupted companies.H3 There is a firing away between the Net Profit Margin ratio of bankrupted companies and non bankrupted companies.H4 There is a difference between the Assets to long term debt ratio of bankrupted companies and non bankrupted companies.H5 There is a difference between the Growth rate of bankrupted compani es and non bankrupted companies.1.4 Outline of the StudyThe query structured as follows. Chapter one based on the creation of the thesis, which consists of the close to introduction of the prognostic of bankruptcy by different authors, the statement of problem, scope and objectives, hypothesis etc. Chapter two consists of literature review given by different authors, theories on omen of bankruptcy and financial factors affecting the choice of decision to file for bankruptcy or not. Chapter three described methodology which is composed of justification of the excerpt of the variants apply in analysis en essay, the info, technique and hypothesis, excessively estimate amaze utilized in analysis. In chapter four, analyses of the results were on that point which were interpreted aft(prenominal) the entropy processing. Chapter atomic number 23 contained the net results, conclusions and recommendations. References are included in chapter number six.CHAPTER 2LITERATURE REV IEWA number of researches have been carried on the presage of bankruptcy formal studies linked with failure of business were conducted in 1930s. A study conducted by Simth and winakor (1935) said that ratios of the failing firms were significantly change from the continuing firms. In addition to that an other study was related to the financial ratio of large size corporation that suffered in meeting fixed liability (Hickman 1958). Recent studies took potential ratios given in annual financial statements like profitability, solvency, and liquidity ratios considered as the most predictive indicator and these ratios were matched with failed and well worth firms for analysis. A stem of financial and economic ratios were examined in the prediction of bankruptcy through multiple discriminant statistical technique, highest contributor ratios were profitability, operational profit/ total assets and very low contributor ratio was working capital/Assets (Altman, 1968).A study conducted by S andin and Porporato (2007) on corporate bankruptcy prediction work applied to emerging economies. The aim of this study was to find the predictability of bankruptcy by utilize the financial ratios given in the financial statements and these financial statements were taken from the Buenos Aires Stock Exchange. To test the hypothesis twenty two bankrupt and non bankrupt companies were examined by development the multiple discriminant analysis technique, resulted that financial ratios were very useful in predicting the bankruptcy. Actu each(prenominal)y this study was somewhat the prediction model and smorgasbord of the distressed and failed companies in the Argentina.William work (1996) conducted a study that Financial Ratios As Predictor of Failure, wherein ratios were tested for a specific conception. The purpose was to forecast the failure. Since ratios were mostly examined for the prediction of failure. The aim of the study was to fail the status quo that was depended on th e financial statements make under the reporting standard and this study was conducted as a bench mark for further studies in bankruptcy area. Sample of data was selected on the basis of industry, firm size and power point, Walworth companies should have taken from the same industry where from failed companies taken along with same firm size based on firm value and equal fourth dimension duration then reliable result can be obtained said by genus Castor (1996). This study pointed out and directed to the asset size and relationship among ratios, assets size and failure, study implicated that larger firms were to a greater extent solvent than smaller firms, however if ratios were same. To examine the hypothesis, a paired analysis was employ.According to Pastena and Ruland (1968), the bankruptcy was defined in the literature review in various ways. Among those one was in a condition of negative worth where the market value of assets was less than the total value of liabilities. An d the other was that the firm was not in a condition to fix stake its liabilities as it became due. This term could also be utilize in a legal condition under which the firms continued to operate under cost protection. nuclear fusion reaction and BankruptcyBased on the literature review in the different research studies, it was found that the shareholders of the distressed firms were getting more benefit from mergers than from the bankruptcy. thence, the investors unbroken the corroborative number of the firms stocks up as a consequence of the merger. Contrastingly, the stakeholders received nothing in fount of the bankruptcy. Shrieves and Stevens (1979) managed to let off all of the possible reasons for preferring merger everyplace bankruptcy and those principles included (1) to avoid the bankruptcy legal and administrative costs, (2) possible loss of tax carry forwards of the loss firm incurred on liquidation, (3) the value of the going- trouble in the merger was more th an liquidation value if the firm bankruptcy progressed towards the liquidation, and (4) the bankruptcy created the bad effects on the revenues including gross revenue and income due to the customer fears of softness contracts, give replacement parts, etc.Bulow and Shoven (1978) noticed based on the research that the investors have always been avoiding the bankruptcy and this tendency always benefitted the creditors as a wholly and that theoretically, the bankruptcy occurred because of the disagreement between the concerned parties. This was treated in a literature that the merger was the beat out possible alternate of the bankruptcy with the as center of attentionption in the promontory that it was more easy for the distressed firms to find a merger partner at some price as long as the net asset value was positive and this was also under the assumption of a well-functioning market for information. When the situation was aggravated toward a condition of less or negative net asset value, the possibility of merger was reduced.Hong (1983) do an empirical as well as theoretical model which make loveed among three different categories of financially cut into firms and it was organized in three ways such as firms which filed bankruptcy but reorganized successfully, firms which filed for bankruptcy but were liquidated ultimately, and also the firms which continued trading operations with out even filing for bankruptcy. Author further make a hypothesis that the intangible assets, the value of the firm as in a going concern and the value of the same firm in liquidation was different, were the main describing factor which affected the eventual outcome. The firms which had greater intangible assets were possibly having a sustainable economic growth and that growth allowed a firm to survive rather than be liquidated.LoPucki (1983) made an explanatory study of about 41 firms which filed the bankruptcy address of the Hesperian District of Missouri. In this study, t he successes? were defined as the firms which have verified its various reorganization strategies that kept it on to survive for about three eld after the date of petitioning the bankruptcy. Failures according to the author were those firms which had stopped in operation(p) functions beforehand February 1983. LoPucki (1983) further could not try to make a method with discriminatory power, but in fact simply scrutinized the associations between the results of reorganization process and numerous individual variables. These individual variables included size, age, and type of the businesses, the survival of creditors opposition to the reorganization strategy, and somatic geographic location. The relationships which were found during the research were significantly higher success rate was associated with the manufacturing firms more successful firms were only when the larger firms success was not significantly associated with the age of the firms the bulls eye opposition of the cr editors was mainly at the more successful firms and lastly, the physical geographical location was not a significant describing variable.In short, only a bounded amount of research was conducted on the topic of differentiating between failures and successes in bankruptcy, and outcomes have been open to doubts or inconclusive. The one published study conducted by the LoPucki (1983), scrutinized the firstly rear correlations and could not struggle to build the model of classification. The other published research study conducted by Hong (1983), scrutinized the comparative importance of numerous individual variables and had not study the classification authentication of the multivariate model. As it was already discussed in detail, this present study scrutinized the classification authentication of the multivariate model by exploitation data from both analysis sample and a holdout sample 113 firms.Bordman, Bartley, and Ratliff (1981) noticed that firms went bankrupt only when its capital resources were not enough to pay back the obligations of the business. Thus it became the more important challenge for the new comers in the industry to maintain and establish such valuable resources and capabilities which could ultimately leaded to the production of positive cash in flows before starting asset resources were exhausted (Levinthal, 1991). According to DAveni (1989), and Hambrick and DAveni (1988), both researches have noticed that most of the attention has been paid to the early failures and dramatic research has also been conducted in the literature.A macro view of the bankruptcy was given as a known strategy and an empirical examination of factors associated to successful reorganization (Moultan, and Thomas, 1993) and however, the structures of corporate brass instrument were not incorporated in the analysis. An extensive data was available relating the intensity to which the officers and directors of the firm which was bankrupt were more possibly resigni ng or were being replaced (DAveni, 1990 Fizel Louie, 1990 Gilson, 1989). some(prenominal) researchers used the multiple discriminant analysis MDA technique to develop a linear model to predict those firms which failed could be differentiated from the non-failed firms in UK (Taffler, 1977). This model resulted in an overall classification authentication for the year before the failure as comparative to three or two prior years of failure. The major contribution made by Taffler was the establishment of a Z-score model which was used for the prediction of company failures in the UK and furthermore, the author claimed of 100 percent predictive authentication in the model. In addition, in the consequent studies, Taffler (1982, 1983) discussed the pairing technique which was used in the prediction of corporate failure studies proved no more successful technique than any choice by the other tool or technique. Multiple Discriminant epitome MDA models were dependable to certain intensity in the prediction of corporate failure.CHAPTER 3 search METHODS3.1 manner of Data CollectionData was selected from Karachi Stock Exchange KSE 100 powerfulness as given by State Bank of Pakistan in result symmetricalness Sheet Analysis of Joint Stock Companies Listed on the KSE (2004-2009). The period of study covers six years, 2004-09. The opted sample size of 44 firms was taken from KSE 100 big businessman and all of the firms listed on KSE 100 Index were selected for the samples which were going to bankruptcy in the past and some were also the present functioning firms which were currently working so, only 44 firms included in the sample period of 2004-09. The objective behind the inclusion of these selected firms in the sample was that the inclusion of bankrupt and non-bankrupt firms in the analysis made it easier to distinguish the critical financial ratios of these both firms in order to predict for corporate bankruptcy.The data approachability was the major issue faced in this research study. The auxiliary data sources were adopted for the collection of the data during this research study. Both of the empirical and theoretical aspects regarding the prediction of corporate bankruptcy were analyzed in this research study. For the purpose of the collection of the secondary data, external data sources were used, such as the data was collected from State Bank of Pakistan, general business publications, newspapers and journal articles, annual reports, lucre and books. The data required for this study was completely dependent on the published data sources, such as the published sources listed above.3.2 Sample SizeA sample of 44 firms from KSE 100 Index was selected and in addition, out of these firms 22 firms were bankrupt and the remaining 22 were not bankrupt which was taken as the holdout sample for the prediction of the corporate bankruptcy. Only firms were used in the samples which were either became bankrupt due to the impact of the some of the f inancial factors or the ratios or the firms which were in operations during the research study was conducted and these firms were listed on the KSE 100 Index form 2004-2009. The impact of the different financial factors or ratios, which were listed in the previous chapters, on the prediction of corporate bankruptcy was analyzed on all of the firms selected as the sample.3.3 Research Model DevelopedThere are various financial factors or the ratios of the firms which affected the prediction of the corporate bankruptcy of the firms. This research study analyzed the impact of different factors or ratios already listed in the previous chapters on the prediction of corporate bankruptcy. The model create was a binary logistic model and its specifications are provided below liquidness = a0 + a1Firm Size + a2DEBT + a3LTD + a4LSALES + a5OI/S + a6OI/TA+a7IGP/TA+ a8Market to Book Ratio + whereLiquidity = the sum of cash and marketable securities divided by total assetsFirm Size = natural log of the book value of total assetsDEBT = the ratio of shorter period plus longer period debt to total assetsLTD = the ratio of longer period debt to total assetsLSALES = natural log of the annual salesOI/S = the ratio of operating income to salesOI/TA = the ratio of operating income to total salesIGP/TA = the inventory plus gross fixed assets to total assets ratio = the error term3.4 statistical TechniqueBinary put downistic Regression Analysis technique was used for this research study to examine the impact of the typical financial characteristics or the financial ratios of the firms on the prediction of corporate bankruptcy of the selected firms Statistical Package for the sociable Sciences (SPSS) was used for the examination of the secondary data.Binary Logistic Regression Analysis technique was used for the purpose of prediction of of corporate bankruptcy or the prediction of the firms decisions to file for bankruptcy. The selected technique was used to study the impact of the different individual variables (financial factors as listed in the previous chapters) on the dependent variable i.e., prediction of corporate bankruptcy. The binary logistic regression analysis was selected for this study. It showed the intensity of the impact on the prediction of corporate bankruptcy during year 2004-2009 on the basis of several independent variables.CHAPTER 4 allow forSThe sample of 44 firms from the Karachi Stock Exchange KSE 100 Index was taken Binary Logistic Regression Analysis technique was used for this research study. Research examined the distinctive financial characteristics or financial ratios of firms which filed for the bankruptcy. The selected technique was used to study the impact of the different independent variables (financial factors as listed in the previous chapters) on the dependent variable i.e., the prediction of corporate bankruptcy. Statistical Package for the Social Sciences (SPSS) was used for the analysis and examination of data.4.1 Fi ndings and Interpretation of the resultsInitially, the binary logistic regression technique was applied on the data collected using SPSS. Now, it was a enough time to proceed with the analysis of the results because the data was collected and ready to be examined. The interpretation and analysis is presented in the next sections of this research study.Case Processing thickUnweighted CasesaNPercentSelected CasesIncluded in Analysis19291.4Missing Cases188.6Total210100.0Unselected Cases0.0Total210100.0This table explains the total population in the data file that is the 210 observations or the cases for the analysis of the bankruptcy prediction. This table further elaborates that the there were also some of the cases missing in the data because of the issue of data availability and some of the cases were the figures of zero.Dependent Variable EncodingOriginal ValueInternal ValueBankrupt0Non-Bankrupt1The above table shows that there are only two variables in the dependent variable of bankruptcy that are the being bankrupt or non-bankrupt.Model SummaryStep-2 Log likelihoodCox Snell R SquareNagelkerke R Square1234.707a.144.192This table elaborates the predictability of the complete model of the logistic regression which meant that to what extent the model predict the translation in the predicted group of bankruptcy. According to Cox Snell, the total predictors jointly explained variation in the groups of bankruptcy was 14.4%. While according to Nagelkirki, the all independent variable explained the group prediction of about 19.2%.Hosmer and Lemeshow TestStepChi-squaredfSig.132.7158.000This table checks the overall model fit which means that the model is at its best in predicting the group variation from non-bankrupt to bankrupt. The hypothesis of the above table is that the test model is fit. The hypothesis is rejected because the sig value is less than .05 which concluded that the test model was not fit in this case of predicting the group variation.Classifica tion TableaObservedPredictedBanckruptcyPercentage CorrectBankruptNon-BankruptStep 1BanckruptcyBankrupt762972.4Non-Bankrupt434450.6Overall Percentage62.5The classification table is the most important table in case of the logistic regression because this table explained the pay off denomination of the cases correctly identified. The percentage of correctly identified cases is 62.5% which is also commonly known as the hit ratio which means that to what extent the numbers of cases were correctly identified.Variables in the EquationBS.E.WalddfSig.Exp(B)95% C.I.for EXP(B)LowerUpperStep 1aDA-1.219.5105.7251.017.295.109.802AtoLTD-.002.0011.5831.208.998.9961.001CR.938.3487.2421.0072.5541.2905.056NPM.037.073.2621.6091.038.8991.198SG.161.232.4821.4881.175.7451.852 continuous.066.579.0131.9101.068This is the final most important table in the logistic regression because this is the only table which shows the function of different predictors in significantly explaining the role in the predict ion of group variations. Those important significant variables were only two that were DA, and CR because the sig value of only these variables were less than .05.4.2 Hypotheses Assessment SummaryThe hypothesis of the study was distinctive financial ratios have significant impact on the non firms decision to file for bankruptcy. These financial characteristics were current ratio (CR), debt ratio (DA), net profit margin (NPM), assets to long term debt ratio, and growth rate. In this study each of the financial characteristics or financial ratios of firms was tested and concluded the results.TABLE 4.4 Hypotheses Assessment SummaryS.NO.HypothesesSIG.RESULTH1There is a difference between the Current ratio of bankrupted companies and non bankrupted companies.0.9380.007AcceptedH2There is a difference between the Debt Ratio of bankrupted companies and non bankrupted companies.-1.2190.017AcceptedH3There is a difference between the Net Profit Margin Ratio of bankrupted companies and non ban krupted companies.0.0370.609rejectedH4There is a difference between the Assets to long term debt ratio of bankrupted companies and non bankrupted companies.-0.0020.208RejectedH5There is a difference between the Growth rate of bankrupted companies and non bankrupted companies.0.1610.488RejectedCHAPTER 5DISCUSSIONS, CONCLUSION, IMPLICATIONS AND FUTURE RESEARCH5.1 ConclusionIt was concluded based on the results of this research study that current ratio and debt ratio were only the independent variables which were showing significance in Pakistani market and these variables were highly significant in acting the decisive role explaining the variation in the dependent variable of the prediction of corporate bankruptcy and the remaining independent variables could not explain the variation in the prediction of corporate bankruptcy. These results were not matching with the study conducted by Altman (1968). These results were varying because in various countries, there was difference in env ironments and circumstances and firms usually made decision accordingly.5.2 DiscussionsCurrent ratio played a significant role in defining the variation in the prediction of corporate bankruptcy and this was also the case with the research study conducted by Altman (1968) because in his study the firm size was also playing a significant role. The variation in the prediction of corporate bankruptcy was not explained by the net profit margin ratio while it was significant in the study done by Altman (1968). The assets to long term debt ratio, and growth rate were not significantly explaining the variation in the prediction of corporate bankruptcy and study analyzed by Altman (1968), concluded the different results with some addition.5.3 Implications and RecommendationsThis research was limited to the various firms listed on Karachi Stock Exchange of Pakistan only. The data taken from 44 firms which were took through various sectors of the KSE 100 Index for the year 2004-09 which were antecedently bankrupt and which were currently operating. It suggested that such type of study should be carried out in other countries of Asia as well, as to have comprehensive motif about the choices of the firms decision to file for bankruptcy. Moreover, it also suggested that other factors except ones examined in this study should be researched as to have perfect idea about the extract of the prediction of corporate bankruptcy. Besides that, this study can also be replicated in other developing countries.5.4 Future ResearchThis study helped various investors, worry and other research conductors in analyzing and observing the behavior of firms regarding their decisions to file for the bankruptcy. Research students who want to work further on the prediction of bankruptcy can be benefited by this research study. Further more, the firms will become advantageous from this study because the study clarifies the distinctive financial characteristics or the financial ratios of differe nt firms which significantly explain the variations in the prediction of corporate bankruptcy.Study on the Prediction of Corporate BankruptcyStudy on the Prediction of Corporate BankruptcyCHAPTER 1A number of researches have been carried on the prediction of bankruptcy formal studies linked with failure of business were conducted in 1930s. A study conducted by Simth and Winakor (1935) said that ratios of the failing firms were significantly changed from the continuing firms. In addition to that another study was related to the financial ratio of large size corporation that suffered in meeting fixed liability (Hickman 1958). Recent studies took potential ratios given in annual financial statements like profitability, solvency, and liquidity ratios considered as the most predictive indicator and these ratios were matched with failed and well worth firms for analysis. A group of financial and economic ratios were examined in the prediction of bankruptcy through multiple discriminant st atistical technique, highest contributor ratios were profitability, operational profit/ total assets and very low contributor ratio was working capital/Assets (Altman, 1968).According to Pastena and Ruland (1968), the bankruptcy was defined in the literature review in various ways. Among those one was in a condition of negative worth where the market value of assets was less than the total value of liabilities. And the other was that the firm was not in a condition to pay back its liabilities as it became due. This term could also be used in a legal condition under which the firms continued to operate under court protection.1.2 Problem StatementIn the corporate finance, the prediction of corporate bankruptcy was considered to be one of the most important issues. The main objective behind the study of the prediction of corporate bankruptcy was that this was the most important issue for the present firms to either file for the bankruptcy or not.The rationale of the study was to examin e whether the financial ratios given in detail by Altman (1968) presented the detail regarding the factors of the firm which were helpful in the prediction of corporate bankruptcy in Pakistan. The capacity of study was to investigate the distinctive financial ratios which impacted the firms decisions to file for the bankruptcy or not and on the basis of firms financial ratios, the research study found the different significant ratios which were useful in determining the prediction of any of the organization.1.3 HypothesesThe main problem of the different firms was to identify those financial factors or the most important ratios which could lead to the filing of bankruptcy or those factors which were useful in determining the prediction of the corporate firms. A central query in front of firms which wanted to file for bankruptcy was why the firms filed for bankruptcy or what financial factors helped out in taking decision to file for bankruptcy. Various financial factors or ratios im pacted the decision regarding the filing for bankruptcy. These financial characteristics or the most important ratios were current ratio, debt ratio, net profit margin, assets to long term debt ratio, and growth rate. Many authors as Altman (1968) discussed these characteristics in research. The hypothesized relationship of these listed financial factors with bankruptcy was provided belowH1 There is a difference between the Current ratio of bankrupted companies and non bankrupted companies.H2 There is a difference between the Debt ratio of bankrupted companies and non bankrupted companies.H3 There is a difference between the Net Profit Margin ratio of bankrupted companies and non bankrupted companies.H4 There is a difference between the Assets to long term debt ratio of bankrupted companies and non bankrupted companies.H5 There is a difference between the Growth rate of bankrupted companies and non bankrupted companies.1.4 Outline of the StudyThe research structured as follows. Chap ter one based on the introduction of the thesis, which consists of the some introduction of the prediction of bankruptcy by different authors, the statement of problem, scope and objectives, hypothesis etc. Chapter two consists of literature review given by different authors, theories on prediction of bankruptcy and financial factors affecting the choice of decision to file for bankruptcy or not. Chapter three described methodology which is composed of justification of the selection of the variables utilized in analysis sample, the data, technique and hypothesis, also estimate model utilized in analysis. In chapter four, analyses of the results were there which were taken after the data processing. Chapter five contained the final results, conclusions and recommendations. References are included in chapter number six.CHAPTER 2LITERATURE REVIEWA number of researches have been carried on the prediction of bankruptcy formal studies linked with failure of business were conducted in 1930 s. A study conducted by Simth and winakor (1935) said that ratios of the failing firms were significantly change from the continuing firms. In addition to that an other study was related to the financial ratio of large size corporation that suffered in meeting fixed liability (Hickman 1958). Recent studies took potential ratios given in annual financial statements like profitability, solvency, and liquidity ratios considered as the most predictive indicator and these ratios were matched with failed and well worth firms for analysis. A group of financial and economic ratios were examined in the prediction of bankruptcy through multiple discriminant statistical technique, highest contributor ratios were profitability, operational profit/ total assets and very low contributor ratio was working capital/Assets (Altman, 1968).A study conducted by Sandin and Porporato (2007) on corporate bankruptcy prediction model applied to emerging economies. The aim of this study was to find the predic tability of bankruptcy by using the financial ratios given in the financial statements and these financial statements were taken from the Buenos Aires Stock Exchange. To test the hypothesis twenty two bankrupt and non bankrupt companies were examined by using the multiple discriminant analysis technique, resulted that financial ratios were very useful in predicting the bankruptcy. Actually this study was about the prediction model and classification of the distressed and failed companies in the Argentina.William Beaver (1996) conducted a study that Financial Ratios As Predictor of Failure, wherein ratios were tested for a specific purpose. The purpose was to forecast the failure. Since ratios were mostly examined for the prediction of failure. The aim of the study was to analyze the status quo that was depended on the financial statements made under the reporting standard and this study was conducted as a bench mark for further studies in bankruptcy area. Sample of data was selected on the basis of industry, firm size and period, Walworth companies should have taken from the same industry where from failed companies taken along with same firm size based on firm value and equal time duration then reliable result can be obtained said by Beaver (1996). This study pointed out and directed to the asset size and relationship among ratios, assets size and failure, study implicated that larger firms were more solvent than smaller firms, even if ratios were same. To examine the hypothesis, a paired analysis was used.According to Pastena and Ruland (1968), the bankruptcy was defined in the literature review in various ways. Among those one was in a condition of negative worth where the market value of assets was less than the total value of liabilities. And the other was that the firm was not in a condition to pay back its liabilities as it became due. This term could also be used in a legal condition under which the firms continued to operate under court protection.Mer ger and BankruptcyBased on the literature review in the different research studies, it was found that the shareholders of the distressed firms were getting more benefit from mergers than from the bankruptcy. Thus, the investors kept the positive number of the firms stocks up as a consequence of the merger. Contrastingly, the stakeholders received nothing in case of the bankruptcy. Shrieves and Stevens (1979) managed to explain all of the possible reasons for preferring merger over bankruptcy and those principles included (1) to avoid the bankruptcy legal and administrative costs, (2) possible loss of tax carry forwards of the loss firm incurred on liquidation, (3) the value of the going-concern in the merger was more than liquidation value if the firm bankruptcy progressed towards the liquidation, and (4) the bankruptcy created the bad effects on the revenues including sales and income due to the customer fears of inability contracts, give replacement parts, etc.Bulow and Shoven (19 78) noticed based on the research that the investors have always been avoiding the bankruptcy and this tendency always benefitted the creditors as a whole and that theoretically, the bankruptcy occurred because of the disagreement between the concerned parties. This was treated in a literature that the merger was the best possible alternate of the bankruptcy with the assumption in the mind that it was more easy for the distressed firms to find a merger partner at some price as long as the net asset value was positive and this was also under the assumption of a well-functioning market for information. When the situation was aggravated toward a condition of less or negative net asset value, the possibility of merger was reduced.Hong (1983) made an empirical as well as theoretical model which distinguished among three different categories of financially upset firms and it was organized in three ways such as firms which filed bankruptcy but reorganized successfully, firms which filed fo r bankruptcy but were liquidated ultimately, and also the firms which continued operations with out even filing for bankruptcy. Author further made a hypothesis that the intangible assets, the value of the firm as in a going concern and the value of the same firm in liquidation was different, were the main describing factor which affected the eventual outcome. The firms which had greater intangible assets were possibly having a sustainable economic growth and that growth allowed a firm to survive rather than be liquidated.LoPucki (1983) made an explanatory study of about 41 firms which filed the bankruptcy court of the Western District of Missouri. In this study, the successes? were defined as the firms which have verified its various reorganization strategies that kept it on to survive for about three years after the date of petitioning the bankruptcy. Failures according to the author were those firms which had stopped operating functions before February 1983. LoPucki (1983) furthe r could not try to make a method with discriminatory power, but in fact simply scrutinized the associations between the results of reorganization process and numerous individual variables. These individual variables included size, age, and type of the businesses, the survival of creditors opposition to the reorganization strategy, and physical geographic location. The relationships which were found during the research were significantly higher success rate was associated with the manufacturing firms more successful firms were only the larger firms success was not significantly associated with the age of the firms the target opposition of the creditors was mainly at the more successful firms and lastly, the physical geographical location was not a significant describing variable.In short, only a finite amount of research was conducted on the topic of differentiating between failures and successes in bankruptcy, and outcomes have been open to doubts or inconclusive. The one published study conducted by the LoPucki (1983), scrutinized the first order correlations and could not struggle to build the model of classification. The other published research study conducted by Hong (1983), scrutinized the comparative importance of numerous individual variables and had not analyzed the classification authentication of the multivariate model. As it was already discussed in detail, this present study scrutinized the classification authentication of the multivariate model by using data from both analysis sample and a holdout sample 113 firms.Bordman, Bartley, and Ratliff (1981) noticed that firms went bankrupt only when its capital resources were not enough to pay back the obligations of the business. Thus it became the more important challenge for the new comers in the industry to maintain and establish such valuable resources and capabilities which could ultimately leaded to the production of positive cash flows before starting asset resources were exhausted (Levinthal, 1 991). According to DAveni (1989), and Hambrick and DAveni (1988), both researches have noticed that most of the attention has been paid to the early failures and dramatic research has also been conducted in the literature.A macro view of the bankruptcy was given as a known strategy and an empirical examination of factors associated to successful reorganization (Moultan, and Thomas, 1993) and however, the structures of corporate governance were not incorporated in the analysis. An extensive data was available relating the intensity to which the officers and directors of the firm which was bankrupt were more possibly resigning or were being replaced (DAveni, 1990 Fizel Louie, 1990 Gilson, 1989).Several researchers used the multiple discriminant analysis MDA technique to develop a linear model to predict those firms which failed could be differentiated from the non-failed firms in UK (Taffler, 1977). This model resulted in an overall classification authentication for the year before t he failure as comparative to three or two prior years of failure. The major contribution made by Taffler was the establishment of a Z-score model which was used for the prediction of company failures in the UK and furthermore, the author claimed of 100 percent predictive authentication in the model. In addition, in the consequent studies, Taffler (1982, 1983) discussed the pairing technique which was used in the prediction of corporate failure studies proved no more successful technique than any selection by the other tool or technique. Multiple Discriminant Analysis MDA models were dependable to certain intensity in the prediction of corporate failure.CHAPTER 3RESEARCH METHODS3.1 Method of Data CollectionData was selected from Karachi Stock Exchange KSE 100 Index as given by State Bank of Pakistan in publication Balance Sheet Analysis of Joint Stock Companies Listed on the KSE (2004-2009). The period of study covers six years, 2004-09. The opted sample size of 44 firms was taken fr om KSE 100 Index and all of the firms listed on KSE 100 Index were selected for the samples which were going to bankruptcy in the past and some were also the present functioning firms which were currently working so, only 44 firms included in the sample period of 2004-09. The objective behind the inclusion of these selected firms in the sample was that the inclusion of bankrupt and non-bankrupt firms in the analysis made it easier to distinguish the critical financial ratios of these both firms in order to predict for corporate bankruptcy.The data availability was the major issue faced in this research study. The secondary data sources were adopted for the collection of the data during this research study. Both of the empirical and theoretical aspects regarding the prediction of corporate bankruptcy were analyzed in this research study. For the purpose of the collection of the secondary data, external data sources were used, such as the data was collected from State Bank of Pakistan , general business publications, newspapers and journal articles, annual reports, internet and books. The data required for this study was completely dependent on the published data sources, such as the published sources listed above.3.2 Sample SizeA sample of 44 firms from KSE 100 Index was selected and in addition, out of these firms 22 firms were bankrupt and the remaining 22 were not bankrupt which was taken as the holdout sample for the prediction of the corporate bankruptcy. Only firms were used in the samples which were either became bankrupt due to the impact of the some of the financial factors or the ratios or the firms which were in operations during the research study was conducted and these firms were listed on the KSE 100 Index form 2004-2009. The impact of the different financial factors or ratios, which were listed in the previous chapters, on the prediction of corporate bankruptcy was analyzed on all of the firms selected as the sample.3.3 Research Model DevelopedTh ere are various financial factors or the ratios of the firms which affected the prediction of the corporate bankruptcy of the firms. This research study analyzed the impact of different factors or ratios already listed in the previous chapters on the prediction of corporate bankruptcy. The model developed was a binary logistic model and its specifications are provided belowLiquidity = a0 + a1Firm Size + a2DEBT + a3LTD + a4LSALES + a5OI/S + a6OI/TA+a7IGP/TA+ a8Market to Book Ratio + whereLiquidity = the sum of cash and marketable securities divided by total assetsFirm Size = natural log of the book value of total assetsDEBT = the ratio of shorter period plus longer period debt to total assetsLTD = the ratio of longer period debt to total assetsLSALES = natural log of the annual salesOI/S = the ratio of operating income to salesOI/TA = the ratio of operating income to total salesIGP/TA = the inventory plus gross fixed assets to total assets ratio = the error term3.4 Statistical Techn iqueBinary Logistic Regression Analysis technique was used for this research study to examine the impact of the distinctive financial characteristics or the financial ratios of the firms on the prediction of corporate bankruptcy of the selected firms Statistical Package for the Social Sciences (SPSS) was used for the examination of the secondary data.Binary Logistic Regression Analysis technique was used for the purpose of prediction of of corporate bankruptcy or the prediction of the firms decisions to file for bankruptcy. The selected technique was used to study the impact of the different independent variables (financial factors as listed in the previous chapters) on the dependent variable i.e., prediction of corporate bankruptcy. The binary logistic regression analysis was selected for this study. It showed the intensity of the impact on the prediction of corporate bankruptcy during year 2004-2009 on the basis of several independent variables.CHAPTER 4RESULTSThe sample of 44 fir ms from the Karachi Stock Exchange KSE 100 Index was taken Binary Logistic Regression Analysis technique was used for this research study. Research examined the distinctive financial characteristics or financial ratios of firms which filed for the bankruptcy. The selected technique was used to study the impact of the different independent variables (financial factors as listed in the previous chapters) on the dependent variable i.e., the prediction of corporate bankruptcy. Statistical Package for the Social Sciences (SPSS) was used for the analysis and examination of data.4.1 Findings and Interpretation of the resultsInitially, the binary logistic regression technique was applied on the data collected using SPSS. Now, it was a nice time to proceed with the analysis of the results because the data was collected and ready to be examined. The interpretation and analysis is presented in the next sections of this research study.Case Processing SummaryUnweighted CasesaNPercentSelected Cas esIncluded in Analysis19291.4Missing Cases188.6Total210100.0Unselected Cases0.0Total210100.0This table explains the total population in the data file that is the 210 observations or the cases for the analysis of the bankruptcy prediction. This table further elaborates that the there were also some of the cases missing in the data because of the issue of data availability and some of the cases were the figures of zero.Dependent Variable EncodingOriginal ValueInternal ValueBankrupt0Non-Bankrupt1The above table shows that there are only two variables in the dependent variable of bankruptcy that are the being bankrupt or non-bankrupt.Model SummaryStep-2 Log likelihoodCox Snell R SquareNagelkerke R Square1234.707a.144.192This table elaborates the predictability of the complete model of the logistic regression which meant that to what extent the model predict the variation in the predicted group of bankruptcy. According to Cox Snell, the total predictors jointly explained variation in t he groups of bankruptcy was 14.4%. While according to Nagelkirki, the all independent variable explained the group prediction of about 19.2%.Hosmer and Lemeshow TestStepChi-squaredfSig.132.7158.000This table checks the overall model fit which means that the model is at its best in predicting the group variation from non-bankrupt to bankrupt. The hypothesis of the above table is that the test model is fit. The hypothesis is rejected because the sig value is less than .05 which concluded that the test model was not fit in this case of predicting the group variation.Classification TableaObservedPredictedBanckruptcyPercentage CorrectBankruptNon-BankruptStep 1BanckruptcyBankrupt762972.4Non-Bankrupt434450.6Overall Percentage62.5The classification table is the most important table in case of the logistic regression because this table explained the correct identification of the cases correctly identified. The percentage of correctly identified cases is 62.5% which is also commonly known as the hit ratio which means that to what extent the numbers of cases were correctly identified.Variables in the EquationBS.E.WalddfSig.Exp(B)95% C.I.for EXP(B)LowerUpperStep 1aDA-1.219.5105.7251.017.295.109.802AtoLTD-.002.0011.5831.208.998.9961.001CR.938.3487.2421.0072.5541.2905.056NPM.037.073.2621.6091.038.8991.198SG.161.232.4821.4881.175.7451.852Constant.066.579.0131.9101.068This is the final most important table in the logistic regression because this is the only table which shows the role of different predictors in significantly explaining the role in the prediction of group variations. Those important significant variables were only two that were DA, and CR because the sig value of only these variables were less than .05.4.2 Hypotheses Assessment SummaryThe hypothesis of the study was distinctive financial ratios have significant impact on the non firms decision to file for bankruptcy. These financial characteristics were current ratio (CR), debt ratio (DA), net profit margin (NP M), assets to long term debt ratio, and growth rate. In this study each of the financial characteristics or financial ratios of firms was tested and concluded the results.TABLE 4.4 Hypotheses Assessment SummaryS.NO.HypothesesSIG.RESULTH1There is a difference between the Current ratio of bankrupted companies and non bankrupted companies.0.9380.007AcceptedH2There is a difference between the Debt Ratio of bankrupted companies and non bankrupted companies.-1.2190.017AcceptedH3There is a difference between the Net Profit Margin Ratio of bankrupted companies and non bankrupted companies.0.0370.609RejectedH4There is a difference between the Assets to long term debt ratio of bankrupted companies and non bankrupted companies.-0.0020.208RejectedH5There is a difference between the Growth rate of bankrupted companies and non bankrupted companies.0.1610.488RejectedCHAPTER 5DISCUSSIONS, CONCLUSION, IMPLICATIONS AND FUTURE RESEARCH5.1 ConclusionIt was concluded based on the results of this resear ch study that current ratio and debt ratio were only the independent variables which were showing significance in Pakistani market and these variables were highly significant in playing the vital role explaining the variation in the dependent variable of the prediction of corporate bankruptcy and the remaining independent variables could not explain the variation in the prediction of corporate bankruptcy. These results were not matching with the study conducted by Altman (1968). These results were varying because in various countries, there was difference in environments and circumstances and firms usually made decision accordingly.5.2 DiscussionsCurrent ratio played a significant role in defining the variation in the prediction of corporate bankruptcy and this was also the case with the research study conducted by Altman (1968) because in his study the firm size was also playing a significant role. The variation in the prediction of corporate bankruptcy was not explained by the net profit margin ratio while it was significant in the study done by Altman (1968). The assets to long term debt ratio, and growth rate were not significantly explaining the variation in the prediction of corporate bankruptcy and study analyzed by Altman (1968), concluded the different results with some addition.5.3 Implications and RecommendationsThis research was limited to the various firms listed on Karachi Stock Exchange of Pakistan only. The data taken from 44 firms which were took through various sectors of the KSE 100 Index for the year 2004-09 which were previously bankrupt and which were currently operating. It suggested that such type of study should be carried out in other countries of Asia as well, as to have comprehensive idea about the choices of the firms decision to file for bankruptcy. Moreover, it also suggested that other factors except ones examined in this study should be researched as to have perfect idea about the selection of the prediction of corporate bankru ptcy. Besides that, this study can also be replicated in other developing countries.5.4 Future ResearchThis study helped various investors, management and other research conductors in analyzing and observing the behavior of firms regarding their decisions to file for the bankruptcy. Research students who want to work further on the prediction of bankruptcy can be benefited by this research study. Further more, the firms will become advantageous from this study because the study clarifies the distinctive financial characteristics or the financial ratios of different firms which significantly explain the variations in the prediction of corporate bankruptcy.

Banning Smoking in public places

Banning Smoking in public placesBanning Smoking in public placesHave you ever halt to think about the dangers of locoweed? Did you ever put in your guessation about other nation who are affected by your smoking? many a nonher(prenominal) people think that smoking gives them the feeling of satisfaction and happiness. Other people thinks that smoking makes the to calm down when they are angry, but all of this is nonsense. Smoking is a picked up as a habit by the youth in an unusual way. This habit risks their life and makes them prone to dreadful diseases. So if you are bemoan to kill yourself, then its your own choice, but, your freedom ends when other peoples freedom begins. Smoking is a very dangerous activity done by most of the people around the world which causes several health, environmental, and social problems. As a result, the WHO decided to pass a law to prohibit smoking in public places to foster people and the environment from the dangers of smoking.Heavy roll of tobaccors are likely to health problems which they jargon figure it out until it reaches a late stage. Researches made by scientists proofed that tobacco is one of the most addictive substances on earth. Every cigarette a smoker smokes supplies the body with great amounts of carcinogen and hazardous chemical substances. A heavy smoker, who ordinarily consume from 1 to 2 packs of cigarettes per day inhales 150,000 dose of poisonous smoke yearly. 1These Toxic compounds are create when tobacco reacts with fire. Therefore Nicotine and the other dangerous chemicals formed while smoking are the most harmful things in cigarettes which causes severe health problems.Another Dangerous health problem caused by smoking is Lung Cancer. This is because cigarettes nurse over 80 different cancer-causing substances. Scientists showed that these substances could damage the bodys DNA and change their characteristics. This process leads the cells to grow and multiply out of control causing blockage in racy areas in the lung. A smoker could avoid all of these problems and live at ease just by giving up smoking.The pollution caused by cigarettes does not stop in our bodies or the air it also affects the land we live on and the water that we drink. Millions of cigarette butts are thrown onto the ground every day. They at long last end up in the rivers and lakes where fish and animals -by mistake- eat them and quite often die from it. The rest are left on the ground to decompose which allow for take an average of 25 years while all of the chemicals and additives leach into the ground and pollute the soil and the plants. If you are going to smoke please consider this information before your throw your cigarette butts on the ground. It looks unattractive, it is a major fire hazard in dry weather, and it is extremely harmful to the environment.Another impacting sentiment of cigarettes is in producing them. The land used for growing the tobacco all over the world could be used fo r planting trees or food for starving children in the 3rd worlds countries. Tobacco plants are often sprayed with great amounts of harmful pesticides and chemicals as tobacco is very fragile plant and likely to pick up disease. A lot of trees are also used in producing and packing cigarettes. They use 4 miles of paper an hour just for rolling and packaging cigarettes. Every carbon cigaratte produced waste in the other hand a tree. The trees used in the cigarettes could be filtering out the pollutants found in the air instead of being shredded down for manufacturing a new cigarette. Cigarette smokers have a common belief between them. They think that they only endure themselves, in fact they hurt all the people surrounding them, people they love and the environment. Your freedom ends when the freedom of others begins. This say delivers the meaning directly. It means that its prohibited to go beyond others scope. This means smokers cant take the action of smoking only if he isnt g oing to hurt anyone around him.As a result, some countries governments decided to completely ban smoking in public areas to protect the environment and people from the dangers caused by the smokers. A great number of mostly developed countries have enacted bans on smoking in public places or workplaces since the early 2000s. The first building in the world to have a smoke-free policy was the Old Government Building in Wellington, New Zealand in 1876. In 1990, the city of San Luis Obispo, California, became the first city in the world to ban indoor smoking at all public places, including forbid and restaurants. Arguably by allowing smoking in public places it is the freedom of non-smokers that is being restricted. By smoking in public the smoker is forcing everyone in the vicinity to smoke too, perhaps there should be signs everywhere reading public smoking area only. This banning prohibition law was in the interest of non-smokers as they are able now to go to public buildings and f inish their official transactions without being hurt by smokers around them.There are 3 billion victims yearly in the world die on account of cigarette smoking. Why dont we all start from today to reduce the numbers of smokers to protect our lives from the muted killing substance called tobacco? Why dont we start living green and stop affecting ourselves and our environment from the smoking circumstances? A problem that necessarily to pause and re-calculations so that we would not target and regret for what happened as a result of smoking.1 http//healthcare-guidance.blogspot.com/2009/11/effect-of-cigarette-in-our-health.html

Events Of The Year 1793 Essays -- essays research papers

Laurie Halse (rhymes with "waltz") was born on October 23, 1961 in Potsdam, New York, to Methodist minister Frank A., Jr. and manager Joyce Holcomb Halse. The agent says that she unconquerable to become a bring outr in second grade. Her teacher taught the class how to write haiku. She enjoyed it a lot and hopes that every second grader will learn to write poetry. Halse soon started reading library books for hours. The magic of the elementary school library came alive in life. Heidi, one of Halses favorite books, sparked her interest in foreign cultures. Halses creative thoughts began as a child. For instance, Halse recalls trudging through the snow on her way to school. She imagined that she had changed into an enormous polar bear. Writing also started as a little girl for Halse. She enjoyed ceremonial occasion her father write poetry and read the comics spread out on his office floor. She used her fathers old typewriter for hours, writing newspaper columns, stories, an d letters. Halse declares that the dictionary is her favorite book. As a senior in high school, Halse visited Denmark as an American Field Service exchange student. She lived on a pig farm and learned to communicate Danish. The author obtained an associate of arts degree in 1981 from Onandaga County Community College. She married Gregory H. Anderson, chief executive officer of Anderson Financial Systems, on June 19,1983. They raised two children Stephanie and Meredith. The author earned a bachelor of science in Languages and Linguistics (B.S.L.L.) degree in 1984 from Georgetown University. Laurie Halse Anderson belongs to the Society of Childrens Book Writers and Illustrators (SCBWI). Anderson organized the SCBWIs Fall Conference in Philadelphia from 1994 to 1996. She persistent to write Fever 1793 after reading a newspaper article in August of 1993 that explained the chicken fever epidemic that devastated Philadelphia 200 years earlier. The U.S. working capital in 1793, Philadel phia served as the political and cultural hub of the nation. Anderson believed these ingredients would create a story with strong elements of conflict and a rich background. She also suspected that stories written around this period were rare. Moreover, her upbringing near Philadelphia inspired her to begin research. Researching the idea proved to be no simple task, however. It took two years. Anderson read about the periods archit... ...is quickly becoming. When Nell contracts yellow fever, Mattie and Eliza follow the French medical treatment fresh air, rest, and fluids. Her suggestion works, and Nell heals. Nell symbolizes hope for a brighter future, and she enables Mattie to take on the responsibility of adulthood. At the novels resolution, Matties character transforms as she realizes that she must persevere. She knows that she has many choices. Mattie cultivates her friendship with Nathaniel. She opens up the coffeehouse with Eliza as her partner and begins to implement her d reams for a growing business. Nell stays with her and depends on her like a mother. Lucinda returns from the country in ill health. Forced into a life of leisure, Lucinda must also depend on and trust in Mattie to run the coffeehouse. Lucinda begins to respect Matties choices and to treat her with respect and compassion. Anderson does an glorious job of developing believable characters with realistic themes set against a rich background. She shows how this disease changed her characters, much as horrific life events change real lives. three-year-old adults will emerge from this novel with a sense of hope, perseverance, and understanding.

Power for Women in Alcestis and Hippolytus Essay -- Greece Greek Play

Is it feasible that by the loss of ones liveliness and being, one would be able to gain influence and power? Does this fatal gain of power show a previous lack of it? Does forgoing ones invigoration for an echt cause better a womans reputation in turn giving her more power? Through our studies, we have observe that typically women exhibit a limited amount of agency in ancient Greece. Women occasionally assert dominance in the household although, even inwardly the home they posses limited influence over their maintains. An interesting theme runs though Euripides theatrical tragedies Alcestis and Hippolytus. In each play the lead female character forgoes her life for the sake of love. In Alcestis, Alcestis willingly gives her life to prevent her husband Admentus death. In Hipplytus, Phaedra chooses to commits suicide as a result of falling in love with her husbands son and refusing to be deceitful to her husband. Consequently, is self-professed death a venue for the women to assert authority and gain status and agency? How do their reputations and the reputations of their households affect this step-up of power? In ancient Greece, women, through sacrifice of their lives, uphold and improve their reputation through which they increase their influence and power in society, yet although they atomic number 18 praised by society because of these valiant deeds, they are unable to actively reap the benefits of this powerful reputation. Numerous sources including Euripides tragedies show that reputations are held with the highest regard in ancient Greece. It is through peoples perceptions that one is judged in that locationfore, reputation should be upheld at the greatest of costs. Laws of classic society allow for a ma... ... Phaedra preserves her reputation and altered her husbands behavior through her suicide. Yet, there was a trade off each woman acquired a greater amount of power, yet lost her life in the process. Ho wever, because Greek society praised honorable death, this was an appropriate societal action and a proper way for the women to acquire power. Women did assert power in choosing to take their own lives and improve their reputations.Small summaryGreek tragedies show that women, through sacrifice of their lives, uphold and improve their reputation through which they increase their influence and power in society. Yet, there was a trade off each woman acquired a greater amount of power, yet lost her life in the process. However, because Greek society praised honorable death, this was an appropriate societal action and a proper way that women acquired power.

Film Analysis of All That Heaven Allows Essays -- Papers Movie Film Ci

Film Analysis of All That Heaven Allows Chosen sequence Golden rain Tree/Carys bedroom scene. Before the emergence of auteur theory the director Douglas Sirk was a renowned exponent of continent Hollywood narrative, particularly in the genre of romantic melodrama, of which his take up All That Heaven Allows is a classic example. However, he is now regarded as a master of mise-en-scene, one of the few tools left to a director working within the constraints of the Hollywood studio/institutional system who is now thought to have been highly critical of American mainstream culture and society in this fortunate era. 1, 2 The Golden Rain Tree sequence occurs early on in the film after the crack panoramic, establishing shot - showing the scene of the action, a small middle-class New England town in autumn. The main protagonists are soon introduced of which the prime causal agent is an unsettled woman, Cary Scott (Jane Wyman), in keeping with romantic mel odrama. As a widow, she is a victim of circumstance who is eager to change her life. Her friend visits (Mona) and hopes to persuade Cary to take a conventional route out of widowhood but, by chance, she meets the gardener, Ron Kirby (Rock Hudson) and from the mise-en-scene in these opening scenes it is obvious that she would prefer a romantic affiliation of some sort with Ron. After a few pleasantries, some tea at the table foreign Carys house and some profound references to gardening, Ron goes to hand Cary a small brance/twig, evidently a token of deep affection. The film so far displays all the conventions of classical narrative and maintains all the dominant ideologies o... ...lassical narrative cinema. In Being There, the character and motives of Gardiner are made much clearer to the viewer through the imaginative use of mise-en-scene, as illustrated above. NOTES 1. Carroll. Essay The Moral Ecology of Melodrama The Family Plot and Magnificent Obse ssion. p. 170. 2. Cook. p. 76-79. BIBLIOGRAPHY An Introduction to Film Studies Jill Nelmes (ed.) Routledge 1996 Anatomy of Film Bernard H. Dick St. Martins Press 1998 Key Concepts in Cinema Studies Susan Hayward Routledge 1996 give lessons Yourself Film Studies Warren Buckland Hodder & Stoughton 1998 Interpreting the Moving Image Noel Carroll Cambridge University Press 1998 The Cinema Book Pam Cook (ed.) BFI 1985 FILMOGRAPHY All That Heaven Allows Dir. Douglas Sirk Universal 1955 Being There Dir. Hal Ashby 1979

Hypnosis :: essays research papers

Hypnosis     The British Medical Association and the Ameri give notice Medical Association hascalled it "a temporary causation of change attention in the overmaster that maybe induced by another person," (Comptons Multimedia Encyclopedia) but there isstill much almost hypnosis that is not understood. Because it resembles normalsleep, it was studied and was found that the brain waves of hypnotized peopleare more similar to the patterns of deep relaxation than eachthing else. Ratherthan a psychic or mystical idea, hypnosis is now looked upon as a form ofhighly focused concentration in which outside influences are ignored.     The most known feature of the hypnotic trance is that hypnotized personbecomes easily influenced by the suggestions others-usually the hypnotist. Theyretain their abilities to act and are able to walk, talk, speak, and respond toquestions but their perceptions can be altered or distorted by externalsuggestion s. At the command of the hypnotist, subjects may lose all feeling ina place on the body, and any kind of pain will not cause them any pain. Theheartbeat can be slowed or quickened, and a rise in temperature and perspirationcan be created. They can be commanded to experience visual or auditoryhallucinations or live the past as if it were the present. Also, recently ascientist discovered that the dash the subjects mind experiences time can bealtered so that hours or even weeks can pass in second, from the subjects pointof view. Subjects may forget stir up or all of the hypnotic experience or recallthings that they had forgotten. The hypnotist may also make "posthypnoticsuggestions" that are instructions to the subject to respond to a something later on awakening. For example, the hypnotist might suggest that, after thesubject wakes up he will have an urge to remove his left shoe, and the more thesubject resists, the greater the urge to remove it will be, and once it isremove d the urge leaves. These suggestions are sometimes used by specialiststo repress or suggest off symptoms in a patient such as anxiety, itching, orheadaches.     Hypnosis is produced essentially by creating a deep relaxation andfocused concentration in the subject. They past become mostly unresponsive toordinary forms of stimulation, and although they are sometimes told to sleep,they are also told to listen and be ready to respond to commands made by thehypnotist. The rallying cry sleep is used in hypnosis not to induce actual sleep, butin practice it is understood that sleep is simply the hypnotic trance. Theprefix hypno- is named after the Greek god Hypno which means "sleep.