Green Fluorescent Protein (GFP) Mutants

Green Fluorescent Protein (GFP) Mutants GREEN FLUORESCENT PROTEIN (GFP) MUTANTS WITH ALTERED FLUORESCENCE INTENSITY AND EMMISSION SPECTRA Introduction: Now-a-days GFP is creating revolution in the field of science by its applications and properties.GFP is a stable protein extracted from the photo organs of the jellyfish Aequoria victoria by Shimomura et al in 1962. In 1992 the cloning of GFP has done. It is found in a variety of coelenterates (both hydrozoa and anthozoa) and it emits light by utilising energy from the Ca2+ activated photoprotein aequorin [1]. Energy transfer and the emission spectra of GFP can be affected by dimerization. Structure of GFP is cylindrical ÃŽ ²-can structure and has a chromophore located centrally. The chromophore is responsible for the fluorescence and the formation is independent of species but mainly depends on oxygen. GFP is a small protein and has been made up of 238 amino acids. Deletion of any seven amino acids either from C-terminus or N-terminus may result in the loss of fluorescence. Amino acid replacement is responsible for the change in colours of GFP. It has a molecular weight of 27 KDa an d has an absorption range at 488 nm and an emission range at 509 nm. It can accomplish high temperatures (65 ÌŠc) and basic PH range of 6-12 [2]. Increase in PH results in the decrease of fluorescence. Increase in the fluorescence and photo stability can be achieved by single point mutation at S65T. Fluorophore of the GFP is generated by using auto-catalytic process of continuous mechanisms. Visible excitation is one of the optical properties of GFP. Its derivatives are produced from the mutagenesis experiments like random and directed mutagenesis [3]. GFP is majorly used as a reporter in expressing genes. Protein and chromophore folding also constitutes as a major advantage of GFP. It can also be used in protein fusion by applying recombinant DNA technology. Aim of this research is to analyze properties of GFP by cloning, mutations, expression of proteins and purification. Objectives of this research are to sub-clone GFP into a vector and mutations are carried out by various mutagenesis experiments followed by expression of proteins and purification. Finally after purification properties are analyzed. Materials and methods: Initially DNA is isolated and GFPuv is sub-cloned into the pET28c vector from pET23 plasmid by speectrophotometric analysis. 5 µg of pET23GFPuv DNA is digested by using NdeI and HindIII restriction enzymes. And the digests are analysed by using Agarose gel electrophoresis. GFP fragment is extracted and purified using QIA quick gel extraction kit from QIAGEN and the recovered DNA is estimated. Recombinant protein is expressed in E.coli by ligation and transformation. To confirm the presence of GFP in the pET28c plasmid, colony PCR is used. Further mutagenesis experiments are carried out by designing oligonucleotide primers which will alter the spectral properties of the protein. Complementary primers containing same mutations are generated. Mutagenic primers are prepared with a melting temperature of ≠¥ 78 ºC, length between 25 and 45 bases and primers longer than 45 bases are generally used. Introduction and identification of mutations within GFPuv gene: Mutations are created in the GFPuv insert by site-directed mutagenesis Site-directed mutagenesis: 5 µl 10 x PCR buffer 5 µl 20 mM dNTP mixes 15 ng GFPuv-pET28c template DNA 125ng oligonucleotide primer F+ 125ng oligonucleotide primer R+ 2 µl 25mM MgSo4 32 µl sterile water 1 µl KOD hot start polymerase (1U/ µl) * All the above are added to 0.2ml PCR tubes and incubated in a PCR machine for 24 cycles: 94 ºC 30s 94 ºC 30s 55 ºC 1min 68 ºC 4min 20s 68 ºC 10 min * Reaction is then kept on ice for 2 min and 1 µl (1U) of Dpn1 is added and incubated for 60 min at 37 ºC Alignment of amino acid sequences is carried out using: http://www.ebi.ac.uk/Tools/clustalw2/index.html Product of site-directed mutagenesis (pET28c DNA) is transformed into XL-1 supercompetent cells. Transformed colonies are extracted using QIAprep Mini prep kit Qiagen [5]. Concentration and purity can be checked by using Agarose gel electrophoresis. For this 5 µl of plasmid preparation and 10U HindIII are digested at 37 ºC for 1h. Sequencing is then carried out by using 10 µl of DNA at a concentration of 50ng/ µl. E.coli BL21 (DE3) cells are prepared and are transformed into the pET28cGFPuv plasmid for expression Auto-induction method: Wild type protein (GFPuv) and the mutant protein are expressed in the expression vector [BL21 (DE3)] using auto-induction method. For this transformed colonies are inoculated into 3ml of LB-1D + antibiotic media and incubated at 37 ºC at 300 RPM for 6 hrs and O.D is taken. Inoculum is taken into the flask containing SB-5052 auto-induction medium along with antibiotic and incubated at 28 ºC at 300 RPM for 20 hrs. Cultures are then cooled for 1 hr. Total induced sample is prepared by taking 100 µl of cooling culture and 900 µl of SB-5052 media. Cells are then pelletized by centrifuging it with both total induced and non-induced samples and are resuspended in 100 µl of SDS-PAGE (sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis(PAGE)) sample buffer. 12% of polyacrylamide gel is prepared and the Soluble and insoluble samples are prepared by cell fractionation using BUGBUSTER. For this 1 µl of DNAase1 is used along with reagents. Cell suspension is then centrifuged at 13000rpm for 20mins. Supernatant is then used as soluble sample and insoluble is prepared by resuspending the pellet in 2ml binding buffer. SDS-PAGE buffer and binding buffer are added to the soluble and insoluble fractions. At 95 ºC all samples are heated for 5 min. Gel is then loaded as: Molecular weight standard-5 µl Uninduced sample 5 µl Induced total sample 5 µl Soluble sample 5 µl Gel has to run for 1 hr. And is transfered to a box of Coomassie blue stain. Western blotting: GFP protein presence can be verified using western blotting technique. Protein samples are first seperated by SDS-PAGE and are transferred to the nitrocellulose membrane. GFP bound to nitrocellulose membrane is then visualised by incubating the blot with His-probe which is linked to a HRP (horse radish peroxidase) enzyme (HisprobeTM-HRP solution is diluted to 1:5000 (1 µl in 5ml) ). His-tag of GFP protein is bound to probe. Blots are kept in TBST and probes and thus probes are visualised by chemiluminescence and these are photographed by chemiluminescent reader. Ni-NTA chromatography: His tagged GFP can be purified by Ni-NTA (nickel nitrilo triacetic acid) chromatography method. In this, sample of soluble protein is loaded on column packed agarose resin and the non-specific protein binding is removed by washing resin with buffer and is eluted by high concentrated imidazole of elution buffer. After elution the purification of protein is done by SDS-PAGE and Coomassie staining. The concentration of the protein is measured by Bradford assay. Fluorimetry and mass spectrometry: Properties of GFPuv protein are analysed by Fluorimetry and mass spectroscopy. Fluorimetry: In this wavelength and intensity of a molecule at specific wavelength are measured using fluorimeters. Perkin Elmer LS50B is the fluorimeter used to measure GFP. Quartz cuvettes are placed in a chamber to measure the concentration and intensity. The parameters set to measure GFP are: Excitation 440nm Emission 460-550nm Slit widths 4 and 4 Accumulation 5 20 µg/ml of protein concentration is used. The emission and excitor wavelengths are set at 509nm and 395nm. Mass spectrometry: GFPuv properties and molecular mass can be analysed by mass spectroscopy. The type of mass spectroscopy used here is electron spray ionization (ESI). ESI is a type of atmospheric pressure ionisation technique (API) which is used for biochemical analysis. JEOL HX110/HX110A equipped with electron ion source tandem mass spectrometers are used to analyse structural properties [7]. 1-10 pmol/ µl of protein concentration is used. Solvents used are: MeOH MeCN TFA During ionisation sample is dissolved in a solvent and is pumped through a steel capillary at a rate of 1 µl/min and voltage of 3 or 4KV is applied [8]. Ion current is amplified by the detector and the data system will record signals in the form of mass spectrum. RESULTS: Site-directed mutagenesis: Primers used for site directed mutagenesis (Mutant) Forward primer: 5-CACTTGTCACTACTTTCTCTTGGGGTGTTCAATGCTTTTCC-3 Reverse primer: 5-GGAAAAGCATTGAACACCCCAAGAGAAAGTAGTGACAAGTG-3 Alignment of the amino acid sequence of the mutant with the GFPuv amino acid sequence GFPuv MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTL 60 mGFPuv MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTL 60 ************************************************************ GFPuv VTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGNYKTRAEVKFEGDTLV 120 mGFPuv VTTFSWGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGNYKTRAEVKFEGDTLV 120 *****:****************************************************** Y66W GFPuv NRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKANFKIRHNIEDGSVQLAD 180 mGFPuv NRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKANFKIRHNIEDGSVQLAD 180 ************************************************************ GFPuv HYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK- 238 mGFPuv HYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK- 238 ********************************************************** Amino acid substitution: Y66W Belongs to Class 5, indole in chromophore (cyan fluorescent proteins) [6] eCFP CATATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGAT 60 GFP ATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGAT 57 ********************************************************* eCFP GGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATAC 120 GFP GGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATAC 117 ************************************************************ eCFP GGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACA 180 GFP GGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACA 177 ************************************************************ eCFP CTTGTCACTACTTTCTCTTGGGGTGTTCAATGCTTTTCCCGTTATCCGGATCACATGAAA 240 GFP CTTGTCACTACTTTCTCTTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCATATGAAA 237 ******************* ******************************** ****** Mutation eCFP CGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAACGCACTATATCT 300 GFP CGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAACGCACTATATCT 297 ************************************************************ eCFP TTCAAAGATGACGGGAACTACAAGACGCGTGCTGAAGTCAAGTTTGAAGGTGATACCCTT 360 GFP TTCAAAGATGACGGGAACTACAAGACGCGTGCTGAAGTCAAGTTTGAAGGTGATACCCTT 357 ************************************************************ eCFP GTTAATCGTATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTCGGACAC 420 GFP GTTAATCGTATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTCGGACAC 417 ************************************************************ eCFP AAACTCGAGTACAACTATAACTCACACAATGTATACATCACGGCAGACAAACAAAAGAAT 480 GFP AAACTCGAGTACAACTATAACTCACACAATGTATACATCACGGCAGACAAACAAAAGAAT 477 ************************************************************ eCFP GGAATCAAAGCT 492 GFP GGAATCAAAGCTAACTTCAAAATTCGCCACAACATTGAAGATGGATCCGTTCAACTAGCA 537 ************ eCFP GFP GACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCAT 597 eCFP GFP TACCTGTCGACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGCGTGACCACATGGTC 657 eCFP GFP CTTCTTGAGTTTGTAACTGCTGCTGGGATTACACATGGCATGGATGAGCTCTACAAATAA 717 SDS-PAGE : Coomassie staining gel of (Sample 6): Marker GFP protein (soluble sample) Western blotting (Sample 11): Induced total sample GFP protein Ni-NTA chromatography: Fluorimetry: Mass spectrometry: Wild-type: Mutant: Discussion: Site-directed mutagenesis: In the site-directed mutagenesis mutation is carried out at the right place i.e., at 197 and 198 places. Tyrosine (TAT) is mutated to tryptophan (TGG), Y W. During this mutation protein undergoes many changes especially in the fluorescence. GFP turns into CFP (Cyan fluorescent protein) hence the light emitted will not be exactly green. CFP will have many peculiar features like rather than single excitation and emission peaks it possess double humping. Tag CFP possess some properties like: Structure monomer Molecular weight 27KDa Polypeptide length 239aa Fluorescence colour Cyan Maximum excitation 458nm Maximum emission 480nm Excitation coefficient 37000M-1 cm-1 Pka 4.7 Quantum yield 0.57 Brightness 21.1 Brightness is produced by the quantum yield and extinction coefficient. Dual colour visualisation of the protein expressed is enabled by the CFP. This has led to the Fluorescence Resonance Energy Development (FRET). SDS-PAGE: SDS-PAGE is carried out to separate proteins according to their electrophoretic mobility and experimental repeats will result in the purity assessment of the protein. Four wells are loaded with samples and 2 and 4 wells show protein result and as 1 and 3 wells dont contain protein they will be normal without any bands. Results shows that little amount of GFP has been observed in the insoluble and large amount of protein has been observed in the soluble sample. Uninduced sample cannot find GFP. Western-blotting: Western-blot is performed to make sure the presence of protein. Histidine tagged probe is added to confirm the protein present was GFP or not. pET28c plasmid contains T7 RNA polymerase promoter sequence. But this promoter is blocked by the repressor. Hence lactose containing medium is required for E.coli growth. Because lactose is used as carbon source, glucose is converted into allolactose. This allolactose will bind to repressor by unblocking promoter, and expresses GFP. Hence presence of glucose will result in Lac-I and is binds to the operator. Band observed in the blot is probably GFP and it has high level of intensity after induction. And it is necessary to confirm this by performing blotting technique using His probe to detect His tagged GFP. Bands are observed in the induced and soluble samples after performing western blotting confirming the presence of GFP. Ni-NTA chromatography: Purification of GFP can be done by Ni-NTA chromatography. For a recombinant protein the amino acid binding site with 6 or more His residues in a row acts as metal binding site. So hexa-his sequence is called as His-tag. His-tag sequence is present in the N-terminal of the target protein and is located in the promoter region adjacently to the GFP gene. During this process enzyme HRP is also bound to the probe. This HRP-probe will react with luminal 4 peroxidase buffer which is further used for purifying GFP by Ni-NTA chromatography. Purification by His-tagged GFP can be done by using several methods like Ni2+-poly (2 acetomidoacrylic acid) hydrogel. Displacement of GFP can be done by binding nickel to imidazole. This is mainly because of high affinity of nickel towards imidazole compared to GFP.Distinctive bands are supposed to observe in the elute1, elute 2 and also in the total soluble fraction. Bands formed states the presence of the GFP mutant. Absence of the bands states mutant a bsence. In the results bands are observed at the total induced and the soluble samples which state the protein presence. Even small amounts of bands are also observed in the insoluble sample. GFP protein produced in the induced total sample is approximately at 27KDa. Slight bands are observed in the insoluble sample as it may be because of some impurities. Finally the GFP protein has been detected. References: 1. Davenport D, Nichol JAC: Luminescence in Hydromedusae. Proceedings of the Royal Society, Series B 1955, 144:399-411 2. Ward. W., Prentice, H., Roth, A. Cody. C. and Reeeves.S.1982.Spectral perturbations of the Aequoria green fluorescent protein. Photochem. Photobiol. 35:803-808 3. Cormack, B. P., Valdivia, R. H., Falkow, S. (1996). FACS-optimized mutants of the green fluorescent protein (GFP). Gene, In press 4. Darelle Thomson , Greg Smith. (2001).PCR-based plasmid vector construction for generation of recombinant viruses. Journal of Virological Methods 94, 7-14 5. Vogelstein, B., and Gillespie, D. (1979) Preparative and analytical purification of DNA from agarose. Proc. Natl. Acad. Sci. USA 76, 615-619. 6. HEIM, R., PRASHER, D. C. TSIEN, R. Y. 1994. Wavelength Mutations and Posttranslational Autoxidation of Green Fluorescent Protein. Proceedings of the National Academy of Sciences of the United States of America, 91, 12501-12504. 7. HARUKI NIWA, SATOSHI INOUYE et,al., Chemical nature of the light emitter of the Aequorea green fluorescent protein. Vol. 93, pp. 13617-13622, November 1996. Proc. Natl. Acad. Sci. USA. 8. â€Å"Mass Spectrometry: A Foundation Course†, K. Downard, Royal Society of Chemistry, UK, 2004.

Foundation Certificate in Human Resource Practice Essay

1. Collecting and recording HR data is vitally important to an organisation. The collecting of the data could be to monitor that laws and regulations are being adhered to for example the Health and Safety at work act 1974, ensuring that all staff are maintaining high health and safety awareness and complying to the law. The data would need to be collected to enable the organisation to prove that it is adhering to current law and legislation. Another example could also be to monitor employee absence levels across the organisation and looking for any pattern or trend relating to individual absences. This data could be used in Absence review meetings and having all the correct and accurate data could be vital in a dispute with an employee. It could highlight issues with employee welfare and enable the company to offer support in order to support the employee back to work. 2. Storing Records There are many methods of storing records, an example is: Electronic which includes hard disks drive – PC, CD – recorder, DVD, databases and spreadsheets, internet or intranet, USB devices, emails and virtual learning environments. Electronic storage can have pros and cons. Advantages can be the speed and accuracy that it provides, spellcheckers etc can all help the documents to be stored accurately. Vast amounts of data can be stored on a computer software system and therefore not take up and physical office space. The electronic way of storing data can also be protected by a password meaning that it is secure and accurate at the same time and protected from anyone outside the HR function, and it means that a variety of colleagues can have access to update and amend the records at the same time, even updating at the same time as colleagues. Manual Storage. Manual storage can be personnel files, absence forms, reports, filing cabinets etc There are lots of benefits to manual storage including having documents which need a physical signature and provide proof of identity like bank details etc. Also should a computer system crash or wipe the documents the paper copy is always accessible. Manual storage is easy to move around and is easy to keep protected and confidential via a lock/key etc although staff with access must ensure it is securely locked away. 3. UK Legislation The Data Protection Act 1998 is about respecting individual rights when processing/collecting and storing their personal information. This is achievable for the company by being honest with employees about the use of their information and by following good data handling procedures. The act is compulsory and all organisations that hold or process personal data must adhere to this. Personal data should be processed fairly and lawfully, the data should be adequate, relevant and not excessive, it should be accurate and where necessary kept up to date, any data should not be kept for longer than necessary, data should be kept secure. All staff has responsibilities under the Act to ensure that their activities comply with the Data Protection Principles Employees do have a right legally to access information that an organisation may hold on them. This could include information regarding any grievances or disciplinary action, or information obtained through performance monitoring processes. Processes should be in place to deal with a data request from an employee as a 40 day time limit is compulsory. The health and safety at work at 1974 is legislation relating to protecting employees from injury or illness as a direct result of their job. All data relating to health and safety must be recorded and stored securely, including accident books. This data may be called upon many years after an employee has left the organisation so staff should ensure documents and information are kept in a secure adequate accessible place. The Freedom of Information Act which came into force in 2000 gives you the right to ask any public sector organisation for all the recorded information they have on any subject. Anyone can make a request for information – there are no restrictions on your age, nationality or where you live. If you ask for information about yourself, then your request will be handled under the Data Protection Act 1998. Recording, Analysing and using Human Resources information is highly important and ensuring it is accurate and efficient will support the organisation strategy in many ways. The Analysis can change the way the organisation moves forward and affect future plans/decisions.

Memorandum: Net Present Value and Apex Investment Partners

MEMORANDUM To Apex Investment Partners: According to my analysis of the Accessline’s proposed term sheet, I do not believe that Apex would serve its own interests, or those of its investing partners, by investing in Accessline according to the terms proposed. By investing at the proposed valuation, according to the proposed control and incentive structure, Apex would be shouldering a disproportionate share of the risk should Accessline fail to meet its performance targets, or require fresh inflows of capital from future investment rounds.Nor can Accessline take the sort of steps necessary to protect its investment in the case of management failure. Should Apex make a counter-offer, I would suggest the following terms: Valuation: Accessline’s projected revenues in 1999 are $208m. Using the average price/revenue ratio of 3com and Boston Technologies, it seems reasonable to expect an IPO valuation at 3. 67 times revenues, producing gross proceeds of $764m with a present va lue of $116m (using our 60% discount rate).Assuming that Accessline meets this revenue target, and that no future funding is required, Apex will take a slight loss on its required rate of return, barring the voluntary distribution of the dividend from the board of directors, on which we are not offered a seat. The present price per share at such an exit would be approximately $7. 84. However, given Accessline’s historical burn rate, it seems unreasonable to expect the $16m investment produced in Series B to last Accessline until 1999.Assuming Accessline will need another $32m to reach its revenue targets by 1999, Apex takes a much more severe loss relative to its required rate of return. The present price per share at such an exit, assuming the new shares are also offered at $8 per share, would be $6. 18 per share. I therefore suggest using $6 per share as a point for a new valuation of the company, assuming the inclusion/revision of terms as described below. Rights and Prefe rences Apart from the valuation, other elements of the term sheet must be adjusted to allow Accessline to protect its interests and motivate or replace management in the case of performance failure.First and foremost, Apex must insist on the right to elect one director to the board. Series A investors already have one seat, and the current voting clauses allow Series A to effectively retain control of decision making by requiring 2/3rds majority for many key decisions. Should future funding rounds be required, those investors may insist on seats on the board. Apex must remove antidilution protection from employee shares, as this removes a significant incentive for employees and management to reduce Accessline’s burn rate.However, as Series A investors retain a veto over the deal, their shares must be allowed to retain anti-dilution protection. Additionally, we may propose a point at which additional investment rounds (above and beyond $32m of fresh capital) would cause diluti on of ESOP shares at an accelerated rate. Dividends should be made cumulative and issuable upon a liquidation event or an IPO. Such dividends may be converted, if the holder desires, to common shares. This will encourage management to seek a quicker exit. Liquidation preference must be strengthened in other ways.In my opinion, the current arrangement allows management and employees to receive unjustified returns in the case of a liquidation. I suggest a ratio of 1. 5 times the Series B purchase price, applicable to Series A shares, with the remainder to be distributed among Series A, Series B, and common shareholders/ESOP on an as-if-converted basis. In an IPO, Series B shares should auto-convert at a ratio of one-to-one at a target price of $12 until June 30th and $15 after June 30th 1996. After that, the targets must continue to ratchet upwards.The written consent of 3/4ths of Preferred shareholders could override this requirement while preserving Apex’s ability to veto aut o-conversion. This voting ratio should also be employed in the voting clause, since without it Apex lacks any ability to control future funding rounds. Series B must be allowed to redeem all of their shares upon the failure of Accessline to come within 5% of its revenue and income projections for 2 consecutive years. Alternatively, Apex could require that unvested management/ESOP shares be returned to Series A and Series B on a pari passu basis in the case of performance failure.Alternatively, Accessline could insist on a right to replace management in the case of this eventuality. Given the large number of competitors already present in the market, it is likely that if Accessline’s business fails, it will do so quickly and drastically. Negotiation considerations It is important to note that a counterproposal from Accessline that strengthens or enhances any of these provisions in Apex’ favor in exchange for a higher issue price of the Series B shares should be consider ed.However, there are limits to the premium we should pay for enhanced control, and firm limits for how far such control can be reduced. A board member and the voting rules are non-negotiable. The dividend and the autoconversion terms, however, are places in which we can demonstrate flexibility. At this price, with these changes to the term sheet, we are still exposed. Significant competitive, regulatory, or technological changes in the marketplace could quickly destroy Accessline’s profitability.This is, as it stands, a strong counterproposal that is bound to meet resistance from management and employees, but provided we preserve Series A’s valuation, I believe Series A investors will be inclined to allow us more control and latitude provided the performance requirements for management are strengthened. Since I believe our competitors will also propose lower valuations based on a view of these same numbers, we must act tactfully. Perhaps some sort of parachute can be arranged for senior management in the event of a takeover.

The Philosophical Branch Of Personal Identity - 1884 Words

In the philosophical branch of personal identity there exist several approaches to the question what is it for the same person to exist over time. It is important to stress that we are referring to numerical identity here, i.e. the identity of a person over a period of time. What is it on the basis of which we say that a person on time point 1 is same as that on time point 2? In general, there exist three approaches to this question: the physical theories (the brain view and the body view), the brain and the bodily identity as the possible criterion of Personal Identity as well as the mental theory such as the psychological continuity view. The latter one determines that a person at an earlier and at a later time are the same person if and only if there is a continuous chain of overlapping direct psychological connections. In his paper â€Å"The Self and the Future†, Williams presents two thought experiments. 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