Nuclei And Mitochondrial Fraction

Nuclei And Mitochondrial Fraction The objective of this experiment is to prepare a nuclei and mitochondrial fraction using differential centrifugation, from a rat liver homogenate sample. The amount of activity of mitochondria in the fractions can be measured using succinate dehydrogenase (SDH) as a marker. To measure the percentage recovery of the SDH of Mitochondrial, Nuclei and supernatant fractions in comparison to the Homogenate and to Calculate the specific and relative activity of SDH in each fraction. Figure 1: Shows a typical animal cell with the individual organelle components. Figure 2: Shows the typical features and functions of the organelles of interest in this report. Figure 1 + 2 Created on Microsoft paint with reference to Essential Biology (2004) Individual organelles differ in size but are all usually around 10nm in diameter. There is a small surface area and size/density depends on the organelle, the smaller organelles being lysosomes and ribosomes. Mitochondria differs in cell type depending on the energy demand of that organ, the more ATP that is required in a particular organ the more mitochondria found. E.g. more mitochondria found in heart and liver cells than in a white blood cell like a lymphocyte. Smaller organelles include lysosomes and ribosomes. Metabolism can be detected using various methods such as use of inhibitors. These can be both competitive and non-competitive, an example is seen with arsenic with inhibits pyruvate dehydrogenase. Another method is with the use of radioisotopes to measure activity aswell as histochemistry, immunocytochemistry and electromicroscopy. Preparation of the homogenate occurs in various stages. Firstly the homogenisation of liver cells. This can be done using a Potter Elvehjem homogeniser to extract organelles without damaging the actual cell. This is a simple and effective homogenisation method. A small gap is made within the cell wall which is then pressurised which forces the contents i.e organelles, cytoplasm etc. out of the cell. This occurs at a low temperature and mild pH, and to keep the isotonic solution a sucrose buffer is used, therefore since there is the same water potential inside the cell and outside the cell there is no net movement of water (osmosis) and thus the cell remains the same size. Homogenized cells also must be kept at low temperatures to prevent autolysis (the degradation of a cell by its enzymes). (www.bookrags.com). Figure 3 shows a classic Potter Elvehjem homogeniser Image taken from (umwcellbiology.org) The second stage is fractionation of the homogenate sample. This process is called centrifugation and can be further split into either a differential centrifugation or a density gradient centrifugation. The differential centrifugation splits the impure fraction into separate compartments due to the size of the various organelles in question and there density. The centrifuge applies a gravitational force onto the sample to separate components. The rate of centrifugation is determined by the acceleration or speed applied to the homogenate and is usually measured in revolutions per minute (RPM) or g. Depending on the density of the organelles will determine their isolation at a given speed. The higher density organelles and the bigger organelles separate at a lower speed centrifugation. (K. Wilson 2005). The separation forms a pellet which is the precipitate proportion of the sample and the component of interest and a supernatant which is the liquid component. The supernatant readily de canted from the sample without removing the precipitate. Diferemces in centrifugation occur due to the techniques used, differential centrifugation is based upon the sedimentation rate of particles and thus the sedimentation rate separates them based on size and density. After initial sedimentation the largest particles separate first into pellet and supernatant (K. Wilson 2005). Density gradient centrifugation separates organelles using a media. Various media can be applied and depending on the particles will be best for certain types and may not work well for others. (K. Wilson 2005). The 4 fractions we will obtain are nuclei, mitochondrial, supernatant and homogenate. Various tests can be carried out to distinguish between fractions and to determine their actual purity, testing for specific enzymes can code for the activity occurring in the cell fractions therefore indicating the most abundant component. Some tests include: Testing for DNA in both nuclei and mitochondrial fractions. This is because DNA is contained within the nucleus but also within the mitochondria. This is because relating to the endosymbiotic theory mitochondria was a separate aerobically respiring bacterial cell which was later engulfed by an early eukaryotic cell to merge into one aerobically respiring cell. Mitochondria is maternally inherited in the case of the majority of multicellular organisms, this is due to the higher number of mtDNA molecules in the ooecyte and much fewer in a sperm cell which are mostly degraded before fertilization takes place. Test for histones which indicate nuclei fraction as well as testing for various enzymes such as ATPase found in cytoplasmic (supernatant) and mitochondrial fractions and phosphotase kinase indicating microsomes and golgi apparatus are present. Some enzymes are exclusive to the citric acid cycle which occurs in the mitochondria, therefore testing for these enzymes indicates the presence of mitochondria in a fraction. The enzyme marker to test for mitochondria which we use is succinate dehydrogenase which is exclusive to the inner mitochondrial membrane. Succinate dehydrogenase is formed only during the citric acid cycle so is only given as an indication of mitochondria. However, since during the homogenisation process the mitochondria could potentially burst spilling their contents into the cytoplasm (supernatant fraction), this does not therefore give an accurate indication of mitochondria present in a fraction. Succinate dehydrogenase breaks down succinate into fumurate, therefore t he measurement of formazan indicates presence of succinate dehydrogenase. Measuring Succinate Dehydrogenase Activity (Red Formazan assay) This occurs in 2 reactions: 1: succinate + FAD à ¨ fumarate + FADH2 SDH breaks down succinate into fumarate. This is an oxidation reaction since the succinate loses 2 electrons, in addition a reduction of the enzyme flavin adenine dinucleotide occurs (FAD gains 2 electrons) (FAD + 2 electrons à ¨ FADH2) Figure 4: Shows the redox reaction which occurs with succinate and FAD. Image taken from natuurlijkerwijs.com SDH activity is measured by the formation of formazan a deep red compound formed from the reduction on a tetrazolium salt. The reduced FADH2 reduces tetrazolium salt (INT). 2: FADH2 + INT à ¨ FAD + formazan Centrifugation and calculating the relative centrifugal field. (K. Wilson 2005) G = W2r = 4 II2 r (rPM)2 = 1.1110-5r (rPM)2 3600 G= Relative centrifugal force (RFC) r = Radical distance from axis of rotation w = Angular velocity rPM = Revolutions per minute. T = 9 É ² (In Rt/Rb) 2 W2rp2 (Pp -P) É ² = Viscosity of medium rp = Radius of particle Pp = Density of particle P = Density of medium Rt = Radius to top of centrifuge tube Rb = Radius to bottom of centrifuge tube. There are many differences in types of centrifuges available and results depend on the speed of the centrifugation and whether a vacuum is present and the type of rotor used. (K. Wilson 2005) Analysis of marker enzymes in subfractions determines the recovery of subcellular organelles, with comparison to previous tests, quantative data can be used to assess contamination of fractions. Showing whether the subfractionation method has been successful or not. These tests also hold health benefits and implications e.g. microsome C causes cell death and can be found in mitochondrial fractions, however in cancer patients no microsome c is present, indicating no cell death will occur a common feature of cancer cells. Enzyme measurement in subcellular fractions however does hold some implications such as the solubility of the environment which may cause differences in enzyme function. Another implication is latency of enzymes, this refers to whether proteins are bound to the enzyme which in turn activates them once bound signalling enzyme function. There may also be low recovery of enzymes in the fractions due to poor recovery of the organelles which they come from, in particular if the enzyme is confined only to a specific region. Over the 3 week period centrifugation will separate the fractions according to size/density and separating the sample into the pellet and supernatant fractions. The speed of the centrifuge determines whether the pellets will separate. A lower speed is needed to separate the nuclei fraction due to the higher density, whereas the higher speed is needed to separate the supernatant due to the smaller density remaining organelles. (K. Wilson 2005). The protein content is also measure for each fraction using the biuret assay, absorbance values are given which determine the protein content of each fraction. Finally succinate dehydrogenase is measured. This causes a redox reaction and causes e- ions are released, using formazan as an indicator this changes the colour of solution red, showing a redox reaction has taken place. From this research I can predict that the mitochondrial fraction is expected to have the highest results in specific activity due to fewer proteins present in that fraction. Results: Calculations: Formazans molar extinction coefficient (E490nm) = 20,100 M-1 cm-1 The specific activity and relative activity of the fractions can be determined by measuring the concentration using Beer- Lamberts Law: (www.chemguide.co.uk) A = ÃŽ µ x l x C A = Absorbance (no units) ÃŽ µ = Epsilon. The adsorbtion coefficient M -1 cm -1 l= Cuvettes light path length, this is the length of solution a light passes through (always 1 cm) C= Concentration of substance in M (moles in 1 litre) Rearrange to give concentration: C = A / ÃŽ µ x l Units: M-1 x cm-1 = 1 / M x cm C = A / ÃŽ µ x l Gives units: ( 1/ (1/M x cm) x cm). This can be simplified to give 1/ (1/M) And further simplified to give units: M (moles per litre or dcm -3) Know values: ÃŽ µ = the formazan adsorption coefficient is 20,100 M -1 cm -1 A = refers to the absorbance at 490nm values for each fraction are found in the mean-control table section. Using the equation: C = A / ÃŽ µ x l We can work out the concentration of formazan formed in the reaction. The concentration value is for 1 litre, therefore we must calculate the actual concentration from the actual assay volume used. Concentration = amount/volume rearranged to give A = C x V The final assay volume from week 3 is 6 ml* due to the addition of ethyl acetate. * Note by mistake 6ml of ethyl acetate was added instead of 4 ml giving a different final volume to the other groups. Converting 6ml into its litre value and x by the concentration gives the accurate mole product of formazan produced. Reaction time needs to be included to give the accurate units. Activity units can be determined using the following equation. Activity = Moles of formazan/reaction time (12 minutes) This gives the activity in M -1 Calculating total activity and specific activity of the fractions. Table 1: Total volumes from each cellular fraction. Fraction Total Volume (ml) Homogenate 12 Nuclei Fraction 12 Mitochondrial Fraction 12 Supernatant Fraction 26 To do this we need to take into the account: The total volume The total protein of the fraction. Dilution factor The total volume values for each fraction can be found in table 1. The sample of each fraction used was 0.2ml, therefore the amount of moles of formazan calculated is in 0.2ml. (0.2 / total volume) x moles of formazan in 0.2ml X by the dilution factor of each fraction to give the total activity for each fraction, the values are given in table 4. To determine the specific activity we must consider the total protein of the fraction. Values are given in table 3. Specific activity = Total activity of fraction/ total protein of fraction Table 2: Bovine serum albumin (BSA) solution concentrations Volume (ml) BSA (10mg/ml BSA 0 0.2 0.4 0.6 0.8 1.2 1.6 0.1m NaOH 2.0 (blank) 1.8. 1.6 1.4 1.2 0.8 0.4 Table 3: Values for BSA standard curve. See Graph 1 for the results from the corresponding fraction absorbance. Protein Amount (mg) 0 (blank) 2 4 6 8 12 16 Absorbance at 550nm 0 0.105 0.184 0.275 0.354 0.511 0.531 Table 4: Protein amount in homogenate and subcellular fractions. Homogenate 0.05ml Nuclei 0.2ml Mitochondria 0.2ml Supernatant 0.2ml Average Absorbance (550nm) 0.169 0.054 0.174 0.199 Protein amount in samples aliquot (mg) 3.6 1.18 3.8 4.15 Protein concentration in fraction (mg/ml) 72 5.9 19 20.75 Protein amount in fractions total volume (mg) 864 70.8 228 539.5 Graph 2: Shows the difference in protein amount amongst cellular fractions. Table 5: Actual concentration of fraction after dilution. Dilution Factor Actual concentration (mg/ml) Homogenate 20 3.6 Nuclei 3 2 Mitochondrial 20 0.95 Supernatant 1 20.75 Table 6: Formazan content absorbance at 490nm. Fraction Control Test 1 Test 2 Mean-Control Homogenate 0.132 0.58 0.52 0.42 Nuclei 0.21 0.352 0.326 0.13 Mitochondrial 0.057 0.391 0.265 0.27 Supernatant 0.132 0.52 0.33 0.29 Results for Homogenate: From table 5, we have the absorbance of homogenate as 0.42 this divided by the adsorption co-efficient gives: 0.42/20,100 = 2.1 x 10 -5 M The units for concentration are left as moles per litre (M). To get this into moles in the actual volume used (6ml not 1 litre) 2.1 x 10 -5 M x 0.006 lite = 1.3 x 10-7 M Include the reaction time of 12 minutes to give moles per minute. 1.3 x 10-7 M /12mins = 1.010-8 M -1 To determine total activity and specific activity. The total volume from table 1: for the homogenate is 12ml, however the sample used was only 0.2ml we therefore divide actual volume / used volume x concentration of H x dilution factor (20 in the case of the homogenate from table 5 values) Total activity = (12/0.2) x1.0x10-8 M -1 x 20 = 1.2 x 10 -5 M -1 specific activity = 1.2 x 10 -5 M -1/ total amount protein in homogenate from table 4 1.2 x 10 -5 M -1/864= 1.3 x 10-8 M min-1 Results for nuclei fraction: 0.13/20,100 M-1 cm-1 = 6.5 x 10-6 In 0.006 litre : 6.5 x 10-6 x 0.006 = 3.9 x 10-8 M 3.9 x 10-8 M / 12 = 3.2 x 10-9 M min-1 Total activity = 3.2 x 10-9 M min-1 x (12/0.2) x 3 = 5.8 x 10 7M min-1 Specific activity = 5.8 x 10 7/ 70.8 = 8.2 x 10 -9 M min-1 Results for mitochondria: C = 0.27/20,100 m-1 cm -1 = 1.3 x 10-5à ¢Ã¢â€š ¬Ã¢â‚¬ËœM 1.3 x 10-5à ¢Ã¢â€š ¬Ã¢â‚¬ËœM x 0.006 = 7.8 x 10-8 M 7.8 x 10-8 M / 12 = 6.5 x 10-9 M min-1 Total activity = 6.5 x 10-9 M min-1 x (12/0.2) x 20 = 7.8 x 10-6 M min-1 Specific activity = 7.8 x 10-6 M min-1/228 = 3.4 x 10-8 M min -1 Results for supernatant: C = 0.29/20,100 m-1 cm -1 = 1.4 x 10-5à ¢Ã¢â€š ¬Ã¢â‚¬ËœM 1.4 x 10-5à ¢Ã¢â€š ¬Ã¢â‚¬ËœM x 0.006 = 8.7 x 10-8 M = 8.7 x 10-8 M / 12 = = 7.3 x 10-9 M min-1 Total activity = 7.3 x 10-9 M min-1 x (26/0.2) = 9.4 x 10-7 M min-1 Specific activity = 9.4 x 10-7 M min-1/539.5 = 1.7 x 10-9 M min -1 Percentage recovery of Succinate Dehydrogenase for the fractions This is done by dividing the amount of Succinate dehydrogenase in the individual fractions by the original homogenate and then multiplied by 100 to give a percentage. Table 7: Shows the total activity for each of the fractions. Fraction Total Activity Homogenate 1.2 x 10 -5 M -1 Nuclei 5.8 x 10 7M min-1 Mitochondria 7.8 x 10-6 M min-1 Supernatant 9.4 x 10-7 M min-1 Nuclei fraction: (5.8X10-7/1.210-5 ) x 100 = 4.8% Mitochondria fraction (7.810-6/1.210-5 ) x 100 = 65% Supernatant fraction (9.410-7/1.210-5) x 100 =7.8% Relative Specific Activity of Succinate Dehydrogenase This is found by dividing the specific activity of the fractions (found above) by the specific activity of the homogenate (found above). Table 8 shows the specific activity for each of the fractions: Fraction Specific Activity Homogenate 1.4 x 10-8 M min-1 Nuclei 8.2 x 10 -9 M min-1 Mitochondria 3.4 x 10-8 M min -1 Supernatant 1.7 x 10-9 M min -1 Nuclei fraction 8.2 x 10 -9 M min-1 /1.4 x 10-8 M min-1 = 0.586 Mitochondrial fraction 3.4 x 10 -8 M min-1 /1.4 x 10-8 M min-1 = 2.429 Supernatant fraction 1.7 x 10 -9 M min-1 /1.4 x 10-8 M min-1 = 0.121 Discussion: Note: There was very little protein found in the nuclei fractions total volume, this is abnormally low since we would expect this to be higher. From the results we can determine that the this supports our prediction that The mitochondrial fraction is expected to have the highest results in specific activity due to fewer proteins present in that fraction. Organelles have been isolated from each other as seen with the differing proportions of protein found in each fraction as well as the differing values for specific and total activity calculated. However the homogenate is expected to have the highest total activity due to the higher amount of protein since all fractions are present. However since protein was found in the cytoplasm or supernatant fraction, this indicates that there was an error in the separation of the fractions as SDH is present where it usually isnt found. Succinate dehydrogenase works by transferring 2 electrons from succinate which transfers it to fumerate, which blocks the rest of the reaction when it binds to FAD, from the measurement of formazan gives the value of activity. Results show that the relative specific activity is highest in the mitochondrial fraction, as well as the percentage recovery of the fractions. Therefore demonstrating that the fractions were purified and that the homogenisation and centrifugation has been relatively successful in separating fractions. However there were some inaccuracies from the results, this includes the very low protein amount found with in nuclei fraction, this was however predicted to contain a higher amount of protein due to the nature of the organelle and the enzymes contained within it. Another inaccuracy in this experiment is that SDH was found within the supernatant. This is primarily a marker for mitochondria so would not usually be found within the cytoplasm, however due to mitochondria bursting and releasing its contents into the cytoplasm during the homogenisation stage and centrifugation the enzyme succinate dehydrogenase was present. Since the test was carried out under the same conditions in a neutral pH buffer we can conclude that this was a fair test, however is it often found that the more molecules present in a The separating of the homogenate could be improved by using another method of homogenisation, in this experiment we used a Potter Lethem homogeniser which is a glass and plastic hand homogeniser. This perhaps isnt the most accurate at pressurising cells with the force needed to accurately release cell content. Alternative homogenisers include ultrasonic and rotor based homogenisers which may provide more accurate. (www.proscientific.com) A different centrifugation method used. During this experiment differential centrifugation was used, however density gradients may provide more accurate at purifying a sample (www.coleparmer.co.uk). This method works by placing various layers after layer of gradient media such as sucrose in a tube with the heaviest layer at the bottom and the lightest at the top. The cell fraction to be separated is placed on top of the layer and centrifuged. Density gradient separation can be classified into two categories. Rate-tonal (size) separation. Isopycnic (density) in which organelles separate until their density matches the surroundings of the media in which they are. A very good medium for separating organelles is an iodinised media. (www.coleparmer.co.uk). Accuracy of the absorbance and accuracy of obtaining the protein amount. Results are slightly low indicating inaccuracy in both collecting the samples and also measuring the absorbance, this could be due to error in homogenisation and centrifugation techniques but could also be due to error in the reading of absorbance using the Spectrophotometric assay since U.V wavelength has different absorbance levels if either oxidised or reduced enzymes absorb light therefore giving innacurate indication to enzyme present (www.millipore.com) . This may affect the absorbance levels in the fractions if specific enzymes are affected thus giving an altered absorbance level and therefore undermined protein amount. Another method to measure enzyme assay could be to use a caliometric method which measures heat radiance given off instead of the absorbance levels. Some of the organelles which remain in the supernatant fraction are the smaller and less dense proportions of the cell such as ribosomes and lysosomes. Further centrifugation at a higher speed can be used to separate these smaller less dense organelles into pellets. This can also be used to further purify bacteria. In conclusion we see that as predicted, the specific activity is highest in the mitochondrial fraction and the total activity is highest in the homogenate. The % recovery of each fraction and the relative specific activity for each fraction calculated shows a higher proportion in the mitochondrial fraction also. Overall the results indicate accurate laboratory skills and results conclude what was intended, however some slight changes to laboratory equipment would mean that some of the results such as SDH found in the supernatant may not come about in a future test.

What Is the Future of Social Media

What is the future of social media? In research for this discussion, I came up with a few insights on what I foresee coming up next in the world of social media. †¢ The physical and digital worlds will be more highly connected than ever before – already today we are able to run in the park and track our progress online while sharing it with our friends or track our weight loss, or even our ovulation (well, some of us, that is) with iPhone apps that connect to our Facebook and twitter profiles and enable us to keep track of our progress as well as share the data with our friends. Facebook, Twitter and other major social networks will become increasingly what Fred Wilson coins â€Å"Social Dashboards†. In essence, Facebook and Twitter are social channels on which other companies can grow and develop their own technologies and businesses. Both Facebook and Twitter have created economies far larger than many nations. †¢ Until now, brands have been very concerned w ith bringing as many people as possible to their pages. Consumer brands can now finally reap the fruits and build social commerce stores where Facebook users (all 700 Million of them) can purchase products on their favorite social network without needing to go to any destination site. Facebook will become one of the major channels of future online shopping. †¢ Companies like Google, Facebook and Amazon are currently collecting information about each and every one of us: Our likes and dislikes, our interests and disdains. Soon in an age of Web 3. 0, an age of Semantic Web, we will no longer need to search for information on the Web as information will find us based on all this data which companies are collecting. The right information will be served to the right people at the right time, saving us all a lot of time, effort and energy. †¢ Mobile technology will become more dominant and NFC technology will be developed further enabling it to offer us special promotions, coupons and ips based on our geographical location and the interest graph. †¢ Human Relationships will no longer be as physically dependent and we will befriend and hang out with people from all over the world and all walks of life, all ethnicities and all beliefs, creating a worldwide melting pot. †¢ We will no longer be passive media consumers. Media will interact with us in dynamic ways on all platforms. Just like gamers playing WOW today, we will all become a part of a virtual world unknown to us yet where we will all be avatars in the game of life. †¢ As the Web is overloaded with more information, the content that we are exposed to will become more and more customized to our needs as companies will large sums of money to companies like Facebook and Google, making sure that the information we are exposed to is highly targeted to our interests. Rather than experiencing information overload, we will actually experience the opposite effect. †¢ Companies will understand better how to measure the ROI of social media and realize that social media is not about the number of people brands have in their communities but rather the amount of engagement that they see on their page and the overall online sentiment they faced this month as opposed to the last. †¢ Services will become increasingly crowd sourced. Whether it be the way that we get from point A to point B (Waze), the way that we find answers to our questions (Quora), the manner in which we test our Websites (uTest), the way that we get things done (Fiverr) or the way that we share information (Wikipedia). Source: www. http://thenextweb. com http://blog. hubspot. com/blog/tabid/6307/bid/7850/What-Is-the-Future-of-Social- Media-Marketing-Marketing-Cast. aspx http://www. slideshare. net/derickson/the-future-of-social-media-marketing http://irclay. hubpages. com/hub/The-Future-of-Social-Media-for-Hotel-Marketing-Travel-and-Tourism

Succubus on Top CHAPTER 10

Jerome didn't seem very happy to hear from me the next morning. â€Å"Do you have any idea what time it is, Georgie?† he growled into the phone. â€Å"Why are you whining? You don't even need to sleep.† â€Å"Make this fast.† I told him about my experience at the concert and my inability to ID the mystery immortal. â€Å"He wasn't one of us. Er, I mean, you know†¦not part of our†¦pantheon,† I finished lamely. â€Å"‘Pantheon?' I've never heard it put quite like that – outside of an introductory mythology class, of course.† â€Å"So?† â€Å"So what?† â€Å"So isn't that weird? I've met hundreds of different immortals and never felt one like this. He didn't feel†¦normal. I mean, he did feel like an immortal, but it was just weird.† â€Å"Well, hard as it is to believe, there are still a lot of things out there you haven't experienced – despite your vast age. â€Å" â€Å"Yeah, yeah, I know I'm an infant, all right? But doesn't this worry you at all?† He yawned. â€Å"Not in the least. Something angelic ordemonic would, but some random demigod or satyr? Hardly. They're not part of the game. Well, they're all part of the Game. What I mean is, they're not part of our game. They don't have to get permission to be here. As long as they don't interfere with our business, I don't really care. They do their own thing. We'll just catalog them and move on.† â€Å"Catalog? You've got a record then?† â€Å"Well, I don't, of course. That's one of Grace and Mei's things.† No surprise there. Jerome wasn't really big on†¦well, work. Grace and Mei were subordinate demonesses who did a lot of the dirty jobs he didn't want to. I hardly ever saw them. â€Å"I'll have to page them,† I murmured, mind spinning. â€Å"You know, I suppose it goes without saying that there are a hundred other more useful projects you could be channeling your energy into. Like, say, helping your incubus friend. From what I hear, he's stuck high and dry out in the suburbs. Emphasis on the high.† â€Å"Hey,† I said, defensive of Bastien's honor, â€Å"he's just taking his time. You can't rush quality work. Besides, he learned everything he knows from me. â€Å" â€Å"Somehow that doesn't reassure me.† Jerome disconnected. I hunted down Grace and Mei's number. I waited for the tone, punched in my call-back number, and hung up. A minute later, a Fourth of July worthy shower of sparks appeared in my living room and the two demonesses stood before me. For having chosen two very different bodies, the pair looked remarkably alike. Grace was slim in an all-business, non-nubile sort of way, enhanced by the designer black skirt and jacket she wore. She had pale blond hair cut bluntly at chin length, brown-black eyes, and skin that never saw the sun. The only true color on her was the fire engine red lipstick she wore. Mei dressed exactly the same, down to the red lipstick. Her hair, also chin-length, was a deep blue-black. Despite the softer lines, higher cheekbones, and delicate almond shape of her dark eyes, she radiated no more warmth or friendliness than her counterpart. The two always stuck together, and I assumed they must be friends. Sort of. I had no doubt they'd claw each other's eyes out – or Jerome's, for that matter – if an opportunity for power or promotion was on the line. â€Å"Georgina,† said Mei. â€Å"Long time no see,† said Grace. Both watched me expectantly. Aubrey watched them from the back of my couch, her hair on end and tail poofed out. â€Å"Hey guys,† I replied uneasily. â€Å"Thanks for coming over so fast. Slow day?† They both stared at me. â€Å"Um, so, okay. Jerome said you keep records of immortals who pass in and out of the city. Immortals who are outside of our†¦Ã¢â‚¬  â€Å"Game?† suggested Grace. â€Å"Pantheon?† suggested Mei. â€Å"Yeah. Sure. So†¦do you?† â€Å"Who are you looking for?† asked Mei. â€Å"What kind of immortal?† asked Grace. â€Å"That's the problem.† I told them everything I knew about him, which mostly included appearance and other encounters when I'd felt that weird sensation. Describing his signature was harder. I couldn't exactly say he felt like an incubus or an angel or a nymph or an oni. I hadn't run across his type before. The demonesses processed this information, glanced at each other, and then shook their heads. â€Å"He doesn't sound familiar,† said Grace. â€Å"But we can double-check the records,† said Mei. â€Å"Thanks,† I told them. â€Å"I'd really appreciate it.† They nodded curtly and turned as if to leave. Mei suddenly glanced back at me. â€Å"You should hang out with us sometime,† she said unexpectedly. â€Å"Cleo's in Capitol Hill has great specials on Ladies Night.† â€Å"There are so few of us girls around here,† added Grace. â€Å"We need to stick together.† They smiled and disappeared. I shivered. Going to a bar with those two sounded only marginally more appealing than stamping with Dana's CPFV friends. Speaking of which, I decided to visit Bastien later that afternoon. I hadn't heard from him in a few days. â€Å"Do you have any idea how much I don't care about your mortal friends?† he snapped when I told him about the whole bizarre situation surrounding Doug, Alec, and the mystery man. â€Å"I have real problems here. I'm dying. I'm getting nowhere with Dana. I keep seeing her, she's nice, and that's it! It's like she only wants – â€Å" â€Å"To be friends?† He stopped pacing around his kitchen and cut me an arch look. â€Å"Women are never just friends with me.† He leaned against the counter and closed his eyes. â€Å"I just can't think what else to do. If I don't act fast, one of our superiors is going to find out how bad things are.† I decided not to mention Jerome's â€Å"high and dry† comment just then. â€Å"Well, jeez, take a break and do something fun. Peter's having another poker game. Come over and play with us. I'm going to bring Seth.† â€Å"I thought you said this was going to be fun.† â€Å"Hey! Who was that a dig at? Peter or Seth?† â€Å"Pick one, Fleur .Although, admittedly, Peter does make a pretty decentsouffle. What can the author do?† â€Å"I wish you'd stop picking on Seth. You don't even know him.† Bastien shrugged. â€Å"Sorry. You just make it so easy.† â€Å"You're jealous.† â€Å"Hardly,† he snorted. â€Å"I've had my share of mortal infatuations, thank you. So have you, if memory serves. And you've also had a number of immortal boyfriends you seemed to have liked reasonably well. None of them ever gave you as much grief as this guy.† â€Å"Seth's different. I can't explain it. Being with him just feels so†¦right. I feel like I've known him forever. â€Å" † Fleur , I've known you forever. You've only known this guy for a couple months.† We had gotten involved pretty quickly, and it did bug me sometimes, but I truly believed in the strength and depth of my feelings for Seth. They were neither superficial nor transient – I hoped. He had once told me there was no one else in the world for him but me. When I'd pointed out that was a bold statement in light of how long we'd known each other, he'd simply said, â€Å"Sometimes you just know.† It was remarkably similar to what my husband, Kyriakos, had told me when we'd first met, back in my long-ago, dust-covered days as a mortal. I'd been fifteen at the time, and my father had sent me down to the docks of our town with a message for Kyriakos, father. Sending me alone was a bit unorthodox, but my father hadn't thought much about it since he was only a short distance away at the market. Nonetheless, I found it a frightening walk. Sweaty, dirty men worked ceaselessly, unloading and loading in the hot sun while the turquoise Mediterranean shimmered beyond them. I got directions from a short, bald man who leered up at me when he finished. â€Å"You're a tall girl,† he observed. â€Å"Bet that might bother some men, but not me. You're just the right height as far as I'm concerned.† He laughed, and some of his companions laughed too. The man's face came up right to the height of my chest. I hurried past them with lowered eyes, honing in on the indicated ship. Relief flooded me when I found Kyriakos checking lines and talking to some of the workers. I'd never spoken to him, but I knew who his father was and knew he was trustworthy. He looked up at my approach and smiled. â€Å"You're Marthanes, daughter, right? Letha?† I nodded. â€Å"I'm supposed to tell your father that the shipment can be ready this evening if he wants it early.† â€Å"I'll let him know. He's not here.† â€Å"All right.† We stood there awkwardly for a moment. I could sense him studying me out of the corner of his eye while pretending to study the workers. He looked like he wanted to say something, but when nothing came, I made motions to go. â€Å"Well, thanks. I should get back.† â€Å"Wait, Letha.† He reached out a hand to stop me from turning, then shyly pulled back before actually touching me. â€Å"You†¦didn't walk here by yourself, did you?† â€Å"My father said it wasn't that far. And that I wasn't in much danger of attracting interest. â€Å" Kyriakos made a harsh sound in his throat. â€Å"Your father's a fool. Let me walk you back.† He hesitated. â€Å"But don't tell your father I called him a fool.† He exchanged a few curt words with one of his men and then set out back to town with me. He was older than me, his face tanned from sun and sea. His hair was black and messy, about chin-length, and he stood almost – but not quite – as tall as I did. â€Å"I saw you at that wedding a few days ago,† he said after a long stretch of silence. â€Å"You were dancing with some other girls. You know†¦you're really good.† The compliment surprised me. â€Å"I think the wine helped.† â€Å"No. The wine helped the other girls – or hindered, maybe. I'm not sure.† He glanced over at me, and I nearly stumbled at the intensity in his dark eyes. â€Å"But you†¦dancing lives inside of you. The music spoke to you, and you understood it.† â€Å"You were playing a flute,† I recalled, trying not to blush at the regard in his voice. â€Å"Yes.† He sounded happy that I remembered. Silence fell again. We were almost to the market; the sounds of people and commerce drifted down to us. Kyriakos clearly wanted us to keep talking. â€Å"So†¦I heard your sister got married last spring.† â€Å"Yes.† â€Å"What about you?† I eyed him. â€Å"I didn't get married last spring.† A smile turned up the edges of his lips. â€Å"What about next spring?† â€Å"Are you offering?† â€Å"Just checking. I heard my father say†¦Ã¢â‚¬  I stopped walking near the edge of the market, so I could look him in the eye again. People and animals moved around us, and across a walkway I could see my father talking to a fruit vendor. â€Å"Look,† I said brusquely, â€Å"I heard my father say it too – how they're thinking about making a marriage between our families. It'd create good trade deals. But if you're trolling for that, you should talk to your father about one of my sisters, not me.† â€Å"What? Don't you want to get married?† His smile faltered. â€Å"Or is someone else lined up for you?† I stared incredulously. â€Å"No, of course not. You just don't want to marry me, that's all.† â€Å"I don't?† â€Å"No. You want one of my sisters.† â€Å"I do?† â€Å"Yes. They're shorter, prettier, nicer – and softer spoken.† â€Å"Can they dance?† I considered. â€Å"No. They're terrible.† His shy smile returned. â€Å"Then I want you.† â€Å"You're crazy. You don't know what you're talking about. You don't know anything about me. † Of course, in those days, most people knew little about their betrothed. What I found remarkable was his conviction that we were compatible. â€Å"It doesn't matter. I can just tell that you're the one. Can't you feel it?† I met his eyes and felt a shiver go through me, like I'd stumbled into something bigger and more powerful than both of us. For just a moment, I allowed myself to consider that this man from a highly respected family might legitimately be interested in me. It was a heady feeling, and not just from the honor involved. It was from the way he looked at me and spoke to me, like I was both worthy and an equal. Something built between us, drawing me to him, and it confused me. â€Å"You don't know anything about me,† I repeated quietly, my mouth feeling dry. His tentative smile grew bolder. â€Å"I know plenty. I know that you dance and that you're smart – too smart, according to my father. And I know that your family is banned from Lais, bakery because you called her daughter a – â€Å" â€Å"That wasn't my fault,† I interjected quickly. Across the way, my father caught sight of us. I held up a hand of greeting, and he impatiently gestured me over. â€Å"My father wants me.† Kyriakos cast an uncertain look over there and hastily turned back. If I was known for a sharp tongue, my father was reputed to be worse, and however love struck and brazen, Kyriakos apparently wasn't quite up to facing him yet. â€Å"I'll have my father talk to yours.† The earlier joking was gone; Kyriakos was all seriousness now. But there was more than just that. His eyes were looking at me in a way I'd never been looked at before. I felt hot, then cold, and then hot again. A tingle played along my flesh. I couldn't take my eyes away from his. â€Å"This isn't about trade deals,† I whispered. â€Å"No. This is about you and me. You're the one.† I stared, uncharacteristically short on words. My shock now came more from that crazy feeling swirling inside of me, not from the preposterous nature of his proposal – one he shouldn't have even brought up without the involvement of our families. Later I'd learn what a leap this whole conversation had been for him. He was not given to long speeches or bold behavior. He said little, as a general rule, more content to express himself through his eyes and his music, and later†¦after we were married, his lovemaking. â€Å"Look,† he said, suddenly growing nervous as he misinterpreted my silence and expression, â€Å"I've saved. We can get a nice house. You won't have to live with so many people anymore. I'll be gone a lot, but you can probably run things and make deals better than me anyway. Not being able to buy bread will be problematic, but we might be able to afford a servant, or you can learn to – â€Å" â€Å"Shut up,† I said. He stared. â€Å"What?† â€Å"Just shut up. You're wasting time. Go tell your father to talk to mine. And,† I added wryly, â€Å"I know how to make bread.† He caught his breath. â€Å"You're sure?† â€Å"About the bread? Yes, I'm sure.† A slow smile bloomed across his face, spreading up into his eyes, making them smolder. I felt my pulse quicken and smiled back. Nothing else needed to be said. My father yelled again, and I ran off to join him. Pondering this memory and what was now happening with Seth, I stared dazedly out the front window and caught sight of Jody checking the mail. â€Å"Hey,† I told Bastien. â€Å"I want to go say hi to her.† I ran outside and waved, making her break out into one of her big, beautiful smiles. To my surprise, she even hugged me. â€Å"Ooh! I'm so glad to see you. How have you been?† We exchanged a few pleasantries, and then she grabbed my arm excitedly. â€Å"Are you busy today? You want to go to the mall?† To my surprise, that actually sounded like fun. More fun than listening to Bastien bitch and moan. â€Å"Sure.† â€Å"Great. I'll go tell Dana.†

Invisible Man - 11097 Words

According to Goethe, We do not have to visit a madhouse to find disordered minds; our planet is the mental institution of the universe. Despite the hyperbolic nature of Goethe s statement, it holds some truth. Because of this element of truth, society looks to psychoanalysis as an important tool for understanding human nature. Furthermore, psychoanalytic criticism of authors, characters, and readers has a place in literary criticism that is as important as the place of psychoanalysis in society. This is because of the mimetic nature of much of modern literature. In fact, the psychoanalyst Jacques Lacan wrote, If psycho-analysis is to be constituted as the science of the unconscious, one must set out from the notion that the unconscious†¦show more content†¦Despite the limitations of his theories, their usefulness still exists, especially as a background for Jung and Lacan. The Freudian text at work in this analysis will be Civilization and Its Discontents. In this text, Freu d s theories about aggression and the death drive are related to societal tensions that isolate the individual. Carl Gustav Jung was somewhat of a son to Freud, but he quickly outgrew his father s theories, and, in an ironically ÂÅ'dipal conflict, overthrew Freud as the leading psychotherapist.(8) The buzzword of Jungian theory is archetype, so the text of his being used in this study is Four Archetypes. In The Critical Tradition, the editor gives the description of archetypes as structures deep in the human unconscious.(9) The editor continues and says, In Jungian analysis, the patient recapitulates his life and looks for the ways in which symbols of the above-mentioned archetypes have been embodied within its texture.(10) From Four Archetypes, the section on rebirth will be the most useful to this study. Jung s essay Rebirth includes descriptions of five different forms of rebirth along with their psychological implications. Jacques Lacan, a more recent theorist than Freud or Jun g, based his works on a revision of Freudian ideas. Lacan is the father of the philosophy of psychoanalysis. That is, he believed that psychoanalysis was a valid field of thought independent of its use as a medicinal therapy.(11) InShow MoreRelatedInvisible Man1346 Words   |  6 PagesJanelle Clovie Dr. Blanchard AP Literature 3 November 2017 Familial Connections in Invisible Man Family. It is a very fluid yet rigid idea. It has a wealth of definitions, all of which range in degree and magnitude, and vary from person to person; yet the concept of how a family should work and operate is very concrete in most American minds. Family is a bond that is crafted every second of everyday until it is powerful, and this can shape beliefs, outlooks, and confidence. A study found that childrenRead More Invisible Man Essay: Values of the Invisible Man1267 Words   |  6 PagesValues of the Invisible Man      Ã‚  Ã‚   Ralph Ellisons Invisible Man is the story of an educated black man who has been oppressed and controlled by white men throughout his life. As the narrator, he is nameless throughout the novel as he journeys from the South, where he studies at an all-black college, to Harlem where he joins a Communist-like party known as the Brotherhood. Throughout the novel, the narrator is on a search for his true identity. Several letters are given to him by outsiders thatRead More Invisible Man Essay: Self-Identity in Invisible Man1040 Words   |  5 PagesSelf-Identity in Invisible Man      Ã‚  Ã‚   In the novel, Invisible Man, the main character carries around a briefcase throughout the entire story. All of the possessions that he carries in that briefcase are mementos from learning experiences. Throughout the novel, the Invisible Man is searching for his identity and later discovers that his identity is in those items. As the narrator is leaving Marys house for the Brotherhood, he sees a Negro-doll bank in his room. He is angry that the dollRead MoreImprovisation Of The Invisible Man1392 Words   |  6 Pagesand Composition III February 15, 2017 Improvisational Music In Invisible Man â€Å"My only sin is in my skin, What did I do to be so black and blue?† The protagonist, the invisible man, is stoned from marijuana as he listened to Armstrong s rendition of What Did I Do to Be So Black and Blue and determined that invisibility gives one a slightly different sense of time, you re never quite on the beat. (Prologue.)† The invisible man respected Armstrong for making something beautiful out of invisibilityRead MoreHamlet Invisible Man1412 Words   |  6 Pagesthe need to search for . 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The novel begins with a naà ¯ve young, black man in the South caught under the evil boot of racism. As the novelRead MoreThe Narrator As An Invisible Man1305 Words   |  6 Pageshimself to the reader as an invisible man. The Narrator makes it clear that he is not actually invisible but is considered as such because people refuse to see him. The Narrator is speaking from an underground space illuminated by a ridiculous number of light bulbs underneath a whites-only building. He goes on to tell the reader that he was not always in this predicament and begins to tell the tale of his younger days which led him to his current situation. Invisible Man pleads that the reader bearRead More Invisible Man Essay: Invisible Man and the Pre-Made Identity1559 Words   |  7 PagesInvisible Man and the Pre-Made Identity    Society forms definitions, or stereotypes, of people according to the color of their skin, their economic status, or where they live. Stereotypes define how society believes these people should act and how they should be treated. These stereotypes are, in effect, a pre-made identity. There are three options an individual must face when presented with this pre-made identity. The individual can accept this identity as his/her own. This would maximizeRead MoreThe Brotherhoods in the Invisible Man2033 Words   |  9 PagesThe Brotherhood in the Invisible Man Brotherhoods are associations, usually of men, that unite for common purposes. The members in the brotherhood typically respect one another, defend one another, and cooperate to obtain specific goals. The American Federation of Labor (AFL) was one of the first federations of labor unions in the United States, whose goal is to create better employment opportunities for workers. Kappa Sigma and Sigma Chi are two of the largest university fraternities in the countryRead More The Invisible Man Essay964 Words   |  4 Pages The Invisible Man, by H.G. Wells, is composed of many small themes that combined to form two major themes in the novel. Some of the minor themes are acting before thinking and denial of unexplainable events. It is based on the two major themes of science experiments gone wrong and the ignorance of society. nbsp;nbsp;nbsp;nbsp;nbsp;The most important theme in the novel was the experiment that Griffin, the invisible man, was working and it was not going exactly as planned. The way that the experiment