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luciferase essay MicroRNAs (miRNAs) are small non-coding RNAs predicted to post-transcriptionally regulate the doctoral in computer science, majority of the transcriptome . These 20–24 nucleotide-long RNAs have been involved in numerous physiological processes such as development and differentiation , and have been linked to many human disorders including cancer  and cornell college essay admission, neurodegenerative disease . MiRNAs are recognized mainly as negative post-transcriptional regulators of gene expression. They exert their function by association to miRNA recognition elements (MREs), predominantly within the 3' untranslated region (UTR) of mRNAs. In most cases, miRNA binding to mRNA results in science, the repression of the transcript by one of two mechanisms: mRNA cleavage or translational repression . To understand the role of miRNAs in different physiological processes, it is study of the development of an with, important to utilize experimental approaches that allow for the detection of fine miRNA-induced alterations in mRNA expression. Currently, the functional relevance of MREs is thesis in computer science, extensively assessed using reporter gene assays in cell-based systems.
In the miRNA-adapted version of good score this assay, reporter activity is an indicator of miRNA capacity in doctoral thesis, regulating the study of the of an infant with autism, reporter expression in vitro . Cells are co-transfected with the miRNA and a plasmid containing a Firefly or Renilla luciferase coding sequence upstream of an mRNA 3'UTR from the gene of interest. Thesis Science. If the mRNA 3'UTR is a target of the miRNA, the luminescence variation will be altered, a reflection of the case study of the autism, changes in the transcript’s stability and/or translation efficiency. Recently, we profiled miRNAs from amyotrophic lateral sclerosis (ALS) spinal cord tissues and used miRNA reporter gene assays to test the functionality of dysregulated miRNAs . We observed that the controls and the normalization methods most commonly reported in these assays do not take into consideration the possibility of science direct and/or indirect effects of the email, miRNAs on the luciferase mRNA. Here, we present a comprehensive miRNA reporter gene assay protocol that includes a rigorous normalization of the data. We show that incomplete normalization can lead to erroneous conclusions, not only to the magnitude of the thesis in computer science, miRNA expression regulation, but more importantly to the type of miRNA regulation (up- or down-regulation). Interestingly, these results suggest that miRNA up-regulatory effects are not uncommon and college admission, could reveal previously undetected roles for miRNAs in regulating the transcriptome. Currently, the luciferase reporter gene assay is extensively used to evaluate the functional relevance of miRNAs. Doctoral. We have observed that studies in the miRNA field lack rigorous normalization of the reporter activity data, generally only showing a transfection control (such as Renilla luciferase) and how essays, a control without miRNA or a non- targeting miRNA negative control. These types of doctoral analyses do not consider the possibility of indirect effects of miRNAs on the luciferase transcript and/or the existence of cornell college essay admission functional MREs in the luciferase protein coding region [7,8,9].
Therefore, it is important to doctoral thesis consider that, in addition to their putative effects on their mRNAs targets, miRNAs and other cellular factors could potentially regulate luciferase mRNA, critically affecting its activity. It is important to note that, while miRNAs and other factors could affect both Firefly and chrysalids, Renilla luciferase mRNA levels, it is the regulation of the in computer, luciferase linked to the mRNA 3'UTR ( Firefly in our study) that is relevant for cite essay, the normalization of the reporter gene assay data. Any miRNA or cellular effect over thesis science, the control luciferase ( Renilla in our study) will be cancelled due to the normalization. We performed reporter assays by transfecting HEK293T cells with a plasmid containing Firefly and Renilla luciferase coding sequences (pmirGLO) or the same vector harboring the low molecular weight neurofilament ( NEFL ) mRNA 3'UTR linked to the 3'-end of the Firefly luciferase (pmirGLO- NEFL 3'UTR). Essay. Renilla luciferase was used as transfection control. In these experiments, the independent effect of three miRNAs, miR-507, miR-518e* and miR-let-7a (negative control) were studied. Analyses using miRanda algorithm (see experimental section) showed that miR-507 has four MREs and miR-518e* has one MRE within the in computer, NEFL mRNA 3'UTR. Additionally, miR-507 had two MREs within the Firefly luciferase coding region (Figure 1). Chrysalids Essay. Six wells of each condition were transfected to obtain the mean of luciferase activity of thesis each experiment (for details, see Experimental Section). After normalizing each sample’s Firefly luciferase activity value with its respective Renilla luciferase activity value (data not shown), we calculated the mean of the luciferase activity (Mean Luc) for each experiment ( n = 3; Table 1). In this step of the analysis, we noticed that both miR-507 and miR-518e* down-regulated the luciferase activity when pmirGLO- NEFL 3'UTR was transfected.
Interestingly, the negative control miR-let-7a also appeared to cornell college down-regulate the luciferase activity (Table 1, Mean Luc columns, rows b compared to rows a). We then analyzed the effect of each miRNA on the luciferase mRNA without the 3'UTR (pmirGLO transfection), and again found that miR-507, miR-518e* and miR-let-7a down-regulated the luciferase activity (Table 1, Mean Luc columns, rows d compared to rows c). pmirGLO- NEFL 3'UTR. pmirGLO- NEFL 3'UTR. pmirGLO- NEFL 3'UTR. Abbreviations: Exp: Experiment; Luc: Luciferase; NEFL : Low molecular weight neurofilament; UTR: Untranslated region.
In a second step of normalization, we calculated the effect of miR-507, miR-518e* and miR-let-7a on the Firefly luciferase with or without the NEFL mRNA 3'UTR (Table 1, columns A and B; Figure 2). This normalization is critical to obtain the specific effect of the miRNA over thesis in computer science, the NEFL mRNA 3'UTR. As is described in Figure 3a, the transfection of pmirGLO- NEFL 3'UTR accounts for cellular effects (e.g., RNA binding proteins, endogenous miRNAs, etc. Good Gre. ) on the expression of luciferase linked to NEFL mRNA 3'UTR. Doctoral Thesis In Computer. The co-transfection of the pmirGLO- NEFL 3'UTR and email essay, the exogenous miRNA account for cellular and thesis, exogenous miRNA effects on the expression of luciferase linked to essay NEFL mRNA 3'UTR (Figure 3b). When the normalization of column A is performed (Table 1), cancellation of the cellular effects occurs, leaving only the exogenous miRNA effects on the expression of luciferase linked to NEFL mRNA 3'UTR. Figure 3c,d describe the controls, pmirGLO and pmirGLO plus the exogenous miRNA respectively, which account for doctoral in computer science, the effects on luciferase expression without 3'UTR. When the how essays, normalization of column B is doctoral thesis science, performed (Table 1), we obtain the effect of exogenous miRNA on luciferase expression without 3'UTR. Thus, to obtain the specific effect of the exogenous miRNA on only NEFL mRNA 3'UTR, the effect of this miRNA over the luciferase coding sequence must be eliminated. This is cornell, achieved by dividing column A by B (Table 1, Luc relative variation column; Figure 2). Calculation of the relative variation in luciferase activity showed that only miR-507 down-regulated the activity of luciferase (0.82 ± 0.02, p 0.001).
Interestingly, we found that miR-518e* up-regulated the activity of the reporter (1.66 ± 0.02; p 0.001). Thesis In Computer Science. No relative changes in the activity of the reporter linked to the NEFL mRNA 3'UTR was found in comparison, cells transfected with miR-let-7a control miRNA (1.05 ± 0.03, p = 0.059). Without this normalization procedure, our data would have falsely concluded that miR-518e* and the negative control miR-let-7a could both down-regulate luciferase via the 3'UTR of NEFL . Our results clearly demonstrate that performing incomplete controls or normalization on the data of the miRNA reporter gene assay without considering the impact of cellular factors and exogenous miRNAs on luciferase without 3'UTR could generate miscalculation of the relative variation in luciferase activity, leading to doctoral thesis in computer science erroneous conclusions about miRNA functionality. The most extensive experimental approaches to determine functional relevance of predicted MREs are the electrophoretic mobility shift assay (EMSA) to chrysalids comparison detect interaction between miRNAs and their targets, and the reporter gene assay to study miRNA regulatory effects. Considering that the reporter gene assay is a unique functional tool to study miRNA-based regulation on mRNA expression, it is extremely important to doctoral science perform appropriate data analysis to avoid inaccurate results and admissions application essay, conclusions. Currently, the majority of published miRNA studies perform normalization using only a transfection control reporter (e.g., Renilla luciferase) and controls lacking exogenous miRNA or transfecting a miRNA negative control with luciferase linked to doctoral science the 3'UTR as target. Good Score. However, the effect of the exogenous miRNA and cellular factors on doctoral in computer the luciferase coding sequence alone has not been included as a control, and how essays, as described here, must be considered. Thesis. We demonstrate that normalization against the effect of the miRNA and cellular factors on the luciferase coding sequence alone is essential to obtaining the how essays, specific impact of the miRNA on the 3'UTR target. We propose this normalization method as standard for miRNA luciferase reporter assays. Therefore, errors and misinterpretations in miRNA functionality commonly observed using previous protocols can be avoided. MiRNAs predominantly, but not exclusively, exert their functions through interaction with mRNA 3'UTRs .
There is evidence that miRNAs have functional MREs in 5'UTRs [11,12] and protein coding regions of mammalian transcripts [13,14]. In fact, Argonaute HITS-CLIP and PAR-CLIP studies have shown that almost half of all Argonaute binding sites are located in doctoral science, human protein coding regions [15,16]. In addition, it is cite essay, well known that reporter gene assays reveal specific regulation of miRNAs on their targets that could be both directly mediated through interaction, and indirect for instance through the regulation of other factors involved in mRNA stability. Doctoral Thesis In Computer Science. For example, miR-375 down-regulates the expression of HuD, an RNA-binding protein that participates in mRNA stability and other regulatory processes, and thus governs the fate of many neuronal mRNAs  through an independent mechanism. Prediction analysis showed MREs for both miR-507 and miR-518e* within the NEFL mRNA 3'UTR. Score Gre. The results of the reporter gene assay support the idea that both miRNAs regulate the reporter linked to the NEFL mRNA 3'UTR. After normalizing against both the transfection control ( Renilla luciferase) and the control without the miRNA, miR-507 shows a down-regulatory effect on thesis the luciferase linked to the NEFL mRNA 3'UTR that once normalized for effects on luciferase, is only half as strong as what it was originally detected to be before normalization.
In fact, a manual search revealed two MREs for miR-507 within the Firefly coding sequence, and the reporter gene assay showed that miR-507 regulates Firefly luciferase. Hence, our results suggest that miR-507 could be directly regulating Firefly luciferase. However, it is important to consider that site accessibility and translation efficiency around MREs are two factors that might affect miRNA binding when MREs are located in coding regions . Unlike miR-507, the regulation by how essays miR-518e* changes from negative to positive after the normalization against the effect of miR-518e* over Firefly luciferase. In Computer. Considering that miR-518e* does not have binding sites within the application, Firefly luciferase coding sequence, our reporter assay results suggest an indirect down-regulatory effect of miR-518e*. Thesis. All of this evidence supports the idea that miRNAs might regulate Firefly luciferase in miRNA reporter gene assays, and as a consequence the essay, effect of each miRNA on the luciferase mRNA itself must be included in the normalization of the data.
It is important to note that while the reporter gene assay is a highly sensitive and reliable technique, using confirmatory and complementary methods to study the regulatory effects of doctoral thesis science miRNAs on their targets is greatly recommended. Cite Email Essay. Studies of miRNA effects on mRNA and protein levels and the usage of MRE mutants to determine the doctoral in computer science, specificity of these effects are all valuable approaches. Although the admissions application, actions of miRNAs on mRNA cleavage or translational repression are their more well-known roles, there is increasing evidence of miRNAs being involved in up-regulation of the mRNA expression [19,20,21]. For instance, in thesis in computer, mammalian cells the translation of ribosomal proteins and other proteins involved in protein synthesis is regulated via the 5'TOP motif, which renders transcripts sensitive to stress signals. MiR-10a can interact with the 5'UTR, immediately downstream of the regulatory 5'TOP motif, and enhances the translation of ribosomal proteins . Another example is miR-122, which naturally represses CAT-1 mRNA in hepatoma cells. Application Essay. However, in the presence of HuR protein, the repression of CAT-1 is science, removed due to the interaction of HuR with the cornell essay admission, CAT-1 mRNA 3'UTR.
Thus, HuR acts as a modifier altering the ability of miRNAs to repress gene expression . Recently, we reported a group of dysregulated miRNAs in ALS that regulate the expression of a reporter linked to the NEFL mRNA 3'UTR. We found that a significant group of miRNAs up-regulate the in computer science, expression of luciferase linked to the NEFL mRNA 3'UTR . The novel results shown in this paper and the data we published previously suggest that up-regulatory roles for miRNAs are not an uncommon phenomenon. 4.1. Plasmid Construction, Cell Culture and Transfection. The reporter plasmid was constructed by inserting human low molecular weight neurofilament ( NEFL ) mRNA 3'UTR (1–1838; GenBank NM_006158) between NheI and SalI sites downstream of the Firefly luciferase gene in the pmirGLO vector (Promega, Madison, WI, USA). HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and score, plated 24 h before transfection in 96-well plates at 9 ? 10 3 cells/well.
Cells were transfected with 3.47 fmol of pmirGLO- NEFL 3'UTR or pmirGLO and 100 nM of pre-miRNAs (Life Technologies Inc., Burlington, ON, Canada) per thesis science well using Lipofectamine 2000 reagent (Life Technologies Inc.) according to the manufacturer’s instructions. The total amount of transfected nucleic acid was maintained constant at 350 ng per well by adding an cite email, empty vector without eukaryotic elements. Six wells of each specific condition were transfected to thesis in computer science obtain the cite email, mean of luciferase activity of each experiment. All the experiments were performed in triplicate. Luciferase activity was measured 24 h after transfection using the Dual-Glo Luciferase Assay System (Promega; Madison, WI, USA) in doctoral thesis science, a Luminometer (Turner Biosystems Luminometer, Promega) according to the manufacturer’s instructions.
Normalization of the data included two sequential steps. (1) Firefly luciferase activity was normalized to cite Renilla luciferase activity to thesis in computer science account for variations in admissions application essay, the transfection efficiency among experiments. Normalized values out of the mean range of each experiment were eliminated, always maintaining at least four values from the doctoral thesis, six transfected wells; (2) The luciferase activity from each miRNA over the 3'UTR was normalized against the effects of the miRNA on how essays luciferase mRNA without the 3'UTR to doctoral finally obtain the specific effect of the miRNA on the NEFL mRNA 3'UTR (for more detailed discussion see the results section). Values of relative variation in luciferase activity over 1 were considered up-regulation and values under 1 were considered down-regulation. Score Gre. Quantitative data of the reporter gene assay are presented as mean ± SEM. Student’s t- test was used to determine significant differences between the doctoral science, effect of each miRNA on the NEFL 3'UTR or the Firefly luciferase alone. Comparison between miR-507 or miR-518e* and college, miR-let-7a negative control was performed using ANOVA, Newman-Keuls test. To determine miRNA recognition elements (MREs) within the NEFL mRNA 3'UTR, we used the miRanda algorithm . Doctoral Science. Firefly and Renilla coding regions were examined manually in of an with autism, the prediction of MREs considering the criteria of the seed sequence .
Our results using the miRNA reporter gene assay protocol presented in this paper suggest that: (1) roles of doctoral thesis miRNAs could be masked by miRNA effects on the reporter being revealed only case study development infant with through proper normalization of the data; (2) up-regulatory roles of miRNAs could be a common regulatory mechanism in cells; and (3) there could be unrevealed roles for miRNAs as regulatory elements of the thesis science, transcriptome. We are grateful to Kevin Cheung for his critical reading of the manuscript. Michael J. Strong research is funded by the ALS Society of how essays Canada (Senior Scientist Bridge Funding) and the Canadian Institutes of Health Research (CIHR). DCM and CAD performed the experiments and data analysis. DCM, CAD, KV and MJS wrote the doctoral, manuscript.
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An Introduction to Satire: A Modest Proposal. I was a junior in High School when I was first introduced to satire. I had been fluent in sarcasm for some time, but it was in my AP English class that I first became very, very confused. We were instructed to come into doctoral in computer science, the class, sit at college essay admission, our desks, and read the paper on our desks without speaking to one another. This wasn#8217;t abnormal, as our teacher often conducted weird class experiments. In Computer Science! There on our desks was a thick packet with the cover page facing up, #8220;A Modest Proposal#8221; by Jonathan Swift. A Modest Proposal: The Perfect Example of Satire. While the class read through it, heads turned and concerned eyes met from students around the cornell college admission, room. I remember rolling my eyes at a friend muttering how crazy our teacher was. (I had no idea why we were reading this or what it meant.) Swift#8217;s, #8220;A Modest Proposal#8221; outlines the solution for the famine in Ireland in the early 1700#8217;s. The solution proposed by Swift is that poor families should sell their newborn babies to rich families to eat.
He explains how logical it would be for poor families to make money, have less children to thesis science feed, and for rich families to have a high quality protein source. Swift is as kind as to include different ways and suggested recipes for cooking these babies. When we finished reading our teacher asked us what we thought of the piece. Students began commenting, #8220;What is cite email essay this?#8221;, #8220;Is this real?#8221;, and then #8220;Hey, doesn#8217;t sound like such a bad idea.#8221; Before we got too far into our heated debate, our teacher introduced that this was satire . We still smiled blankly, but he finally began to science explain the essay, concept. Here is the definition of satire according to Google: The use of humor, irony, exaggeration, or ridicule to thesis expose and criticize people#8217;s stupidity or vices, particularly in the context of contemporary politics and other topical issues. In this case, Swift used satire as a way to express the issue of poverty in Ireland and to mock the rich#8217;s view towards the of the infant with autism, poor during the famine. To be clear, Swift is not saying that eating children is a reasonable solution to the problem, rather he is demonstrating the heartless and cruel attitude of the rich, while pointing out the issues he sees with the Irish government. Me too. Looks fun, and you get to make ridiculous arguments. Here are two techniques and tips to write great satire. In #8220;A Modest Proposal#8221; Swift uses an intense, serious tone throughout the thesis in computer, entire piece.
One of the most important things about using tone is that we make sure we choose the correct tone to convey the message we so desire. In satire, most commonly, the most effective tone to comparison essay use is the serious tone. This is in computer science because the admissions application essay, serious tone creates this confusion within the reader, just like my junior class was confused the first time we read satire. If the author was using joking language, we would understand that this wasn#8217;t real and lose interest, but because of the intense serious language, we couldn#8217;t stop talking about it. Be definitive. Say crazy things. Give detail. Eat babies. (Just kidding, don#8217;t do that.) Irony is saying one thing, while meaning the other, or in situations when the doctoral in computer science, outcome is contrary to what is cornell admission expected. #8220;A Modest Proposal#8221; is often hailed as one of the in computer, greatest examples of case of the infant autism sustained irony in the English language. Swift accomplishes this by starting the science, piece highlighting the problem of cite essay starving families in Ireland, and then proposing his solution. A young healthy child well nursed, is, at a year old, a most delicious nourishing and doctoral in computer, wholesome food, whether stewed, roasted, baked, or boiled; and I make no doubt that it will equally serve in a fricassee, or a ragout.#8221; How#8217;s that for essay, irony? If you want to doctoral thesis in computer write satire, one of my biggest tips is that you read satire.
Satire is a different way of thinking, and in order to understand it better and write it better, you must become acquainted with it#8217;s style, prose, and voice. Check out a few of how essays these satirical sites: Do you enjoy satire? Have you ever written it? Let us know in the comments section. Take fifteen minutes and practice writing some satire! Choose a popular subject and make your point. Don#8217;t forget to thesis in computer use irony and tone! Make sure you share your practice in the comments below! I enjoy satire, to read and to view in a play or show, as long as it is #8216;satirical#8217; and not mean or cruel, but subtle and humorous. I agree Lillian! This is a challenge.
I#8217;m curious to read posts from members. Great topic! Here#8217;s how I began a #8220;humorous#8221; speech recently. I guess this is satire: When my mother turned 92, she told me: “If I’m still alive in two years… shoot me.” When she turned 94 she said the same thing. And this time she meant it. “Honour Thy Parents,” they say. It’s the admissions, Fifth Commandment, but wait a minute!
I CAN’T SHOOT MY MOTHER. Or can I? Logically, we should shoot everyone over science, 80. Comparison Essay! The evidence against longevity is overwhelming: 1) Old people wish they were dead, and, 2) Old people can’t afford. I’ve done the research on longevity, done my due diligence, and I’m wondering now if perhaps I should just…go ahead and shoot her. (NOTE: Some people in the audience were totally freaked out. They#8217;re still talking about science, it. They keep asking how my mother is.) Ha! PJ that#8217;s great! This is case of an good, P J Reece. I must confess, my husband and I have been talking about no 1) and 2) and old people. He looked at me and said, #8220;What am I doing here?#8221; What could I say except, #8220;We don#8217;t have a gun.#8221; This is great, PJ.
Old people take up so much space, and it#8217;s only logical to shoot them while they#8217;re ahead. Absolutely. The worst part of this mother business is that my mother is doctoral in computer science now 101 and has no intention of surrendering. She has transcended fate. It#8217;s scary.
Now that is cornell college satire! Was Jonathan Swift your ancestor? Mind you, it would have been apt to shoot my father at thesis, a much longer age! Mind you, I wouldn#8217;t have actually done it, no matter how much he merited an abbreviated life span, but to have written a satire on cornell essay the topic would have been so comforting! But is thesis science this really the wrong approach for how essays, this writing exercise? Pity. In Computer! Wow, awesome topic. I come from Serbia and we LOVE satire irony and dark humor!
And I love your point about writing in #8220;serious#8221; tone. That is the very thing that makes people think and scratch their heads in astonishment. #8220;Wait a minute, this guy is not serious!#8221; That moment of case study of the of an infant with autism confusion and thesis in computer, re-thinking is the precious gift for a reader. Thank you for this call, i will definitely try to write satire and let you know how it went. Milena, I agree! It#8217;s so fun to admissions essay watch people scratch their heads #128578; Can#8217;t wait to doctoral thesis science read your satire! Love this.
Satire can also be an amazing tool for application essay, dealing with personal stuff, as I#8217;ve found out over the past few months. It#8217;s oddly refreshing. It#8217;s interesting to note that Swift tried to get his point across in more conventional essays but was largely ignored. It wasn#8217;t until he wrote A Modest Proposal and used pamphlets to distribute it widely, that it received any notice. Sometimes satire is the most powerful tool. Wow Jay, interesting! I did not know that. Satire can be the most influential it seems.
Thanks for sharing! The United States Air Force has a number of B-17 bombers that could be used to in computer science destroy any ISIS stronghold. Fill the bombers with garbage and drop the case of the autism, waste. If ISIS tries to in computer science shoot down the falling garbage, it becomes more garbage. We now solve the ISIS problem and eliminate the need for garbage landfills. The bombers fly at a very high altitude and comparison, are out of harms way.
Great job Frank! Thanks for doctoral, sharing. Great solution, bury them in garbage! A Proposal of Modesty, spoken in the voice of Foghorn Leghorn. Let us propose to move all the Gays, Lesbo#8217;s and admissions application essay, Queer mutha-fuckas to Canada. Lots of room up there, mostly penguins, deers, and polar bears. Perhaps them polar bears can eat some of those queers and Lesbo#8217;s and help take the penguins off the doctoral, endangered species list.
Ya know whut they say, #8220;If it ain#8217;t got antlers!#8221; Damn son, I kill my self sometimes. Of course if we do this, we will have to find something for Pat Robertson, southern congress-folk #8212; you know them representatives and senators #8212; and nearly all of the how essays, Christian folk to in computer blame things on. Well, not the email, Presbyterians. Doctoral In Computer! Of course there might be room in Canada to ship all the Presbyterians as well. Think we could include the Lutherans? I#8217;m neither gay nor Christian nor a penguin, but there#8217;s something not quite right about this satire. I offer up this criticism, Gary, just to point how difficult it is to write good satire. I#8217;d love to how essays see you rewrite this.. but according to what principles of satire, I#8217;m not sure. Anybody have some insight into this? Mostly I believe if I have to explain things, I got it wrong.
Having said that, I have made some changes, and I too would be interested in other insight. Doctoral Science! What I see mainly in your piece, Gary, is the case development of an infant with, courage to throw something on the page, because it#8217;s a potent starting point. Yes, where#8217;s the insight we#8217;re looking for? Help! I don#8217;t know how much U.S. news you are familiar with. There has been a huge flack over same sex marriages here with the fundamentalist and a couple blaming everything from global warming and thesis, the fall of the economy on homosexuality.
It#8217;s gotten ridiculous. Foghorn Leghorn would have been the spokesman. How Essays! Anyway that was the intent. Here is a short start to some satire I have had in mind for quite some time. Doctoral! I realized something long ago. When I was young boy and my father was telling me how lucky I was that I had my whole life ahead of me. He told me about my schooling ahead and application essay, how much there was going to learn. He told me that I would see the world from north to thesis in computer science south, west to east. He teared up as he told me about the life i had coming. #8220;What I wouldn#8217;t give to chrysalids comparison essay be your age again#8221; he told me. It was then I realized: Life is too long.
I began smoking at once. Two birds one stone cigarettes are, You kill time now AND later. I stared into the light of doctoral my phone, flicking my thumb up, down, left and right. I counted stars. I actually counted how many breaths I took in an hour once. I began to write a book in how essays my late twenties called #8220;mediations on passing time#8221; This is great, I would love to doctoral thesis hear more! I think your irony is great! I chose to use a very light, whimsical tone, almost a caricature, instead of a serious tone in my prose poem on #8220;woman on woman combat#8221; or competition among females. A bowl of gre chili explodes in the microwave. Office girls, run. and see, gather round. Who left it there?
How rude. Quite uncivilized. Remove. it at once. Doctoral Thesis In Computer! Ouch, it’s hot! My hand! Let’s run your hand under water until the essay, paramedic arrives. A creamy white hand holds a pink, blistered one under water. A voice, so deep. and calm, I’m here to save you. Thank you, Creamy Hands, for holding the Blistered. Hand until I got here. You must say that to all the girls.
Not all girls have such. smooth, creamy hands. What about me? I’m the one who’s hurt, Blistered Hand pouts, glancing knowingly at the circle of hands waiting round the cauldron. So you. are, Deep Voice says, turning toward Blistered Hand as Creamy Hands tucks her. tail and retreats to her desk. Threat!
She’s an irredeemable flirt. Her hands are so creamy! She’ll steal our boyfriends. Creamy Hands cannot have both creamy hands and. flirty lips. It’s too much! It’s overwhelming. We must stop her. Doctoral Thesis Science! She’ll take. over the office! Blistered Hand heals all afternoon, secret texts sent and. received by her bandaged hand.
Creamy Hands checks her email one last time, just before five. Subject: Indian night delayed. 7pm instead of 6. Shimmy your. ta-tas to my place for a night of how essays exotic curries and sangria! A creamy hand knocks on the door at one minute to seven, cumin-scented rice balls the only thing brighter than her skin. Stand and in computer science, wait, but no one’s home. The glances, the texts sent from under the desks. Creamy Hands. begins to application essay cry, the coven gathering round the cauldron at the bar down the in computer, lane. Tears in her eyes, she mounts her motorcycle, cumin balls in. her purse, tail tucked once more to retreat home in shame. Crash! Vision. blurred, in essay a ditch, the blood drips from her creamy leg.
She surrenders to doctoral thesis in computer the. dirt, alone and outcast, and begs the siren to college essay leave her be. Deep Voice arrives. Creamy Hands, I think you missed me. How. did you end up in this ditch, m’lady? Deep Voice, is that you? I made these. Doctoral Thesis In Computer Science! rice balls for girls’ night, and the girls ditched me.
Only jealous girls do. the ditching. If you’ll hold my strong hand with your creamy one, I’ll pull you. out of this ditch. Shall we? We shall, Deep Voice. Cornell! Where to, Creamy Hands? To. the bar down the doctoral, lane for sangria. Shall I order for us, Creamy Hands? Say, isn’t that Blistered. Hand there with the of the development of an with autism, ladies from doctoral thesis round the cauldron? Wait a minute. Did you. bring me here to make them jealous, Creamy Hands?
But only as an afterthought, Deep. Voice. I do like you! Creamy Hands, I’ll not be flaunted like a piece of deep-voiced. Essay! meat. Leave me be to nurse my beer at the bar. Ladies, I miss you, and I still have the cumin-scented rice.
balls I made for you. Doctoral Thesis In Computer! Hesitant glances abound, sizing up creamy-handed, flirty-lips. when one of the coven spots Deep Voice leaving. Who is that? A cougar! Blistered. Hand points to grey hair. He’s leaving with a cougar! But she’s so old and. wrinkly, Creamy Hands pouted. There, there, dear, have a sangria.
Some women! So cheap and easy! Blank stares round the cauldron. Creamy Hands breaks the. silence. Did you see what curly hair wore to work today? This is really funny Traci! You did a great job!! Thank you, Kellie! Sad, but true. I think a large dose of humour helps when we confront the dark parts of ourselves.
Great article! How silly! Very cute though. This is a plan that I’m putting forward to maintain the balance in our modern culture of case development with tolerance. There have been numerous legal accounts of business owners being ridiculed, sued, and then forced to respect the acts of clientele that are not of their personal interest. This is doctoral thesis my solution.
At every Gay event, there is good essay a tent meeting for biblical revivals and soul winning. The tent meeting should be allowed to be as large and as loud as they want. Doctoral Science! Christian clothing companies should be allowed to stretch fabric along routes of Gay parades, to essay advertise their products. Thesis In Computer Science! This of course might block the view of the parade itself, but that is a right the companies should have. All businesses, from Starbucks cafes, to Apple’s corporate lobby will have a table available for any bible believer the right to cite drink coffee and hand out thesis in computer science biblical tracts. How Essays! The tracts might condemn the behaviour of homosexuality, and not show any acceptance to in computer science Gay marriage, but these bible believers should have the right. These are just a sample of the many suggestions that I am proposing to admissions application alleviate this burden on science our society. To read the complete proposal please got to essay http://www.thebibleisthewordofgod.com. I am currently writing a modest proposal essay for school and doctoral science, i cant think of anything to write about please help me. OK I#8217;ll bite. Admission! Here#8217;s my practice piece: Well, it looks like the Grammar Police will finally take some action.
Last week they made a proposal to revoke the literary licenses of doctoral people who constantly use the the wrong #8220;there#8221; in there writing. Cornell! There licenses would be torn up and doctoral thesis, there fingers would be taped together, or better yet, Crazy glued together. There serious about this and are confident that this one step will improve the quality of the stories that we read. I believe that this is a good first step, but in cite essay step to, they should go after those who use the wrong to, to! Next they could go after the people who write #8220;Wah Lah!#8221;, problem solved. I#8217;m reading these entries, and stressing out that I#8217;m not going to be able to in computer write satire. Then read Jay Warner#8217;s from a year ago It#8217;s interesting to note that Swift tried to case of the with get his point across in more conventional essays but was largely ignored. Okay, I can work with that. Doctoral Thesis Science! Give me time.
Thank#8217;s Jay, and thanks to Kellie. Essay! I#8217;m really stretching out of my comfort zone!
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