Critically discuss the leadership and change management theories Essay

Critically discuss the leadership and change management theories within the case study - Essay Example Biogen Idec is a global biotechnology company that has transformed greatly in the past 25 years to become one of the largest companies in the world. Through mergers with other companies, it has developed into a big company with presence in most parts of Europe and America. Currently, the company is on the verge of changing its operations in order to improve the performance of the company. Managing change is an important aspect of the organization in order for them to remain competitive by responding the market needs. The concept of organizational change and leadership management require a lot of concern since any mistake may lead to big losses (Styhre, 2002; Charlton & Andrew, 2004). Organizations operate in increasing competitive and chaotic global business environment and thus it should form a major focus of the business management. This is because there is enough evidence that about 70% of organizational changes fail to yield enough fruits as projected by the management (Burnes & Jackson, 2011). A properly structured organizational change should thus take full care of content, people, and process since they are the determinants of success or failure of such an initiative (Myungweon, 2011). Leaders are the formulators and implements of any organizational change; hence, they should be responsible for the success or failure of such initiatives (Aaron & Elizabeth, 2012). Leaders are also important to the change process since they create and develop an acceptance culture of change in the organization and develop the ability to achieve such as change as projected by the company (Northouse, 2012). Change Models Kotters 8 Steps The use of Kotters method is evident in the change undertaken by Biogen Idec Inc since they began their initial step of organizational change by conducting research and creating urgency for change. In this method, organization change can be effectively managed using a dynamic 8-step approach that includes increased urgency, building grid team s, get the right vision, communicate for a buy in, enable action, create a short term win, don’t let up, make it stick. The steps are further divided into three phases whereby the first phase is creating a climate change; the second phase is engaging and enabling the whole organization while the last step entails implementing and sustaining the change (Hayes, 2010). Creating urgency This is the first step of Kotters change model whereby he argues that creating urgency enables the members know what they really want and develop a sense of urgency around the need for change. In the case of Biogen Inc, the headquarters in Paris was responsible for all finance, legal, Human resources, and commercial activities of the organization, making the decisions slow and bureaucratic. The urgency created by the management is upon realization that this type of organizational structure is slowing decision making and at times leading to erroneous decisions. They thus needed a structural model t hat can quicken decision making of the managerial functions to avoid several errors that may inhibit the development of the company. Creating urgency helps spark the initial motivation of the workers so that they respond by getting things moving in the direction of change. It involves showing people the poor performance of the organization and increased competition that threatens the existence of the company. Kotter (2012) argues that about three quarters of the organization management needs to support the change initiative for it to be successful hence, the need for creating urgency must be very strong. Form a powerful coalition After the worker have seen the need for change, they need to work together to achieve these desired changes. This step involves convincing people that change is necessary and it encompasses strong governance and sustenance from individuals within the organization. In order to influence the process of change within the organi

Network Security Research Paper Example | Topics and Well Written Essays - 3750 words

Network Security - Research Paper Example Secondly, internal and physical security will discuss human threats, physical access to server rooms and servers, sensors and sprinklers etc. Moreover, protecting accidently shutting down system will also be discussed. After evaluating vulnerabilities associated with these three domains, controls will be proposed and justified accordingly. Furthermore, incorporation of Scilly University network architecture will also be utilized for better insights. 2 Research Methodology and Literature Review We will utilize Scilly University in our research as a basis of our discussions. Moreover, we will focus on qualitative research on information security, tools, assessments and statistical data in some cases. It has been concluded by some experts that the year 2012 is considered to be the worst year in terms of computer network security breaches (Schirick, 2012). Likewise, the year that has not even passed the half year mark, some of the foremost companies were sufferers of network security bre aches resulting in massive losses (Schirick, 2012). However, the news buzz only highlights Sony and Citibank to be victims of network security breaches, as these companies are popular among the public. The other sides of the picture highlights organizations of all sizes are affected by the consequences of network security breaches. Likewise, it can be concluded that network security risks are continuously evolving, modifying and growing at a rapid pace. Organizations normally install a firewall and even intrusion detection systems that triggers alerts of any suspicious activity, as these two components only covers the technical domain and not the human and physical domain. The current network scenario is utilizing a Virtual Private Connection that is connecting one or more sites. However, the VPN connection is also entitled to allow internet traffic on the same dedicated line from the Internet Service Provider. Moreover, the current network only utilizes a single firewall that is lo cated at the main campus of the university. It concludes that the rest of the two remote sites are only protected via a simple Network address translation function that is incorporated in a DSL modem. Moreover, there are no advanced security appliances such as Intrusion detection systems for analyzing and monitoring any suspicious activity that may possibly become a threat to the University’s computer network. Moreover, there is no patch management for updating security patches in the workstations connected to the network. There are no indications of hardening servers for instance, email server, application server, centralized server and database server must be hardened and needs physical protection as well. The network security vulnerabilities will be accessed in three categories i.e. logical security, internal security and external security. As far as logical security is concerned, we can see that the fig 1.1 demonstrates a firewall, Microsoft Internet Security and Accelera tion (ISA) server and a domain controller with Microsoft Active Directory. The three categories for network vulnerabilities are categorized as below: 2.1 Logical Vulnerabilities The current logical controls for protecting information assets within the network are Microsoft Active directory, ISA server and a Firewall. The Microsoft active directory is not primarily a security control, as

Why Is Gatsby so Memorable to the Reader Essay Example for Free

Why Is Gatsby so Memorable to the Reader Essay The Great Gatsby was first published in 1925 and was one of Fitzgerald’s most well-known novels. Many aspects of this book caused this mass popularity, however the main reasons are his use of romantic modernism and most importantly, his portrayal of the different character. The different portrayals of the characters across Long island manipulate the reader’s opinion. One of the most famous examples for this is Gatsby. Gatsby is a very memorable character for many different reasons, such as the portrayal of him by Nick, his mannerisms, his reactions and doings. Each of these reasons helps him become memorable, however another important aspect of this memorability is the effects the Fitzgerald uses, romantic modernism, imagery, atmosphere, descriptions. One reason why Gatsby is memorable to the reader is because of Gatsby’s role as an exception in Nick’s, the narrator of this story, newly formed opinion on the people in Long Island. While Nick has a strong negative reaction to his experiences in New York and eventually returns to the Midwest in search of a less morally ambiguous environment, even during his initial phase of disgust, Gatsby stands out for him as an exception. Nick admires Gatsby highly, despite the fact that Gatsby represents everything Nick scorns about New York as he merges n with the crowd of West Egg, ostentation, garishness and flashy mannerisms, Gatsby clearly poses a challenge to Nick’s customary ways of thinking about the world, and Nick’s struggle to come to terms with that challenge inflects everything in the novel. One example of this is; ‘ â€Å"I wouldn’t ask too much of her,† I ventured. â€Å"You can’t repeat the past. † â€Å"Can’t repeat the past? † he cried incredulously. Why of course you can! † ‘ This quote shows us how motivated and dedicated Gatsby is to relive the past, with his ‘lover’ Daisy. His undying, untameable passion for her is constantly emphasised throughout the book. However, the most unusual feature of this affair, in comparison to the other various affairs of characters is that Gatsby wants to relive the past. Althoug h his ideas and morals highlight everything that Nick’s contradict, he still always has a sense of dedication towards Gatsby, such as being the only person at Gatsby’s funeral. Nick’s attitudes toward Gatsby and Gatsby’s story are ambivalent and contradictory. At times he seems to disapprove of Gatsby’s excesses and breaches of manners and ethics, but he also romanticizes and admires Gatsby, describing the events of the novel in a nostalgic and elegiac tone. This opinion formed by the narrator of this story now means that Gatsby is more memorable because of the sometimes contradictory opinions on his character but also because of the narrator’s reaction to Gatsby. A second reason why Gatsby is memorable to the readers is because of the use of mystery and withheld information throughout the beginning aspects of the book. This mystery is initially triggered when the readers first see Gatsby. ‘He gave a sudden intimation that he was content to be alone — he stretched out his arms toward the dark water in a curious way, and, far as I was from him, I could have sworn he was trembling. Involuntarily I glanced seaward — and distinguished nothing except a single green light, minute and far away, that might have been the end of a dock. When I looked once more for Gatsby he had vanished, and I was alone again in the unquiet darkness’ In this extract Gatsby is initially portrayed as a very mysterious and enigmatic character and interestingly stands in stark contrast to the other denizens of West Egg. Although Nick is unsure of this green light’s origin or even what significance it represents for Gatsby, the inner yearning which is visible to Nick, mainly because of Gatsby’s posture and emotional surrender to this green light makes him seem the opposite of the previous surrounding, the sarcastic Ivy League set at the Buchanans’. Gatsby is a mysterious figure for Nick, since Nick knows neither his motives, nor the source of his wealth, nor his history, and the object of his yearning remains as remote and nebulous as the green light toward which he reaches. This imagery creates a tense atmosphere for the readers initial perception This use of mystery surrounding Gatsby helps him to become more memorable because he is the character who leads the reader’s curiosity and is always indirectly at the center of everything. This helps Gatsby become more memorable by the use of withheld information and mystery. Fitzgerald delays the introduction of most of this information until quite late in the novel. Gatsby’s reputation precedes him—Gatsby himself does not appear in a speaking role until Chapter three. Fitzgerald initially presents Gatsby as the aloof, enigmatic host of the unbelievably lavish parties thrown every week at his mansion. He appears surrounded by spectacular luxury, courted by powerful men and beautiful women. He is the subject of gossip throughout New York and is already a legendary celebrity before he is ever introduced to the reader. Fitzgerald propels the novel forward through the early chapters by shrouding Gatsby’s background and the source of his wealth in mystery. As a result, the reader’s first, distant impressions of Gatsby strike quite a different note from that of the lovesick, naive young man who emerges through the later part of the novel. This also helps him to be more memorable. An alternative reason why Gatsby is memorable is his effect on other characters, although he is a stark contrast in many ways, this helps the audience to warm to him. Some of the defining characteristics of Gatsby are his theatrical quality as a character and also his charisma. Chapter three is when Fitzgerald creates a close examination on Gatsby and allows the reader to form an opinion alone. ‘He had one of those rare smiles with a quality of eternal reassurance in it, that you may come across four or five times in life. It faced, or seemed to face, the whole external world for an instant and then concentrated on you with an irresistible prejudice in your favor. It understood you just as far as you wanted to be understood, believed in you as you would like to believe in yourself. ’ This description of Gatsby’s smile captures both the theatrical quality of Gatsby’s character and his charisma effectively within its essence. Additionally, it encapsulates the manner in which Gatsby appears to the outside world, an image Fitzgerald slowly deconstructs as the novel progresses toward Gatsby’s death in Chapter eight. One of the main facets of Gatsby’s persona is that he acts out a role that he defined for himself when he was seventeen years old. His smile seems to be both an important part of the role and a result of the singular combination of hope and imagination that enables him to play it so effectively. Here, Nick describes Gatsby’s rare focus—he has the ability to make anyone he smiles at feel as though he has chosen that person out of â€Å"the whole external world,† reflecting that person’s most optimistic conception of him- or herself. This synecdoche of Gatsby’s smile also makes the reader remember Gatsby a lot more than they may not have done as it represents his whole character as an unusual but yet enigmatic person. Another reason why Gatsby is memorable to the reader is because of the various comparisons and parallels drawn between other famous characters in stories. These are effective because they help the reader to further challenge their initial opinion on Gatsby but in an unusual, unfamiliar manner. ‘The truth was that Jay Gatsby, of West Egg, Long Island, sprang from his Platonic conception of himself. He was a son of God—a phrase which, if it means anything, means just that—and he must be about His Father’s business, the service of a vast, vulgar, and meretricious beauty. So he invented just the sort of Jay Gatsby that a seventeen year old boy would be likely to invent, and to this conception he was faithful to the end. ’ In Chapter six, when Nick finally describes Gatsby’s early history, he uses this striking comparison between Gatsby and Jesus Christ to illuminate Gatsby’s creation of his own identity. Fitzgerald was influenced in drawing this parallel by a nineteenth-century book entitled The Life of Jesus. This book presents Jesus as a figure who essentially decided to make himself the son of God, then brought himself to ruin by refusing to recognize the reality that denied his self-conception. Renan describes a Jesus who is â€Å"faithful to his self-created dream but scornful of the factual truth that finally crushes him and his dream†Ã¢â‚¬â€a very appropriate description of Gatsby. Fitzgerald’s devising of this metaphor allows Gatsby to be compared more thoroughly and also provides an association to the other characters throughout the rest of the book. Though the parallel between Gatsby and Jesus is not an important motif in The Great Gatsby, it is nonetheless a suggestive comparison, as Gatsby transforms himself into the ideal that he envisioned for himself (a Platonic conception of himself) as a youngster and remains committed to that ideal, despite the obstacles that society presents to the fulfillment of his dream, such as the fact that Gatsby wants to repeat the past but the situation has changed completely. The final reason why Gatsby is so memorable is his impact throughout the novel. An initial factor of his memorability is that the book is called ‘The Great Gatsby’. Before the readers have even heard of this character they already start to question various aspects of him. However, the biggest impact he had was the dedication he created within Nick for him. ‘Gatsby believed in the green light, the orgastic future that year by year recedes before us. It eluded us then, but that’s no matter—tomorrow we will run faster, stretch out our arms farther. . . . And then one fine morning— So we beat on, boats against the current, borne back ceaselessly into the past’ These words conclude the novel and find Nick returning to the theme of the significance of the past to dreams of the future, here represented by the green light. He focuses on the struggle of human beings to achieve their goals by both transcending and re-creating the past. Yet humans prove themselves unable to move beyond the past: in the metaphoric language used here, the current draws them backward as they row forward toward the green light. This past functions as the source of their ideas about the future, epitomized by Gatsby’s desire to re-create 1917 in his affair with Daisy, and they cannot escape it as they continue to struggle to transform their dreams into reality. While they never lose their optimism, â€Å"tomorrow we will run faster, stretch out our arms farther . . . , they expend all of their energy in pursuit of a goal that moves ever farther away. This apt metaphor characterizes both Gatsby’s struggle and the American dream itself. Nick’s words register neither blind approval nor cynical disillusionment but rather the respectful melancholy that he ultimately brings to his study of Gatsby’s life. Therefore overall Gatsby is a memorable character mainly because of his portrayal by Nick and Fitzgerald’s use of imagery to be described. Throughout, Gatsby changes the atmosphere and always clings to the mind of the reader.

Single Chain Antibody Production

Single Chain Antibody Production ABSTRACT The study aimed to characterise His- and Myc- tagged scFv MFE-23 antibodies produced from transformed E.coli cultures using ELISA and immunohistochemistry assays. Anti-His, anti-myc and anti-MFE secondary antibodies were used in the CEA/PBS coated ELISA plate with horseradish peroxidase-OPD chromatic reaction for detection. Culture 1 was identified to produce MFE-His and culture 2 giving MFE-Myc antibodies. The immunohistochemistry assay confirmed the CEA binding profile of scFv MFE-Myc by the comparison between negative controls, positive anti-CEA binding reactions and 4-stage anti-Myc binding of the scFv MFE-Myc examined. The CEA specificity displayed by tagged scFv MFE-Myc can be utilised in antibody-based cancer therapy by targeting tumour antigens specifically. INTRODUCTION Cancer arises due to the defective regulation of normal cell proliferation and homeostasis. This allows tumours to possess the capabilities including self-sufficiency in growth signal, insensitivity to antigrowth signals, avoidance of apoptosis, unlimited replicative potential, sustained angiogenesis and metastasis.1 In additional to the surgical removal of tumours, conventional cancer therapies such as radiation, chemotherapy and immunotherapy have a focus in inducing cytotoxicity against malignant cells. With better understanding of the molecular biology of carcinogenesis, targeted therapies are being developed to achieve lower toxicity to normal tissues and higher clinical efficacy through disrupting pathways that contribute to the tumours proliferative advantage. Attempts are also being made in developing cancer gene therapies to compensate or repair the mutated genes. All tumours express their unique set of antigens on cell surface which can be a result of genetic alterations, upregulated self-antigens or tissue-specific antigens that can be utilized to distinguish cancer cells from normal cells. Antibodies, one of the effectors in immune response, are Y-shaped proteins that each recognizes and binds to specific antigen (Figure 1). The protein consists of two light chains and two heavy chains, with variable (Fab) and constant (Fc) domains on each of the chain. The region of the antibody at which antigen binds is referred as the Complementary Determining Regions (CDR) present at the variable Fab region. The Fc region is responsible in modulating immune response through activation of the complement cascade and Fc receptor mediated activation of effectors such as phagocytes, mast cells, neutrophils and Natural Killer (NK) cells.3 The initial approach of antibody-based cancer therapy is to tag cancerous cells as foreign and eliminate such targets thro ugh cytotoxic effectors of the human immune system. Antibodies against essential growth factors can be synthesized to sequester such molecules from further promoting tumour growth. The high specificity of antibodies to particular antigens also serves as a vehicle in delivering killing machineries to tumour cells. For example, antibodies can be directly conjugated to radionuclides, toxins or cytokines, or indirectly to the surface of liposomes carrying drugs or toxins.4 Antibody-directed enzyme prodrug therapy (ADEPT) is also available in which the antibody targets an enzyme selectively to the tumor where it converts a relatively non-toxic prodrug to a potent cytotoxic drug.4 These various strategies aim to minimize the systemic toxicity afflicted by the cytotoxic agents administered. Monoclonal antibodies are usually preferred compared to polyclonal ones, as they recognize specific epitope of an antigen and hence have greater specificity. Carcinoembryonic antigen (CEA) is first identified as a glycoprotein in the human colon cancer tissue extract and fetal gut, and plays a role in cell adhesion.5 Although CEA can also be detected in normal gastrointestinal tissue, the glycoprotein is overexpressed on the plasma membrane of colon cancer tissues.5 CEA level has also been found to be highly elevated in various cancers of an epithelial origin such as breast, lungs and pancreas.5,6 Moreover, normal CEA is localised on the luminal surface of columnar epithelial cells lining the crypts of the intestine so the glycoproteins are not directly accessible to the blood flow.6 However CEA is usually found on all sides of the cell membranes in tumours. Thus, CEA can be a useful target on cancer cells in immunotherapy using anti-CEA antibodies. The objectives of this lab practical are to introduce the applications of antibodies in cancer therapy and diagnosis through immunohistochemical staining of tissues. It also serves to familiarise students with the enzyme linked immunosorbent assay (ELISA). This is achieved by the production of MFE-23 single chain Fc antibody fragment (scFv) against CEA from transformed E.coli cultures. The scFv MFE-23 can be linked to either a His- or a Myc- tag. The unknown antibodies are characterised in the ELISA assay using appropriate detection antibodies, and the chromatic reaction between horseradish peroxidise (HRP) and OPD substrate. The scFv MFE-23 obtained by students is also used in the immunohistochemical characterisation in cryostat sections of normal and cancerous human tissues. MATERIALS METHODS E. Coli growth curves E.coli cultures were transformed with pUC119 containing either His- or Myc- tagged scFv MFE-23 (Culture 1 2) and were incubated overnight. The expression of pUC119 was controlled by the lac operon, which could be induced by either lactose or lactose analogue isopropyl-1-szlig;-D-thiogalactoside (IPTG). The vector also encoded for ampicillin resistance. The E.coli cultures were grown with ampicillin selection and 0.05% glucose. Optical density (OD) readings at 600nm were taken at 30min interval until OD=0.9 when IPTG was added to both cultures to induce pUC119 expression and hence scFv MFE-23 production. A negative control of 2xYT was set up, and all three cultures were incubated at 30? overnight. Full experimental procedures are described in Appendix 1. ELISA assay The ELISA assay aims to characterize the identities of the tags conjugated to scFv MFE-23 obtained in the supernatant of the overnight E.coli cultures. Detailed protocols of the assay can be found in Appendix 1. 36 wells of the 96-well ELISA plate were coated with the CEA antigen or phosphate-buffered saline (PBS) as its negative control. Supernatant of the overnight E.coli cultures 12 from the bacterial growth curve assay was obtained which contain either His- or Myc- tagged scFv MFE-23. The supernatant samples 12, the positive control MFE-his-myc antibodies and the negative control 2xYT growth medium were added to the corresponding wells as indicated in the format diagram in Appendix 2. Secondary antibodies rabbit anti-MFE23 polysera, mouse monoclonal anti-HIS tag (TetraHis, Qiagen) and mouse monoclonal anti-MYC (Sigma) were added to the corresponding wells (Appendix 2) to bind the primary antibodies present. Tertiary horseradish peroxidase (HRP) conjugated antibodies against the secondary antibodies goat anti-rabbit HRP (sigma) and sheep anti-mouse HRP (Sigma) in blocking solution were added to the wells (Appendix 2). OPD substrate buffer was applied to each well to detect the presence of HRP, which should give a yellow-orange product in case of positive result. HCl was added to stop the reaction when colour has developed, and the OD at 490nm for each well was measured. Immunohistochemistry assay Five glass slides, each containing 2 colonic adenocarcinoma, 1 normal colon and 1 normal liver tissue sections were fixed and processed for immunohistochemical staining. Avidin-biotin-peroxidase complex (ABC complex) was added following the application of biotin-labelled antibodies. The localisation of antigens was visualised by the formation of brown pigments, as peroxidise reacts with the diaminobenzidene (DAB) substrate. Lattices of several peroxidase molecules were formed to amplify the binding signal from the biotinylated antibody.7 A summary of the treatment given to each slide was illustrated in Table 1, and the complete protocol can be found in Appendix 1. RESULTS E. Coli growth curves Both E.coli cultures transformed with either His-tagged scFv MFE-23 or the Myc-tagged version in a pUC119 vector follow a similar exponential growth curve, as shown by the plot of OD600 against time (Figure 2). The bacterial cultures were in lag phase at t=0-90, and the log phase from t=90. It took approximately 175minutes for the cultures to reach OD600=0.9. The PBS negative controls in wells A-F/7-12 worked relatively accurately with low OD490 readings. Most readings also corresponded to the negative controls in the CEA coated wells (B4-6, D4-6, F4-6) but with 2xYT added instead of primary antibodies. However, the OD490 readings for wells C10-12 and E10-12 were higher than most readings from the negative controls. The higher OD490 readings obtained in certain wells in comparison of their corresponding negative controls indicated the presence of yellow-orange product formation from reaction between HRP and OPD substrate. These positive outcomes found in certain wells are highlighted in the shaded cells of Table Immunohistochemistry Assay Figure 4 shows the immunohistochemical binding reactions for the negative controls (slides 3-5). Slide 3 acts as a negative control for slide 1, treated with the 4-stage anti-Myc technique but omitting the primary Myc-tagged scFv MFE-23 antibody. Thus we should not be able to visualize the localization of CEA antigens due to the absence of MFE-23 binding on slide 3. Slide 4 is a negative control for slide 2, omitting the mouse monoclonal anti-CEA A5B7 antibody treatment in the 3-stage mouse monoclonal technique. No binding reaction is expected on slide 4 as well. Slide 5 was treated like slide 4 but without initial biotin/avidin blocking. As expected, the colonic adenocarcinoma tissue section of slide 3 does not display brown colouration and hence there is no binding reaction to the cytoplasm of tumour cells and connective tissues. Binding reaction to the cytoplasm of cryptal epithelium is not seen for the normal colonic mucosa, although there are strong brown colourations for a few cells in the lamina propria. The normal liver tissue on slide 3 shows some weak reaction with the parenchymal cells. Similarly, the parenchymal cells of the normal liver tissue on slide 4 do not display brown colouration and hence indicates the absence of binding reaction. On slide 5, the normal parenchymal cells are positive for binding reaction, demonstrating the presence of biotin in normal liver. Figure 5 shows the immunohistochemical binding results of slides 1 and 2. Slide 1 was treated with the 4-stage anti-Myc technique and slide 2 is the positive control treated with the 3-stage mouse monoclonal technique (See Table 1). For the colonic adenocarcinoma tissue on slide 1, strong brown colouration is present in the cytoplasm of tumour cells and the basement membrane of malignant acinar structures. Weak positive binding reactions can also be observed in fibrovascular stroma. The normal colonic mucosa of slide 1 shows strong reactions to the cytoplasm of goblet cells in the cryptal epithelium as well as a few cells in the lamina propria. The normal liver tissue shows only weak positive reactions with the parenchymal cells. Thus the scFv MFE-myc antibody was reactive with both normal colonic epithelium and adenocarcinoma, but not the biotin/avidin blocked liver tissue. The colonic adenocarcinoma tissue on slide 2 shows strong reactions with the tumour cell cytoplasm and the basement membrane of malignant acinar structures similar to the reactions seen in slide 1. Weak positive results are obtained in fibrovascular stroma of the positive control slide. As the binding reaction of slide 1 was similar to that of the positive control, this confirms the CEA reactive profile of the scFv MFE-myc antibody from the E.coli supernatant sample 2. DISCUSSION E.coli Growth Curve Both E.coli cultures followed the exponential growth curve as expected. However, the growth curve was obtained in the absence of a negative control i.e. same volume of 2xYT to be treated in the same way as the two cultures. The lack of a proper negative control means that the possibility of contamination cannot be eliminated. Thus it is unknown whether the increase in OD600 readings was partially attributed to culture contamination. ELISA Assay As mentioned in the results, the OD490 readings for wells C10-12 and E10-12 were higher than most readings from the negative controls. This might indicate contamination of these wells with CEA antigens, or insufficient PBS washing following the application of HRP-conjugated antibodies. Wells A-B/1-6 were applied with anti-His secondary antibodies and so would indicate the presence of His-tag by the production of yellow-orange product. Wells A1-3 were treated with supernatant from bacterial culture 1 and wells A4-6 with that of culture 2. As higher readings of OD490 in the positive control B1-3 and wells A1-3 were obtained compared to the negative control B4-6, the culture 1 supernatant contained the His-tagged scFv MFE-23. Wells C-D/1-6 were applied with anti-Myc secondary antibodies and so would indicate the presence of Myc-tag by the production of yellow-orange product. Wells C1-3 were treated with supernatant from bacterial culture 1 and wells C4-6 with that of culture 2. As higher readings of OD490 in the positive control D1-3 and wells C4-6 were obtained compared to the negative control D4-6, the culture 2 supernatant contained the Myc-tagged scFv MFE-23. The anti-MFE antibodies added to wells E-F/1-12 can bind to both His- or Myc-tagged scFv MFE-23. Thus binding should occur against primary antibodies from both cultures 12 and also the positive MFE-myc-his control, as demonstrated by the higher OD490 readings in wells E1-6 and F1-3 compared to the negative controls F4-6. It can be observed that the anti-His antibody gave a stronger signal than anti-Myc and was due to anti-His binding more strongly to its target than anti-Myc (Unpublished results, Kogelberg, H.). Comparing the OD490 of wells E1-3 and E4-6, absorbance of anti-Myc was slightly higher than anti-His despite the lower binding affinity of anti-Myc. Thus there might be a higher concentration of MFE-Myc in the culture 2 supernatant than MFE-His in culture 1, although technical issues like washing times, salt concentration and pH, or structural characteristics affecting the accessibility of antibody can affect the amount of binding. Immunohistochemistry Assay The results obtained from slides 1 and 2 confirm the CEA reactive profile of scFv MFE-myc antibody, as both slides displayed similar binding reaction patterns in colonic adenocarcinoma. The weak positive signal in normal liver tissue in slide 1 is likely to be caused by cross-reaction of secondary mouse anti-Myc antibodies, as similar result can be observed in the negative control (slide 3). Primary antibodies may also cross react and bind to non-target tissues. This demonstrates the possible cross-reactions with antibodies and hence the importance of negative controls to eliminate such artifactual reactivity. The strong binding to goblet cells cytoplasm in cryptal epithelium of normal colonic mucosa in slide 1 is consistent with the findings of CEA present in normal colonic mucosa.5 Although CEA level is lower on normal colonic mucosa, their presence implies that antibodies against CEA in cancer therapy may target normal cells other than malignant cells. Thus it is important to control anti-CEA antibody concentration used to avoid imposing toxicity to normal tissues yet is effective in producing a clinical response on cancer. The negative controls (slides 3-5) allowed the assessment of the level of binding for secondary, tertiary and quaternary antibodies/reagents. The slides also revealed any endogenous background material that might be confused with specific binding of primary antibodies, as well as information about the basic pathology of tissues. The inclusion of normal liver sections helped illustrate the importance in carefully controlling the specific reactions of test antibodies with potential targeting specificity for other cellular proteins. The ABC complex for detecting biotinylated horse anti-mouse antibodies can also react with biotin present in liver and give a positive non-specific result. This unwanted reaction as seen in slide 5 can be prevented by biotin blocking treatment of tissue samples. Moreover, myeloperoxidase white cells containing endogenous peroxidase can react with the DAB substrate and produce brown colouration even in the absence of ABC complexes. The normal colonic mucosa in slide 3 provided examples of such artifact. The naturally occurring bile pigments in liver seen as green/brown granules under high-power bright field microscopy can also be found occasionally. Careful interpretation of slides is required to avoid false judgments as there can be false positive reactions. In addition, it should be noted that adequate fixation of the sample is important in successful localisation of antigens present and hence an accurate representation of antigen distribution profile. The procedure helps to ensure the preservation of tissue morphology, the immobilisation of antigen and the preservation of antigen immunoreactivity. It is also important to ensure optimal fixation for an adequate permeability of the tissue to the immunochemical reagents. Other than mmunohistochemistry, western blotting and immunoprecipitation (IP) can be carried out to confirm scFv specificity. Antigens transferred to nitrocellulose membrane can be probed by specific antibodies in western blot, or precipitated out of lysate using antibodies in IP. Affinity chromatography can also be used which is a method of separating biochemical mixtures based on highly specific biological interaction. Specific antigen can be covalently coupled to a solid support and allow supernatant with the testing antibody to flow through so that only specific antibodies will be bound to the antigens. The assay used in this study can only detect antigens in a non-quantitative way with chromatic display of antigen localisation. Instead radioimmunoluminography (RILG) can be used for quantitative measurements of antigen concentration along with its distribution in histological sections.8 Radiolabelled antibodies against specific antigen can be applied to tissue sections and bound antibodies are mapped by phosphor imaging. Radioactivity detected in each pixel of the digital image will be proportional to antigen concentration if saturating antibody concentration is used. Future Perspective of Antibody Targeted Cancer Therapy The successful application of scFv MFE-23 with a Myc-tag at its C-terminus for detection in immunohistochemistry proves that the attachment of small molecules to the antibody will not affect its specificity for CEA. Thus the scFv chain can be conjugated to cytotoxic reagents or to be used for ADEPT as mentioned previously. The potential clinical efficacy of scFv MFE-23:enzyme fusion protein has been shown in nude mice with human colon adenocarcinoma xenografts by the Bhatia group.9 Moreover, scFv MFE-23 can be used in radioimmunoguided surgery (RIGS) based on the pre-operative injection of a radiolabelled anti-tumor antibody to detect tumour deposits during surgery. A Phase I clinical trial of RIGS using 125iodine-labelled MFE- 23-his scFv has reported good selective localisation at sites of primary colorectal cancer and metastases.10 As illustrated by MFE-23 scFv fragment, antibody targeting has the potential for selective imaging or delivery of anti-cancer molecules. Antibodies can be engineered to modify their biological properties with increased specificity and functionality. This is achieved by reducing antibody size, altering valency, and fusing to different molecules to improve therapeutic efficiency. Scientists have been trying to produce smaller antibody fragments but retaining specific binding to antigens, in order to minimize immunogenicity and achieve better tumour penetration. Continuous research on the specificity and stability of these fragments, and hunting for more tumour-specific antigens are required to further expand the field of antibody-targeted cancer therapies. Slide 3 is a negative control for slide 1 which was treated with the 4 stage anti-Myc technique. Slide 4 is a negative control for slide 2 which was treated with the 3 stage mouse monoclonal technique. Slide 5 is a negative control for slide 2 as well but without biotin/avidin blocking. Slide 1 was treated with the 4 stage anti-Myc technique, and slide 2 was treated with the 3 stage mouse monoclonal technique. REFERENCES Hanahan D, Weinberg RA. The hallmarks of cancer. Cell. 2000 Jan 7;100(1):57-70. Baron, E. J. 1996. Classification. In S. Baron et al., eds. Barons Medical Microbiology, 4th edition. University of Texas Medical Branch. Gura, T. Therapeutic antibodies:Magic bullets hit the target. Nature 417, 584-586 (6 June 2002) Carter, P. Improving the efficacy of antibody-based cancer therapies Nature Reviews Cancer 1, 118-129 (November 2001) Sten Hammarstrà ¶m. The carcinoembryonic antigen (CEA) family: structures, suggested functions and expression in normal and malignant tissues. Seminars in Cancer Biology. Volume 9, Issue 2, April 1999, Pages 67-81 A. Mayer, K. A. Chester, et al. Taking engineered anti-CEA antibodies to the clinic. Journal of Immunological Methods. Volume 231, Issues 1-2, 10 December 1999, Pages 261-273 Hsu SM, Raine L, Fanger H. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem. 1981 Apr;29(4):577-80. GBoxer, SStuart-Smith, et al. Radioimmunoluminography: a tool for relating tissue antigen concentration to clinical outcome. British Journal of Cancer (1999) 80, 922-926. Bhatia J, Sharma SK, et al. Catalytic activity of an in vivo tumor targeted anti-CEA scFv:carboxypeptidase G2 fusion protein. Int J Cancer. 2000 Feb 15;85(4):571-7. Mayer, A., et al. Radioimmunoguided Surgery in Colorectal Cancer Using a Genetically Engineered Anti-CEA Single-Chain Fv Antibody. Clinical Cancer Research May 2000 6; 1711. APPENDIX Appendix 1 Experimental Procedures (UCL Cancer MSc Lab Practical 2 Handout, 12-13Nov09) E. Coli growth curves E.coli culture transformed with pUC119 containing either His- or Myc- tagged scFv MFE-23 (Culture 1 2) were incubated overnight. The expression of pUC119 was controlled by the lac operon, which could be induced by either lactose or lactose analogue isopropyl-1-ß-D-thiogalactoside (IPTG). The vector also encoded for ampicillin resistance. 30 µl of the media was added to 15ml growth media with ampicillin selection and 0.05% glucose. Both tubes on loose caps were placed into a 37? shaker at 225rpm and time was taken as t=0. Optical density (OD) of the culture was measured with a spectrophotometer at 600nm from t=0 at a 30min interval until reading reached OD=0.9. 1mM IPTG was added to each culture and the tubes were incubated at 30? overnight with shaking. A negative control was set up using the same volume of growth media 2xYT with 1mM IPTG for overnight incubation. ELISA assay 100 µl of 10 µg/ml CEA antigen was applied to strips 1-6, A-H of the 96-well ELISA plate. 100 µl of PBS was applied to strips 7-12, A-H as negative controls. The plate was covered with plastic film and incubated for 1 hour at room temperature. Each well was rinsed 4 times with PBS and blocked with 200 µl 5% Marvel milk/PBS. The plate was covered and incubated overnight at 4?. The plate was washed with PBS 4 times after the overnight incubation. Overnight bacterial cultures and the negative control were centrifuged at 4000rpm for 20minutes to obtain the supernatant. 100 µl of each supernatant sample and negative control was added to the ELISA plate according to the format diagram as seen in Appendix 2. The covered plate was left at room temperature for an hour. The plate was then rinsed twice with 0.1% Tween-20/PBS and 4 times with PBS. 100 µl of secondary antibodies rabbit anti-MFE23 polysera, mouse monoclonal anti-HIS tag (TetraHis, Qiagen) and mouse monoclonal anti-MYC (Sigma) with a concentration of 1:1000 in 1% blocking solution was added to the wells according to the format diagram: anti-His A1-12 and B1-12; anti-Myc C1-12 and D1-12; anti-MFE E1-12 and F1-12. The covered plate was left at room temperature for an hour. The plate was washed again twice with 0.1% Tween-20/PBS and 4 times with PBS. 100 µl horseradish peroxidase (HRP) conjugated antibodies against the secondary antibodies 1:1000 goat anti-rabbit HRP (sigma) and 1:500 sheep anti-mouse HRP (Sigma) in 1% blocking solution was added to the corresponding wells. The covered plate was left at room temperature for an hour, and then rinsed twice with 0.1% Tween-20/PBS and 4 times with PBS. 100 µl of OPD substrate buffer was added to each well to detect the presence of HRP, giving a yellow-orange product in the case of positive outcome. 100 µl of 4M HCl was added when the colour has developed, and OD was measured at 490nm with an automated plate reader. Immunohistochemistry assay Five glass slides, each containing 2 colonic adenocarcinoma, 1 normal colon and 1 normal liver tissue sections were removed from the freezer and air dried for 5minutes. Slides were fixed in acetone in 10minutes and rinsed in tap water for 2minutes. Slides were flooded with PBS and Avidin blocking solution was applied to slides 1-4 for 10 minutes. Slides 1-4 were rinsed in PBS and applied with Biotin blocking solution for 10minutes followed by PBS wash. PBS was removed from slide 1 and flooded with 1:20 normal horse serum in PBS for 15minutes. Slide 1 was drained and applied with bacterial supernatant containing Myc-tagged scFv MFE-23 antibodies for 45minutes. Slide 1 was then rinsed with PBS thrice over 10minutes, and 1:20 normal horse serum in PBS was added to slides 2-5 for 15minutes. 20 µg/ml of mouse monoclonal anti-Myc antibody (Qiagen) in PBS was added to slides 13 and left for 35minutes. 20 µg/ml of A5B7 anti-CEA antibody was added to slide 2 for 35minutes. Slides 4-5 were washed in PBS. Slides 1-3 were rinsed with PBS thrice over 10minutes. 1:200 biotinylated horse anti-mouse immunoglobulins in PBS with 5% normal human serum was added to all slides and incubated for 35minutes. All slides were rinsed with PBS thrice over 10minutes. The avidin biotin-peroxidase complexes reagent (ABC reagent) was added to all slides, followed by PBS wash 3 times over 10minutes. 0.03% of hydrogen peroxide in 1mg/ml 3,3,diaminobenzidene tetrahydrochloride solution was applied to all slides immediately and left for 5minutes. Slides were washed with tap water. All slides were stained in Harris haematoxylin for 40seconds, rinsed in tap water and left for 5minutes for colour to develop. Slides were then dehydrated through graded alcohols (70/95/100%), cleared in inhibisol and coverslip using DPX mountant. The slides were then ready for observations under the light microscope.

Java Script :: essays research papers

Equipment –   Ã‚  Ã‚  Ã‚  Ã‚  Backpacking equipment is highly efficient and well-designed to minimize the weight that must be carried. The equipment is also expensive and should be borrowed from someone you know if available to allow you to have more money for your hiking trip. Hiking Boots, Socks, and Gaiters –   Ã‚  Ã‚  Ã‚  Ã‚  Good quality leather hiking boots are essential for serious backpacking because they offer protection and comfort. Sandals may be seen but are not worn when traveling for many miles. Boots that are durable usually last over many miles which may lead into many years. Most boots have soles made of synthetic rubber known as Vibram. Light weight shoes offer protection at a reasonable cost and decreased break-in time.   Ã‚  Ã‚  Ã‚  Ã‚  Beginning Hikers tend to buy boots that are heavier and more expensive than necessary because it is important to have boots that fit properly and be long enough so that the toes don’t contact the front of the boot while walking downhill. Boots must be well broken in before attempting to hike on the trail. They should be worn at every opportunity before making a long hike, to make sure they won’t cause blisters.   Ã‚  Ã‚  Ã‚  Ã‚  Socks are important companion pieces to boots and most hikers use two pairs. The inner pair is usually is lightweight wool, polypropylene, or similar material to wick moisture away from the feet. The outer pair is usually heavyweight wool to provide cushioning of the feet within the boots. Clean socks should be worn everyday and at least one extra set of socks is needed.   Ã‚  Ã‚  Ã‚  Ã‚  Nylon or Gore-Tex gaiters will assist in keeping debris such as dirt and snow from entering the top of the boots. Gaiters usually have side or rear zippers to allow the person to put them on or take them off without having to remove the boot.   Ã‚  Ã‚  Ã‚  Ã‚  Sandals or lightweight tennis shoes are often overlooked as a campsite alternative. A pair of sandals can allow a hiker to air out the feet and dry out the boots at the same time. Sandals or tennis shoes do not offer enough support for carrying heavy loads, but they do have a function around the campsite. Packs -   Ã‚  Ã‚  Ã‚  Ã‚  The pack is a are mounted on external frames. It should be possible to remove the pack from the frame in case one wants to use the frame for carrying loads other than the pack. Frames are made of lightweight metal usually tubular aluminum alloy.

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SoftPot Rotary Potentiometer: Rotary potentiometer is provided at knee joint of the exoskeleton. Whenever the patient is having intent to walk, the rotary potentiometer calculates the respective torque. In response to that a feedback signal is provided to the actuation system to exert equivalent force to interact with the input torque. The feedback system is designed so well that it updates the status of input torque in fraction of a second and as a result exoskeleton keeps working accordingly; and works well with frequent torque changes. Rotary potentiometer works on the principle of resistance change. It is provided with a very fine needle/flip which rotates over the resistive ring. More the angle of rotation more will be the value of resistance observed between the terminal and the rotating flip. It is the measured resistance that tells about the angle of rotation of the joint and torques produced. Therefore, whenever rotational movement is observed the value of resistance of the respective potentiometer is changed and the signal is fed to the controller to take proper action. This helps in maintaining the exoskeleton motion according to the suggested profile and keeping it in the right motion. The rotary potentiometer used in our proposed design is SoftPot rotary potentiometer which is a miniaturized product with slim and sleek design. It can easily be placed along the bearings of the rotary joints and gives output in terms of analog electrical signals which are proportional to the angle of rotation. This sensor is very thin and compact in design with a wide range of operational resistances. Range of resistance change may vary between 100Ohms to 10,000Ohms; also the response of this sensor is much linear; therefore, the positi... ...e with a frequency rate multiplied by the movement. When you rotate the gyroscope, you may notice a so called Coriolis acceleration. In fact, the gyroscope is a spinner, rotating around a vertical axis, fixed in a frame which can articulate around horizontal axis, attached to another frame rotating around the third axis. Thus, we can conclude: no matter how rotate a spinner, it always has an ability to remain in the upright position. The transmitters accepts the signal about the spinner's orientation towards the frames, and the processor defines how the frame must be located with respect to gravity. Regardless to the types of stabilization the system, it can be the power system stabilization (used in two-stage gyroscopes), the tracer system stabilization force (also on a two-level gyroscopes) and the indicator system stabilization (in three-stage gyroscopes). .

John Bunyan: The Pilgrim’s Progress

Bunyan’s ‘Pilgrim’s Progress’ is considered one of most famous allegorical novels in English Literature. The story is about Christian’s spiritual journey going through various phases of temptations and trials. Published in 1678, The Pilgrim’s Progress is an inspirational aid for applying an evangelical view of Protestant Biblical to the trials in life.The value of The Pilgrim's Progress to the Puritans is extremely effective aid to applying an evangelical view of Protestant Biblical theology to the trials faced in human’s life portrayed effectively through various images and allegorical characters. Through its images, allegorical characters (Christian, Evangelist, Help, etc), and allegorical places (â€Å"City of Destruction† = earth, â€Å"Celestial City† = heaven, â€Å"Tophet† = hell) Pilgrim’s Progress Bunyan effectively show readers about spiritual journey in Christianity.Allegories are very popular rega rding that they are the simplest way to educate to and to confirm people’s belief system. An allegory is designed to illustrate a coherent doctrine, which exists outside the fiction. The important matters in allegory is not the adequacy, coherence, or consistency of the doctrine which is being illustrated in the story, however, it is how the words deals with the belief system.Everything in the story bears the point of reference to more specific aspect of the controlling doctrine, which the fiction is illustrating. Allegories do tend to be philosophical, also has its nature of affecting readers psychologically, and Bunyan has succeeded using this convenience to help the readers understanding the spiritual gloom. His success can be measured by its part of metaphorical personifications, which have entered the public vocabulary, as he used these encounters: Slough of Despond, Apollyon, Giant Despair, and Ignorance.Thus we can conclude that Bunyan has been triumphant in playing wi th images and allegory through out the novel effectively. Reference: Henri Talon, John Bunyan: The Man and His Works . Harvard University Press, 1951. Robert G. Collmer, Bunyan in Our Time . Kent State University Press, 1989. Vera Brittain;, Valiant Pilgrim: The Story of John Bunyan and Puritan England . Macmillan, 1950. Galen K. Johnson, â€Å"The Portable Bunyan: A Transnational History of the Pilgrim's Progress . † Christianity and Literature, (2005):