Purification of Alcohol Dehydrogenase From Bovine Liver

Purification of Alcohol Dehydrogenase From Bovine Liver Jekathjenani Ratnakumaran Namrata Verma Introduction: In the world of chemistry, there are millions of enzymes, but in this lab the enzyme used is bovine alcohol dehydrogenase. This enzyme occurs in various mammalian tissues, but generally found in high concentrations in the organs such as liver and kidney. According to its name Bovine alcohol dehydrogenase, which implicates that it is collectively formed from bovine (cow), alcohol and the enzyme dehydrogenase. The protein was extracted from the liver of bovine. Alcohol is an organic compound which contains carbon atom (single bonds) and hydrogen atoms. This alcohol is available in various forms of liquid and used for a variety of purposes. According to its properties, alcohol is a hydroxyl group which has a sweet odor similar to fruit. Alcohol are further divided and identified into different groups and also they are polar. As they possess hydrogen bonding they have higher boiling points. Dehydrogenase is a type of an enzyme which oxidizes a substrate by a reduction reaction that trans fers one or more hydrogen H- to the electron acceptor which is NAD+ /NADP (nicotinamide adenine dinucleotide) or FAD Flavin coenzyme (Shibusawa et al, 2004). Collectively, it forms alcohol dehydrogenase, which is ADH persuaded by ethanol and acetaldehyde as they relate to carbon catabolite repression. It is also zinc containing enzyme which is activated by glutathione and EDTA, which contains heavy metals (Pateman et al, 1983). Many organisms contain an alcohol dehydrogenase enzyme which catalyzes the NADPH dependent of aromatic and aliphatic aldehydes into subsequent alcohols and catalyze the reduction of glyceraldehyde to glycerol (Arslanian et al, 1971). Alcohol dehydrogenase contains a several isozymes which catalyze the oxidation of primary and secondary alcohols to convert into aldehydes and ketones (Arslanian et al, 1971). The molecular weight of this enzyme is 39677.13 Da and it is made up of 374 amino acid sequence. The monoisotopic mass of this enzyme is 39651.32 and its p H value ranges between 8.6 to 9.0 with an extension coefficient of 12.6 and an isoelectric point at 5.4, its theoretical pI is 7.46.The alcohol dehydrogenase is also known for its battle against alcohol , its toxic molecules which negotiates with the nervous system so, the body organs which consist of high toxic of alcohol are liver and stomach which converts alcohol to acetaldehyde which is even more toxic substance and further it is conversted to acetate which is utilized by the cells present within our body (Goodsell et al, 2001). So overall, alcohol dehydrogenase converts potentially dangerous molecule into food ustilized by the cells preent within the body.In human bidy, alcohol dehydrogenase can create upto nine different kinds of alcohol dehydrogenase each having different properties. For example in liver beta3 enzyme(Goodsell et al, 2001).These each enzyme is formed of two subunits and they can be mixed and match to create mixed dimerss which are more active.Alcohol dehdroge nates also modifies certain other alcohols with giving outcome of dangerous products such as methanol. These by products are converted into formaldehyde by help of alcohol dehydrogenase (Goodsell et al, 2001). Catalytic activity of alcohol dehydrogenase: NADPH + an aldehyde NADP+ + an alcohol Methods: In order to conduct this laboratory experiment, All the required apparatus and materials were provided during the lab. Certain precautions and safety rules were followed such as gloves, safety glasses and lab coat. This lab was conducted for about duration of 11-12 weeks. According to the article (Arslanian et al, 1971) most of the steps and procedure was followed. Purification of the enzyme was carried out by following up eight steps. Precautions were made while the experiment was performed. Equipments were rinsed with distilled water before starting the experiment. The reagent and buffer solution were prepared with distilled water. All procedures were carried out at 0-40C. Buffer Preparation: The first step involved in the buffer preparation. The stock solution, 0.1M Tris HCl at pH 7.6 was prepared by dissolving 12.14 g of Tris base with 1000 ml of distilled water and the pH was adjusted to 7.6 by adding diluted HCl. The Tris HCl buffer solution with different concentrations such as 10mM (pH 7.5), 40mM (pH 7.6) and 50mM (pH 7.5) were prepared by diluting the stock solution with distilled water. Sodium Chloride elutant buffer (0.16M NaCl) was prepared by dissolving 9.3504 g of NaCl with 1000 ml of distilled water. Preparation of Homogenate: The second step involved the preparation of the homogenate. The bovine liver was homogenized in a Waring blender in 90 ml of 0.32 M sucrose in 10mM Tris HCl buffer at pH 7.5. Approximately 27.39 g of sucrose was added in 250 ml of 0.01M Tris HCl to make 0.32M sucrose in 10mM of Tris HCl at pH 7.5. The homogenate was centrifuged at 15000 RPM for 30 minutes using centrifuge-Sorvall RC5 refrigerated centrifuge SS 34. Ammonium Sulfate Fractionation: Step three involved ammonium sulfate fractionation. The homogenate was 35% saturated and equilibrated with ammonium sulfate by dissolving 20.9 g of ammonium sulfate in 250 ml of distilled water. The supernatant was centrifuged at 15000 RPM for 30 minutes and the precipitate was discarded. Then, concentration was increased to 60% saturated ammonium sulfate by dissolving 16.4 g in 500 ml of distilled water. The suspension was centrifuged at 15000 RPM for 30 minutes. The obtained gray pellet was dissolved in 40mM Tris HCl buffer at pH 7.6. Then, the solution was dialyzed against 2L of 0.04M Tris HCl at pH 7.6 for 24 hours and again dialyzed with same buffer for another 24 hours. Performing DEAE-Sepharose Chromatography: The fourth step involved DEAE-Sephrose Chromatography. DEAE-Sepharose column that can hold up to 10ml volume was applied. The column was equilibrated by applying four times of 10ml of 40mM of Tris HCl, pH 7.6 buffer. About 10ml volume of centrifuged and dialyzed material was applied through the column. The column was washed with the same buffer (40mM of Tris HCl, pH 7.6) and then eluted by 40mM of Tris HCl with 50mM of NaCl. About 1 ml volume of twenty fractions of enzyme solution was collected using microfuge tubes. Enzyme Activity Assay: Fifth step involved measuring enzyme activity using a spectrometer. The enzymatic activity was initiated with 1mL of volume of blank solution containing 20  µl of distilled water, 10  µl 33mM of ethanol, 10  µl of 0.26mM of NAD+ and 960  µl of 0.1M of glycine buffer. Enzymatic assay activity was measured by taking total volume of 1000  µl containing 20 µl of enzyme solution, 10  µl 33mM of ethanol, 10  µl of 0.26mM of NAD+ and 960  µl of 0.1M of glycine buffer. The wavelength was set up at 340nm and measured using Cary 50s and 60s spectrometer. One unit of activity equals 1 µmol NADH produced per min based on the absorption coefficient of 6220 mol/l/cm for NADH at 340 nm. The above procedure was repeated for kinetic analysis and the range of ethanol concentration used was 20 to 25 mM. The observed data were fitted using Lineweaver -Burk kinetic plots. Gel filtration: The sixth step involved gel filtration. The enzyme was precipitated by 62% saturated ammonium sulfate and dissolve in 10ml of 50 M Tris-HCl, pH 7.5. The suspension was centrifuged for 20 min at 10000 RPM. Then, the column of Sephadex G-50 was run with 10 ml of enzyme solution. The column was equilibrated and washed with 50 M Tris- HCl buffer, pH 7.5. Then, the column was eluted by 50mM of Tris HCl with 50mM of NaCl. Around 10 fractions were collected at the rate of 1 ml/min in a microfuge tube. The highest highest specific activity fractions were precipitated by 62% saturation with ammonium sulfate. In the final step, the enzyme was redissolved in 5 ml of same buffer and apply to the column of Sephadex G-50 under the same conditions. Again, the highest specific activity fractions were precipitated by 62% saturation with ammonium sulfate. Performing CM-Sephadex chromatography: The precipitated enzyme was dissolved in 1 ml of potassium phosphate buffer contain 0.02M of NaCl, pH 7.0 and the enzyme solution was dialyzed against the same buffer for 2 hours. 10ml of non diffusible material was applied to a CM-Sephadex column. Then, the column was equilibrated with the same buffer. Then, the enzyme was eluted from the column with two column volumes of 0.16 M of NaCl (20ml). 10 fractions were collected and precipitated with 62% saturated ammonium sulfate. Bradford Assay: The eighth step involved Bradford Assay. The data (absorbance) observed from Bradford Assay Standards was used for calculating the mass of BSA in  µg. The final step used in this experiment was SDS PAGE method. About 20 µl of enzyme with loading buffer was loaded on the gel and by observing the gel, the mass of the protein was calculated. Results: Bovine Alcohol Dehydrogenase (ADH) was purified by following up few methods. The experimental results were observed and recorded for appropriate methods. Using DEAE Sepharose Chromatography, fractions were collected and all the fractions were appeared colorless. The enzymatic assay activity was measured at 340nm using spectrometer. The figure 1 indicates that the enzyme activity was increased by absorbing the NADH. The highest specific activity was selected based on the graph obtained in the enzyme kinetic activity. However, this method failed, resulting no increased activity. The enzyme kinetic activity had done for all the fractions, but none of them shown the accurate result. The graph obtained from the spectrometer does not show the increased activity of the enzyme to conclude the presence of protein. The result of the enzyme activities of collecting fractions was shown in figure 5, 6, 7, 8 and 9 respectively. However, the Bradford assay method was performed and the absorbance of the standards and the enzyme were recorded in the following tables. Based on these values, the graph of standard curve of absorbance versus mass of BSA was plotted. Table 1: The following table represents the recorded values of absorbance at 540 nm and calculated the mass of the BSA using the Bradford assay method Figure 1: Represents the enzymatic activity obtained from the purified protein ADH after ammonium fractionation method had performed and the peaks are showing that activity is increased. The above plot was obtained at 340 nm using Cary 50s-60s spectrometer and it was run as two parts for 4 minutes. Figure 2: Represents the standard curve of A595 versus mass of ADH protein obtained from the Bradford assay method. From the slope value obtained from the curve, the mass of the ADH protein was calculated. The mass of the protein calculated from the figure 2 is 4.766  µg and concentration of the protein is 4.766  µg / 25  µl SDS-PAGE method: Table 2: Represents the recorded values of SDS-PAGE method for the determination of molecular weight of the ADH purified protein Protein Molecular weight (Dalton) Log (Molecular Weight) Mobility (cm) Strand 1 60000 4.778 4.8 Strand 2 50000 4.699 5.1 Strand 3 40000 4.602 6.5 Strand 4 25000 4.398 7.0 Strand 5 20000 4.301 9.3 Figure 3: Represents the SDS-PAGE analysis of purified protein bovine Alcohol Dehydrogenase (ADH). The graph was plotted with log of molecular weight versus mobility of protein based on the SDS-PAGE values. Figure 4: Represents the single band on an SDS-PAGE gel (9th lane). This figure shown the proof of the protein ADH present in the enzyme solution and mass of the protein was calculated based on the obtained SDS-PAGE results. From the figure 3 and 4, the mass of the protein calculated is 345143.74 Da Figure 5: Represents the enzymatic activity obtained in the fraction 9th of the purified protein ADH and the peaks are obtained at 340 nm using Cary 50s-60s spectrometer. Figure 6: Represents the enzymatic activity obtained in the fraction 10th of the purified protein ADH and the peaks are obtained at 340 nm using Cary 50s-60s spectrometer. Figure 7: Represents the enzymatic activity obtained in the fraction 12th of the purified protein ADH and the peaks are obtained at 340 nm using Cary 50s-60s spectrometer. Figure 8: Represents the enzymatic activity obtained in the fraction 13th of the purified protein ADH and the peaks are obtained at 340 nm using Cary 50s-60s spectrometer. Figure 9: Represents the enzymatic activity obtained in the fraction 14th of the purified protein ADH and the peaks are obtained at 340 nm using Cary 50s-60s spectrometer. Discussion: According to the experimental study, the outcome results were not satisfying, so overall the experiment was not successful it failed. Based on the SDS-PAGE, ADH purified protein was not much visible clearly on the gel. Proteins are viewed as bands. SDS-PAGE results indicates that smaller protein molecules are at the bottom of the gel and larger molecules are at the top of the gel. This is showing that SDS-PAGE gel separate the protein molecules based on the size and mass of the protein. Most of the protein bands are viewed in between the molecular weight, 100 kDa and 30 kDa. Determining mass and purifying the protein, Bovine Alcohol Dehydrogenase using the Bradford assay and SDS-PAGE procedure was conducted successfully using this experiment. The result obtained in the SDS-PAGE and Bradford Assay are differ from the standard value and the concentration of the protein was determined using these methods. Based on the molecular mass on the EXPASY website, the standard molecular mass of the ADH protein is 39677.13 Da. The experimental mass of the ADH protein is 345143.74 Da. The mass difference is a large number. This could occur due to the experimental errors. The experimental errors can be avoided by handling equipments and following the instructions in a proper manner. Predicting the protein band on SDS-PAGE gel could cause the error. Moreover, the purification method such as DEAE Sepharose Chromatography was performed to test the enzyme activity of the protein. The obtained results are shown as a figure 5, 6, 7, 8 and 9 in the results section. The overall results obtained in these figures indicated that the experiment was not turned successful. The figure 5, 6, 7, 8 and 9 shown that enzyme activities are decreasing and wiggling. They are not constantly increasing or decreasing. Therefore, it was concluded that the purification of the enzyme was not turned positive and it could be due to the human errors occurred while conducting the experiment. This could be po ssible due to various reasons such as, during measurements for making the solution at the very beginning may be the concentration required was not appropriate, due to human error it was not properly mixed. It could also be possible that while grinding the liver , certain chunks of the liver were still not properly collected due to which the amount of liver used was not effective to obtain supportive and positive results.The variability of the results presented here is loss of certain atoms during the process of purification as their was no enzyme activity observed.The substrate studies of the alcohol dehydrogenase isolated from the bovine liver have demonstrated the hydrophobic site for binding alcohol (Arslanian et al, 1971). The article mentioned that the buffer that has a low ionic strength is used for the enzyme adsorbtion which caused the incomplete deactivation of enzymes and it was proven evidently (Arslanian et al, 1971). Moreover, as there is no enzyme activity measured in step 5 (DEAE- Sephrose Chromatography), the gel filtration and CM-Sephadex Chromatography method was not performed for this study. The enzyme purification might get succeeded if the study has performed these two methods. The article mentioned that gel filtrations on Sephadex G-100 has successive ability to separate the enzyme from non enzyme protein (Arslanian et al, 1971.) For further studies, more information is required before conducting the study as well as the time allotted was less, due to which it could suggest certain results and test were not done at the appropriate time. In conclusion, the study was conducted by following the method listed in the article. This studys report discussed the properties and successful method for the purification of enzyme, Bovine Alcohol Dehydrogenase. Even though article procedures were followed, errors occurred which resulted in deviations in results. However, the methods of Gel filtration and CM Sephadex Chromatography where successive but could not be conducted in this lab because the enzyme activity was limited after DEAE Chromatography was performed. More caution should have gained while conducting the experiment. It is emphasized that further research on enzyme purification method could improve the results and find success in the study. Appendix: Sample calculation 1: Volume of one microliter= 0.001mL Volume of 20 microliter= (0.001 ml x 20  µL) / 1  µL = 0.02 ml Therefore, mass of protein in 1mL of stock solution= 0.10 mg Mass of protein in 0.02 ml of stock solution = (0.10 mg x 0.02 ml) / 1 ml = 2 x 10-3 mg To convert mg to  µL, multiply by 1000, Mass of protein= 2 x 10-3 x 1000 = 2  µg Absorbance of the ADH purified Protein, y = 0.2544 Slope Line of equation: Y=mx+b Y= 0.0505 x + 0.0137 0.2544 = 0.0505 x + 0.0137 The mass of the protein, x = (0.2544 0.0137) /0.0505  µg = 4.766  µg Concentration of the protein, C = mass/ volume = 4.766  µg / 25  µl = 0.19  µg/  µl Total mass that recovered= Conc. X Total volume = 0.19 x 1000  µl = 190.64  µg SDS- PAGE method: Absorbance of the ADH purified Protein, y = 0.2544 Slope Line of equation: Y= mx+b Y= 6.0902 x + 33.982 0.2544= 6.0902 x + 33.982 X= (0.2544 33.982) / 6.0902 = 5.538 The mass of the protein = 105.538 = 345143.74 Da References: Arslanian,M.J., Pascoe,E,. and Reinhold,J.G., (1971) Rat Liver Alcohol Dehydrogenase.Dept. of Biochem.School of Medicine, American University of Beirut.125,1039-1047. Alcohol Dehydrogenase(ADH)The university of Minnesota Biocatalysis/Biodegradation Database.Calzyme. Lab.inc. Shibusawa,Y.,Fujiwara,T.,Shindo,H., andIto,Y. (2004) Purification of alcohol dehydrogenase from bovine liver crude extract by dye-ligand affinity counter-current chromatography, J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci.799(2):239-44. Pateman,J.A., Doy,C.H.,Olsen,J.E.,Norris,U., Creaser. E.H., and Hynes,M.(1983) Regulation of Alcohol Dehydrogenase (ADH) and Aldehyde Dehydrogenase (ALDDH) in Aspergillus nidulans.Proceedings of the Royal society.Bio.Sci.217, 243-264. Ward,W.W., and Swiatek,G.,(2009) Protein purification.The state University,, Scool of Environmental and Biology Science,Department of Biochem. And Microbio.76,1- 21. Goodsell,D.(2001) Alcohol Dehydrogenase.Molecule if the month. RCSB.Protein Data Bank.doi: 10.2210.

Energy Efficiency: Directives and Legislation

Energy Efficiency: Directives and Legislation 2.1 Introduction The debate is ongoing, but there is now overwhelming scientific evidence that mans activities are causing significant climate change. Climate change has the potential to affect all aspects of life on earth and will have major detrimental social, economic and environmental impacts. The best response to these challenging issues is to change. Change the way we think. Change the way we act. (Get source) 2.2 Background to Directives for Climate Change The International climate change agenda containing the Directives and Legislation that drives for energy efficiency began in 1992 with the United Nations Framework Convention on Climate Change (UNFCCC). The objectives of the UNFCCC were to: stabilise the atmospheric greenhouse gases at a level that would prevent dangerous interference with the climatic system, to be achieved in a time frame to ensure food production is not threatened and to enable economic development proceeds in a sustainable manner. The UNFCCC is the parent treaty of the Kyoto Protocol (1997) which was developed to implement the UNFCCC effectively and properly. (www.euroace.org/reports) Irelands relation to the Kyoto Protocol is outlined in the subsequent sub-chapter. In December 2007, the latest climate change conference took place in Bali, Indonesia and it included representatives of over 180 countries. The two week period included the sessions of the Conference of the Parties to the UNFCCC, as well as the meeting of the Parties to the Kyoto Protocol. The ‘Bali Roadmap was adopted from the conference which charts the course for a new negotiating process to be concluded by 2009 that will lead to a post 2012 international agreement on climate change. The next meeting of the parties to the climate change convention is scheduled to take place on December 2008 in Poland. After the Kyoto Protocol was established, Europe needed to take action to succeed in cutting its greenhouse gas emissions to 8% below 1990 levels by 2008-2012, as required by the Kyoto Protocol. This action was taken by launching the European Climate Change Programme (ECCP) in June 2000 which was then ratified in October 2005. The main goal of the ECCP was to develop all of the necessary elements of an EU strategy to implement the Kyoto Protocol. From this European Climate Change Programme, the Energy Performance of Buildings Directive (EPBD 2003) was developed. This is explained in chapter 2.4 of this text. (www.euroace.org/reports) In order for Ireland to meet its Kyoto target of limiting the increase of greenhouse gas emissions to 13% above 1990 levels by 2008-2012, a National Climate Change Strategy was implemented. 2.3 Ireland and the Kyoto Protocol The Kyoto Protocol was adopted to tackle the threat of climate change. It contains legally binding greenhouse gas emission targets for developed countries for the post 2000 period. The Protocol promises to move the international community one step closer to achieving the Conventions (UNFCCC) ultimate objective of preventing man-made interference with the climate system. As a first step towards tackling the threat of climate change, the United Nations Framework Convention on Climate Change (UNFCCC) required developed countries to put in place policies and measures with objectives of returning emissions of greenhouse gases to 1990 levels by the end of the decade. However, in recognition of the need to take more substantial and urgent action, industrialised or developed countries committed to reduce their combined emissions of greenhouse gases by at least 5% compared to 1990 levels by the first commitment period 2008-2012. The protocol came into force on 16 February 2005. As of November 2007, 174 parties have ratified the protocol. Of these, 36 developed countries are required to reduce greenhouse gas emissions to the levels specified for each of them in the treaty. The EU has an overall reduction target of 8% below 1990 levels and has agreed a burden sharing agreement that recognises the different economic circumstances of each member state.   Irelands target is to limit the increase in its greenhouse gas emissions under the Kyoto Protocol to 13% above 1990 levels by 2008-2012. To date Ireland has struggled to get on target and at this stage looks unlikely to meet the 13% figure. With the help of the National Climate Change Strategy and the Protocol flexible mechanisms, this target may yet be achieved. The National Climate Change Strategy 2007- 2012 provides the national policy framework for addressing greenhouse gas emission reductions and ensuring that Ireland meets its target for the purpose of the Kyoto Protocol. Ireland may achieve their individual targets through domestic actions and use of flexible mechanisms provided for in the Protocol. The Government has decided that it will use the Kyoto Protocol flexible mechanisms to purchase up to 3.607 million Kyoto Units in each year of the 2008-2012 period. (www.environ.ie) 2.3.1 Kyoto Protocol Flexible Mechanisms / Emissions Trading An important part of the Kyoto Protocol was the introduction of three flexible mechanisms to reduce the costs of achieving emission reductions for the member states with emission reduction or limitation targets. The mechanisms enable Parties to purchase Kyoto Units from other Parties or to invest in cost-effective opportunities to reduce emissions. While the cost of reducing emissions varies considerably between projects and between countries, the effect for the atmosphere of limiting emissions is the same no matter where the action occurs. The three mechanisms are outlined below: Joint Implementation (JI) This is provided for under Article 6 of the Protocol, and enables Parties with reduction commitments to implement projects that reduce emissions in other member states with reduction commitments, in return for credits. The tradable unit under the JI mechanism is an Emissions Reductions Unit (ERU). Clean Development Mechanism (CDM) This is provided for under Article 12 of the protocol and enables Parties with targets to participate in projects that reduce emissions in those Parties that do not have targets under the protocol. This mechanism is aimed at developing countries. Credits generated using the CDM mechanism can be used by the investing Party for compliance purposes. The tradable unit under the CDM mechanism is a Certified Emissions Reduction (CER). International Emissions Trading This is provided under Article 17 of the Kyoto Protocol and enables Parties or member states that have a greenhouse gas emissions limitation or reduction target under the Protocol to acquire Kyoto Units from those Parties that have reduced their emissions beyond their target under the Protocol. The tradable unit under emissions trading is an Assigned Amount Unit (AAU). The National Treasury Management Agency is the designated purchasing agent for Ireland and will administer and manage purchases of Kyoto Units on behalf of the Government. A dedicated Carbon Fund has been established for this purpose. All purchases will be made in accordance with the following objectives: That they contribute to the ultimate objective of the United National Framework Convention on Climate Change That risk is minimised, particularly in relation to the timely delivery of credits That they represent good value for money The National Treasury Management Agency will use the following mechanisms to purchase Kyoto Units: Direct purchase of Kyoto Units from other Kyoto Protocol member states Direct investment in joint implementation and clean development project activities Direct market purchases of Kyoto Units Any surplus Kyoto Units held by the State at the end of the 2008-2012 commitment period can be banked and used in a subsequent commitment period of the Kyoto Protocol or any successor treaty. (National Climate Change Strategy 2007-2012, Department of Environment, Heritage and Local Government) Below is a graph illustrating the total greenhouse gas emissions for all sectors of all the member states up to 2005. As we can see, Ireland is somewhat off reaching its Kyoto target. 2.4 The Energy Performance of Buildings Directive (EPBD) 2.4.1 Introduction â€Å"Energy performance demands in the building sector within the EU range from rather demanding energy regulations and already established energy certification schemes in countries like Denmark and Germany, to the situation in countries like France and Spain with low regulation demands and without certification processes established at national level† (Casal, 2006). EU legislation and policies, implemented through the Energy Performance of Building Directive (EPBD), aim to provide a more uniform approach to implementing building energy saving measures and reaching Co2 emission goals. Each member state is required to translate and implement the policies and guidelines within the context of its legal and economic framework. The EPBD was enacted by the European Union in line with the Kyoto Protocol to: reduce European building energy consumption by 10 per cent by 2010 and 20 per cent by 2020; complete energy ratings of 2 million existing buildings by 2010; and cut Co2 emissions by 45 million tonnes by 2010 (Casal, 2006). The directive is the first move to target buildings specifically to reduce emissions and overall energy consumption in the construction sector. 2.4.2 Overview of the EPBD The EPBD is a legislative act of the European Union which requires member states to achieve particular results with respect to the energy performance of buildings. The directive 2002/91/EC (EPBD, 2003) of the European Parliament and Council on energy efficiency of buildings was adopted by member states and the European Parliament on 16th December 2002 and came into force on 4th January 2003. This directive is a very important legislative component of energy efficiency activities of the European Union designed to meet the Kyoto commitment. The directive concerns a large number of participants on all levels with different impacts and different motivations: designers, housing associations, architects, providers of building appliances, installation companies, building experts, owners, and tenants effectively all energy consumers in the European Union. It will greatly affect awareness of energy use in buildings, and is intended to lead to substantial increases in investments in energy efficiency measures within these buildings. The EPBD has created a great challenge for the transformation of the European building sector towards energy efficiency and the use of renewable energy resources. The 4th of January 2006 was the official deadline by which the 25 member states had to transpose the directive. 2.4.3 Objectives and Requirements of EPBD The objective of the EPBD is to improve the energy performance of buildings within the community, taking into account outdoor climate conditions as well as indoor climate requirements and cost effectiveness. The directive lays down requirements regarding: The framework for a methodology of calculation of the integrated energy performance of buildings The application of minimum requirements on the energy performance of new buildings The application of minimum requirements on the energy performance of large existing buildings that are subject to major renovation The energy performance certification of buildings The regular inspection of boilers, an assessment of the heating installation in which the boilers are more than 15 years old and an inspection of air conditioning systems in buildings The requirements for experts and inspectors for the certification of buildings, the drafting of the accompanying recommendations and the inspection of boilers and air conditioning systems. The requirements of each member state are set out in the EPBD under different articles. (EPBD, 2002) 2.4.4 Summary of Articles 2.4.4.1 Adoption of a methodology Each member state is required to have a method of calculating the energy performance of buildings. This calculation method can be set at a national or a regional level. This is an extract of the directive on article 3: ‘Member States shall apply a methodology, at national or regional level, of calculation of the energy performance of buildings on the basis of the general framework set out in the Annex. Parts 1 and 2 of this framework shall be adapted to technical progress in accordance with the procedure referred to in Article 14(2), taking into account standards or norms applied in member state legislation. This methodology shall be set at national or regional level. The energy performance of a building shall be expressed in a transparent manner and may include a CO2 emission indicator (EPBD, 2002) 2.4.4.2 Setting of energy performance requirements These minimum requirements shall be reviewed every five years. Some categories of buildings may be exempted from the requirements. These include: Protected buildings and monuments Buildings used as places of worship Temporary buildings Residential buildings intended to be used for less than 4 months of the year Stand alone buildings with a total useful floor area of less than 50m ² 2.4.4.3 Setting of energy performance requirements for new buildings Each member state will set minimum energy performance requirements for new buildings. For large new buildings with a floor area of over 1000m ² member states should consider alternative energy systems before construction starts. These include: Decentralised energy supply systems based on renewable energy CHP (combined heat and power) District or block heating or cooling, if available Heat pumps, under certain conditions The consideration of the alternative energy systems should take technical, environmental and economic feasibility into account. 2.4.4.4 Setting of energy performance requirements for existing buildings Each member state will ensure that when buildings over 1000m ² undergo major renovation that their energy performance is upgraded to meet minimum requirements. The minimum standards may be applied to the whole building or limited to the renovated part. 2.4.4.5 Energy performance certificate Each member state must ensure that when a building is constructed that an energy performance certificate is made available to the owner. When a building is sold or rented out an energy performance certificate must be made available to the prospective buyer or tenant. The certificate is valid for 10 years. For buildings over 1000m ² occupied by public authorities, an energy certificate must be placed in a prominent place clearly visible to the public. 2.4.4.6 Independent experts Member States shall ensure that the certificate of buildings, the drafting of the accompanying recommendations and the inspection of boilers and air-conditioning systems are carried out in an independent manner by qualified or accredited experts, whether operating as sole traders or employed by public or private enterprise bodies. (EPBD, 2002) Implementing EPBD in Ireland 2.5.1 Building Control Act 2007 The Building Control Act provides for the legal transposition of the EUs Energy Performance of Buildings Directive (EPBD) into Irish law. This will lead to energy efficiency becoming an important aspect of design concern for all buildings, both residential and non-residential. It is essential that the general public and companies involved in the industry understand the impact of the directive on residential and commercial property in Ireland. The Act requires that there will have to be mandatory building energy rating (BER) certificates for some buildings. This means that when a building is constructed, sold or rented out, the owner must provide a BER certificate to the prospective buyer or tenant. The BER will be accompanied by an advisory report setting out recommendations for cost-effective improvements to the energy performance of the building. This is further explained in chapter 3. ‘‘The successful implementation of the directive will require that systems are in place to guarantee the day-to-day delivery of assessment and inspection services by qualified people in a way that is consistent, practical and cost efficient, and with acceptable response times that maintain levels of service in the construction and property markets. (www.lkshields.ie/htmdocs/publications/newsletters) www.sei.ie www.epbd.ie http://www.euroace.org/reports/CIBSE_EUBD.pdf Casal, X.G. (2006), ‘‘Analysis of building energy regulation and certification in Europe: their role, limitations and differences, Energy and Buildings, Vol. 38 No.5, pp.381-92 Energy Performance of Buildings Directive 2002

Pillars of Metaphorical Ambiguity in The Scarlet Letter Essay -- Scarl

Pillars of Metaphorical Ambiguity in The Scarlet Letter Among the multiplicity of arcane elements hidden beneath the words in Hawthorne's "The Scarlet Letter", none is so apparent, yet strikingly subtle to the reader's perception and consumption of characterization than the allegorical play on words within the names of the characters.   Both the protagonist and her rival within the plot are blessed with conveniently appropriate, fitting names.   The four pillars supporting this novel are all cloaked with foreshadowing names, which silently clue the reader into what traits and significance the character holds as the story unfolds.   These pillars that solidify the novel are Hester Prynne, Roger Chillingworth, Arthur Dimmesdale, and Pearl.     Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   The first, possibly strongest column supporting the evolution of themes in the novel is Hester Prynne.   Hester is the young woman who is abandoned by her older, disfigured husband, and falls in love with a young, passionately God-fearing man who subsequently conceives a child, thus revealing her "adultery" and is punished by the Puritan society that he represents.   She is instructed to wear a red letter, hence the title of the book.   Through her punishment, she acquires and applies several motifs that the novel boasts, the most powerful one being represented perpetually throughout the story,   sin.   Apparently, in efforts to stress her significance and origin of decisions in the story, Hawthorne skillfully gave this woman whom the story revolves around the name of Hester Prynne, comfortably in sync with the word she is faced with constantly: sin.   Her last name, rhyming with the word is no mistake, and though subtle in its existence, is ingenious in its implication, and an almo... ...r Dimmesdale divulges the less than resplendent qualities the young minister displayed in his lack of resolve and spirit.   Finally, Pearl implies the costly, lamentable result of a debacle that was ironically conceived from affection and tender ardor. The intricate constituents of this endless metaphor of a novel would vaporize without concrete, stationary components that solidify the plot and stimulate its growth, each reactive and influential upon the other.   Nathaniel Hawthorne’s â€Å"The Scarlet Letter† would crumble into an insipid, low faceted pile of a couple plot twists, monotonous characters, juvenile prose, and a stack of aged papers from Hawthorne’s basement that would have never reached the new millennia without those four pillars of metaphorical ambiguity.   Work Cited Hawthorne, Nathaniel. The Scarlet Letter. Ed. Brian Harding. Oxford: Oxford 1990.

Discussion of Abortion Essay -- Social Issues Abortion Teen Pregnancy

Discussion of Abortion   Ã‚  Ã‚  Ã‚  Ã‚  Abortion is the surgical termination of a pregnancy. How odd that people are able to define something, that is such a controversial issue, so easily. There are hundreds, thousands, and even millions of things to say about abortion. When it comes to abortion, I find myself thinking like a symbolic interactionist. Abortion is a personal social issue and it needs to be seen on a micro level first. Although abortion can also be seen on a macro level, seeing abortion on a micro level lets people see the different symbols of abortion. No social condition creates the same symbol. If abortion is seen on a macro level, all the myths and stereotypes of abortion seem more realistic. For example, some of those myths and stereotypes being, most abortions are from minorities and most people who have abortions are teen girls. If abortion were seen on a micro level it would be evident that these myths and stereotypes are simply not true. Every abortion that occurs has a story beh ind it or a reason behind it. Many of us automatically assume that the person who had the abortion is immoral without even knowing the reason for why the abortion took place in the fist place. And this brings up a series of questions. When is an abortion considered moral or immoral? What should the legal status of abortion be? Should the father have a say if one should have an abortion or not? The answer to these questions are within a persons own mind and how they view this social condition. My answers to these questions are as follows.   Ã‚  Ã‚  Ã‚  Ã‚  I believe that when an abortion is considered moral or immoral all depends on the symbol. For instance, one of my family members had an abortion and I found her decision to have an abortion moral. The symbol behind my family members abortion is as follows. For the purpose of privacy I will refer to my family member as Kate and her first love as Sam. When Kate was fourteen years old, she met her first love. They both went to the same high school but Sam was two years older than Kate. Sam and Kate went out for the next two months and got closer with each passing date. Sam was one of the popular guys in school and could have any girl he wanted so Kate didn’t understand why he was setting his eyes on her. Kate was extremely flattered that Sam even considered going out with her. So as one of the popular guys in school and being... ... in our society for a long long time, maybe even forever. One to two percent of women who have had an abortion will have another one. Twenty percent of abortions are still back ally abortions and thirty nine percent of women that have back ally abortions become infernal. It is really hard for me to believe that there are one point five million abortions every year and that’s only counting the legal abortions. The number one thing that divides moral from immoral when it comes to abortion is the symbol. The symbol basically decides all opinions on abortion. If the symbol is of a woman with a life threatening pregnancy then people would consider this woman’s abortion moral. On the other hand, if the symbol is of a woman that just doesn’t feel like having a baby and wants to get an abortion then people would consider this woman’s abortion as immoral. Like I said before it all depends on the symbol. This is why, when it comes to abortion, I find myself thin king like a symbolic interactionist. There are just too many different symbols of abortion to think of it in a macro level. Symbolic interactionists are completely right when talking about abortion, well at least that’s my opinion.

Free College Essays - Hester as Role Model in Hawthornes The Scarlet Letter :: Scarlet Letter essays

The Scarlet Letter – Hester as Community Role Model â€Å"Woman, it is thy badge of shame!" (107). Governor Bellingham was describing the scarlet letter to Hester while they were discussing if the punishments that Hester had to go through were adequate enough for the crime. Hester was living in the outskirts of the city in a small abandoned cottage for several years with the only thing that had any monetary value in her life, her child and the product of committing adultery, Pearl. She and her little Pearl were shunned from the community for her acts. In the Scarlet Letter by Nathaniel Hawthorne, Hester is punished in more than one way, and she is able to deal with it openly so the community will, over time, forgive her. The most obvious subject of punishment that Hester had to cope with is wearing the scarlet letter. "By the point which drew all eyes and, as it were, transfigured the wearer. . . was the scarlet letter, so fantastically embroidered and illuminated upon her bosom" (51-52). Hester wrought the scarlet letter before she stood on the scaffold. When Pearl asks her why she wears the letter she replies that she wears it for its gold thread. Hester wears the letter for many years, even after the people in the community care anymore, so that she will be fully forgiven for her sin. In the beginning of the story, Hester is faced with serving the temporary part of her sentence, standing on the scaffold in front of the whole town. "It was a circumstance to be noted, on the summer morning when our story begins its course, that the women of whom there were several in the crowd, appeared to take a peculiar interest in whatever penal infliction might be expected to ensue" (48). The citizens of the town had gathered to criticize Hester as she stood on the scaffold, and many of the town’s women were discussing the simplicity of Hester’s sentence, since the usual punishment for committing adultery is the death penalty. Although she had to put up with the remarks about her for three hours while she was standing on the scaffold, the ridicule followed for many years to come. Hester and her daughter were thought upon as sinners long after Hester had served her sentence.

Good Hair

There are three major beliefs that hinder Alice Andrews’s life progression; however one belief hinders it the most in the novel and this is shown in three ways. Alice is young, Black, bourgeois, and in search of everything, as her life unfolds her beliefs and past are challenged. First, Alice maintains the belief that it is important to look the part and not be labeled as low class or impoverished. This is relevant because she becomes a prisoner in a world of pretense. Second, Alice believes she should she should hid the truth about where she comes from and lives her life deeply ashamed of her blue collar New Jersey roots. This is relevant because it causes her to lose herself identity. Third, Alice struggles to fit in the upper echelon class she surrounded herself by and Alice Andrews’s belief that it is necessary to look the part and not be labeled as low class hinders her life progression because she becomes a prisoner in a world of pretense. Based on what she believes about social status, it appears that â€Å"good hair† is a standard of beauty and status amongst the upper echelons of the African American social order in Manhattan. There was pressure from society to promote this standard of beauty in the novel. More specifically, social status and beauty standards are doubtless dependent to a certain extent on physical appearance. As Alice struggled to look the part in order to be accepted, it became clear that the standards of beauty had a significant burden on her notion. In society, African Americans continue to struggle with the concept of good hair. I believe that the lack of proper cultural images and the negative social scripts caused by mainstream media play a significant role in the perception of beauty. Another reason why Alice Andrews’ struggle to maintain an image caused her life progression to be hindered is because she became a prisoner in a world of pretense. Since her entire foundation was built on the claim that

Communication in nursing Essay

Introduction. Communication is a necessary skill to have in the nursing profession. We use communication to exchange information between patients, co-workers and all the people around us (Kozier et Erb 2004). No matter the form of communication we are constantly presenting and interpreting people’s ideas and thoughts. Communication is a method we utilize to connect the gap between knowledge and comprehension (Kozier et Erb 2004).This paper will discuss an observation of the personal care home environment, modes of communication such as; verbal and non-verbal, effective communication methods and barriers to communications. Observation Of Personal Care Home Environment. I visited an urban personal care home in the inner city. I will be identifying this personal care home as PCH â€Å"X†. I walked into the facility and it was very clean and spacious. I conducted my observation during lunch time in the dining room. This area was poorly lit and the temperature was very humid. There was also music playing in the background during mealtime. This personal care facility was very respectful, kind and caring to the residents in their care. The communication I observed in general was very clear and concise. Verbal and non-verbal communication. Verbal communication is a mode of communication that uses both written and spoken words (Kozier et Erb 2004). Here is an example of verbal communication: At PCH â€Å"X†, a nurse was giving medication to a resident and he introduced himself. The resident did not respond to the nurse so the nurse approached him in a different manner. He crouched down to the resident’s level and spoke to him again in a calm voice and lightly touched his shoulder. The resident later complied with his directions to take his medication. The nurse took a different approach with his verbal communication by speaking softly. Verbal communication is largely used due to the fact that people usually chose the words they use (Kozier et Erb  2004). Another mode of communication is non-verbal communication. Non-verbal communication involves other forms of communication such as gestures, facial expressions, and touch (Kozier et Erb 2004, page 423). Most people who use verbal communication use various forms of non-verbal communication simultaneously. The nurse in the example above used non-verbal communication, when he crouched down to the resident’s level and lightly touched his shoulder. This second example involves non-verbal communication: A Health Care Aide was about to assist a resident with lunch. The resident was not aware of his environment and had difficulty feeding himself. The Health Care Aide asked the resident to open his mouth but he did not respond. As a result, the Health Care Aide opened his own mouth and the resident mimicked him and they were able to continue feeding. Effective communication methods. Effective communication is a very important aspect in a nursing career. Here is a summary of some effective communication strategies that I have observed at PCH â€Å"X†: *Health care workers appeared to be neat and tidy wearing uniforms. This will convey a non-verbal communication method of professionalism. Clothing and apparel can be a source of information about a person (Kozier et Erb 2004). *A staff member asked a resident how their lunch tasted. The staff member paraphrased what the client was saying and reinstated the resident’s feelings. This showed the resident that the staff member was actively listening (Kozier et Erb 2004). *Health care workers seemed interested in what clients had to say and differentiated the real from unreal (Kozier et Erb 2004). Health care workers talked to residents about daily activities. One client stated â€Å"I have to go to work; my daughter will be picking me up soon†. The nurse responded â€Å"your daughter will be here to visit but you are retired and you  do not have to work anymore.† These are a few effective communication strategies I have witnessed at PCH â€Å"X†. Communication promotes understanding and can build a constructive relationship with clients and co-workers. As nurses we must be aware of what we say and how we act towards others. Barriers to communication. It is critical to be aware of the good communication methods as well as the bad communications methods. This particular personal care home was very respectful to clients. However, here are some barriers to communication I observed when I was at PCH â€Å"X†: *One resident had a language deficit and had difficulty speaking. The resident was calling out and it seemed as though she wanted something. It was difficult for the staff member to understand what she was saying. He overcame this barrier by asking her yes and no questions until he finally solved what she was trying to say. *Residents were being talked to like children. The clients were often referred to as â€Å"dear, sweetheart, or darling†. During lunch, I also overhead someone saying â€Å"we have to change you diaper you made a pooh-pooh†. This may demean a client however; this communication barrier can be resolved by calling residents by their name and by respecting their dignity as a person. *Residents can also be hard of hearing. A nurse was talking to a resident with his back turned asking him questions and he did not reply back. She continued to speak louder and the resident later asked why she was yelling. The nurse should have faced the client while speaking to him so he could realize that he was being spoken to. Nurses need to recognize the barriers to effective communication. Barriers make communication become non-therapeutic. Non-therapeutic strategies to communication can be detrimental to a healing relationship. Conclusion. In conclusion, communication is extremely important especially to a nurse. To properly assess clients’ needs we must be able to communicate with them verbally and non-verbally. If we can identify the barriers to communication we can find effective strategies to resolve them. We have to be clear when we communicate to our patients. A misinterpretation of instructions, plans, and vital information can be harmful to a client’s health and well being. Caring and comforting is an important aspect of nursing and that can only be demonstrated through effective communication techniques. References Kozier & Erb, Barbara, et al. Fundamentals of Nursing. 7th ed. Upper Saddle River, New Jersey: Pearson Prentice Hall, 2004.