Monday, January 27, 2020
Impacts on Quality of Life Among School Children
Impacts on Quality of Life Among School Children PAVITRA A/P INDRAJOTHY INTRODUCTION It is the truth to say that children are our future and if that is true, more time and energy should be put into the beginning stages of each childs life. The early years of a child’s life are the most important in terms of their general well being, their emotional and social development, and their physical, intellectual and emotional growth. Children develop at different paces and a very high proportion of what they learn takes place in the first five to seven years of life. What happens in the home is extremely important to development in early childhood. There are things that can and should be done to help each child live a life that is productive and beneficial to those that they will interact with in their future. There are many aspects in todays world that make the quality of life of school children better and meaningful. For instance of aspects that can affect quality of life are obesity, bullying, lifestyle, emotional problems, divorce, socio cultural home environment, child abuse, oral health and back pain. First and foremost, there are reports saying that obese children demonstrate more negative self-perceptions, decreased self-worth, increased behavioural problems, lower self-esteem and lower body esteem and perceived cognitive ability. However, because of methodological limitations, definitions of obesity that differ with current recommendations and with non-validated measurements of health related quality of life, it is difficult to generalize the conclusions of these studies (Friedlander et al., 2003). According to research done by Nathan Woon in year 1981, Peninsular Malaysia is a multi-racial country of 12 million people, 41% of whom are less than 14 years of age (Vital Statistics Peninsular Malaysia 1975-Department of Statistics, Malaysia). The population consists of three main ethnic groups: the Malays (53%), the Chinese (35%) and the Indians (11%) (Vital Statistics, Peninsular Malaysia, 1975). Whilst in recent years, there has been an increasing tendency for the various ethnic groups to intermingle and to respect and understand the various cultures and traditions that coexist within the country, in the specific area of child rearing, as with marriages, deep-rooted traditions and customs continue to remain a crucial factor and probably contribute, to some extent, to the pattern and incidence of child abuse in the various races. Bullying has been conceptualized globally as acting in any way that threatens or hurts someone less powerful. Furthermore, bullying also can be distinguished among the different way in which aggressive acts are performed. There are different types of bullying which is physical, verbal, or indirect bullying. Furthermore, distinctions among aggressor’s possible aims can also be made. For instance, bullying physically to hurt the victim or for damage the victim’s relationship with others (Rigby, 2003). Next, parental separation is usually the first major and drastic change in a child’s life. This distressing event drastically alters the family’s future, bringing to a sense of loss due to interference in daily routines as well as absence of daily contact with both parents (Eymann et al., 2009). Lastly, obesity, dental caries and periodontal diseases are among major public health concerns which may affect children’s growth and development. Sedentary lifestyle and unhealthy eating habits could lead to poor oral hygiene statuses and increase the tendency to develop periodontitis and obesity at young ages (Anand et al., 2014). DISCUSSION OBESITY Worldwide estimates of childhood obesity are as high as 43 million, and rates continue to increase each year. Childhood obesity is a growing problem in the present era and it causes serious consequences in the later years (Anand et al., 2014). According to Friedlander et al., childhood obesity is a major public health problem with increasing prevalence; approximately 30% of US school-aged children are defined as overweight or at risk for overweight. Anand and Friedlander states that overweight children are more likely to suffer from a range of chronic health problem such as cardiovascular disease, diabetes mellitus, hypertension, sleep apnoea and asthma. They also concluded that children who are overweight are more likely to become overweight adults. There was a study conducted by Friedlander et al. among 371 students aged 8-10 years old excluding those who had sleep apnoea or who were born prematurely. Based on BMI categories of the study, 17.5% of the children were overweight, 12.4% were at risk for overweight, 8.1% were underweight, and 62.0% were of normal weight. Moreover, children who are overweight have a 2 to 4 times increased odds of having low scores for physiological health, self-esteem, and physical functioning. Similarly, children who are at risk for being overweight have higher odds of having low physical functioning scores. Furthermore, lower self-esteem also was observed in children who are underweight. Children with higher BMIs are more likely than normal weight children to be perceived by their parents as having feelings of anxiety and depression, of exhibiting aggressive or immature behaviour, as having role limitations in their schoolwork and social activities, and as having low self-esteem. And, their parents also report higher levels of emotional distress and little personal time because of their children’s health or behaviour (Friedlander et al., 2003). As a suggestion, if the child or teenager is overweight, further weight gain can be prevented. Parents can help their children in maintaining their weights in the healthy range. In infancy, breastfeeding and delaying introduction of solid foods may help in preventing obesity. In early childhood, children should be given healthy, low-fat snacks and they should be allowed to take part in moderate-vigorous physical activity every day. Older children can be taught to select healthy, nutritious foods and to develop good exercise habits (Anand et al., 2014). BULLYING Bullying invariably involve an imbalance of power in which the aggressor is more powerful than the victim. Moreover, bullying occurs when there is conflict between people of unequally power (Rigby, 2003). As identified by Rigby in year 2003, four categories of negative health conditions are low psychological well-being, poor social adjustment, psychological distress and physical unwellness. Low psychological well-being includes states of mind that are generally considered unpleasant but not acutely distressing, such as general unhappiness, low self-esteem, and feelings of anger and sadness. Next, poor social adjustment normally includes feeling of loathing toward one’s social environment, evident through expressed dislike for workplace or school, manifest loneliness, segregation and absenteeism. Moreover, psychological distress is considered more serious than the low psychological well-being and poor social adjustment categories and comprises high levels of depression, anxiety and suicidal thinking. Last but not least, physical unwellness can determine by precise signs of physical disorder, evident in medically diagnosed illness. Furthermore, psychosomatic symptoms also can be incorporated in this category. Various strategies or treatment should be considered and give a thought to reduce the chances of a child’s further severe involvement in bullying that can worsen the child’s condition. The approach include helping victimized children to evolve self-protective assertiveness skills and working therapeutically with bullying children to create a greater awareness of the repercussion of their antisocial behaviour (Rigby, 2003). LIFESTYLE Television is still the most widely-viewed screen worldwide. Globally, people are spending more time sitting at work and at home, and there’s mounting evidence that this â€Å"sit time†is a major contributor to the obesity epidemic. Sedentary activities not only TV watching, but also working at desk jobs, using computers, playing video games and driving cars. Increasingly, there’s evidence that watching TV and, especially, watching junk food ads on TV promotes obesity by changing mainly as to what and how much people eat (Anand et al., 2014). Too much television viewing among children has been linked with inadequate study patterns. Inappropriate television-viewing has been linked to erratic sleep/wake schedules and poor sleep quality, violent or aggressive behaviour, substance use, sexual activity resulting in decreased school performance or even school drop-out (Karande Kulkarni, 2005). EMOTIONAL PROBLEMS Conditions which cause emotional problems in children such as chronic neglect, sexual abuse, parents getting divorced or losing a sibling might cause long term distress resulting in academic underachievement. Besides that, children can face severe emotional upheavals during the treatment of chronic health impairments such as asthma, cancer, cerebral palsy, congenital heart disease, diabetes mellitus, epilepsy, hemophilia, rheumatic diseases, or thallasemia, resulting in low self-esteem and loss of motivation to study (Karande Kulkarni, 2005). As recommended by Karande Kulkarni in year 2005, children with emotional problems need counselling sessions with a child psychologist or a child psychiatrist. Depending on the severity, at times, appropriate medications (anxiolytics, antidepressants) may be needed. DIVORCE As emphasizes by Eymann et al. in year 2009, divorce is becoming increasingly common in our society, seriously affecting all parties involved, especially children. There were studies conducted in USA and in Europe and roughly calculated that 30 to 50% of children will suffer the effect from a divorce, almost same rates have been reported in Argentina. Eymann et al. conducted a study at the Hospital Italiano, Argentina. A total of 330 families were invited to take part and only 313 completed the questionnaire; 160 from married family and 153 from divorced family. They defined divorced family (DF) as parents who choose to stay at separate residence regardless of legal marital status while married family (MF) as parents who choose to share residence with their children regardless of legal marital status. The aim of this study was to evaluate psychological quality of life among school aged children of divorced parents by using Child Health Questionnaire – Parent Form 50 (CHQ-PF 50). The results showed that CHQ-PF 50 scores were higher in children of MF than in those of DF for various domains, such as self-esteem, time impact on family, mental health, behaviour, social-emotional functioning and behaviour, and lastly the psychosocial summary score joining all these factors. As proved in the results, boys had poorer psychosocial quality of life than girls. This was explained perhaps by the effects of less daily father-child connection on gender-specific identity traits. Furthermore, it is well known that separated mothers who have legal child custody usually suffer a greater slope in their standard of living. Norton Glick and Duncan et al. have clearly proved the unfavourable economic repercussion of divorce on child well-being. In this study, maternal remarriage didn’t appear to influence quality of life of child. Maternal remarriage could impact children who must readapt to new family basis and dynamics. However, a new partner perhaps could provide additional financial support since parental separation usually involves a fall in family income. Moreover, the new couple may construct an improved marital relationship, hence could minimize the distressing emotional burden on children and making the stepfather as a positive and ideal role model (Eymann et al., 2009). Lastly, in this study, separation by mutual agreement because of higher maternal education or maternal university education level and longer the length of time passed since separation has inclined psychosocial quality of life scores, while scores declined in boys and for each one-year increment of maternal age. Eymann et al. proves that higher maternal educational level could give positive impact on psychosocial quality of life, which may be explained as a consequence of the skills build through education years, allowing better and organized control of the marital problems and crisis and greater agreement on conflict issues (Eymann et al., 2009). CHILD ABUSE A prospective study was made of all cases of child abuse seen at the University Hospital, between July 1978 and June 1980. A total of 19 cases, 16 of physical and 3 of sexual abuse were diagnosed during this period. The relatively high (32%) incidence of child abuse amongst Malay families was quite surprised, as Malay children are generally much loved and pampered. Nathan and Woon suspect that the increasing incidence of child abuse amongst the Malays is probably related to their urban migration in recent years with the resultant breakdown of the extended family system and the need to cope with the pressures of life in an unfamiliar urban setting (Nathan Woon, 1981). The commonest injuries found on admission were bruises and injuries to the face and scalp. The majority of the abusers in our series were parents, the ratio of father to mother being. Both social and personal factors in the life of the abuser seem to contribute to the act of child abuse. Marital disharmony appears to feature prominently amongst the interpersonal problems, while two abusers gave a history of having been abused physically during their early childhood (Nathan Woon, 1981). SOCIOCULTURAL HOME ENVIRONMENT It has been recognized that children from poor socioeconomic status families have higher chances of poor school performance. Malnutrition due to poverty coupled with low education and status of parents adversely affect their cognitive development. Such children also have higher chances of experiencing, right from their pre-school years, parental attitudes which do not motivate them to study and an unsatisfactory home environment which does not encourage learning (witnessing domestic violence, family stressors, and adverse life events). Alleviation of hunger, by providing one balanced meal in school is one of the mechanisms to improve academic achievement in undernourished low-income elementary school children (Karande Kulkarni, 2005). Besides that, many of these disadvantaged children are studying in English medium schools as their parents believe that this would help them progress in life. These children face the added burden of language barrier, namely, they are not conversant in English as they came from non-English speaking families, which leads to poor school performance or even school failure. Parents of children with language barrier should be counselled to educate their children in their own language medium schools or to attend a facility for language stimulation if that is available and affordable (Karande Kulkarni, 2005). ORAL HEALTH In the research carried out by Anand et al. in year 2014, they noticed that poor oral hygiene statuses in children who spent more time watching television, playing video games, as their consumption of soft drinks and fast foods was more and due to lack of adequate exercise. BACK PAIN Musculoskeletal pain is exceedingly common in young adults. A questionnaire survey was carried out by Brattberg in year 1994 among children 8, 11, 13 and 17 years old. The prevalence of back pain and headaches among 1,245 Swedish school children was studied. 29% of the students reported back pain and 48% headache. In all age groups studied, both back pain and headaches were more common among girls than boys. Girls also reported more frequent symptoms than boys (Brattberg, 1994). CONCLUSION Children who are at risk for or who are overweight having odds 2 to 4 times greater for lower health related quality of life scores for physiological health, self esteem and physical functioning. Parents of children who are overweight also were more likely to evidence higher levels of emotional distress because of their children’s health. Being relatively underweight also was associated with lower self-esteem (Friedlander et al., 2003). Ken Rigby in year 2003 concluded that being victimized by peers is notably related to relatively low levels of psychological well-being and social adjustment and lastly to high levels of psychological distress and unfavourable physical health symptoms. Eymann et al. in year 2009 concluded that children’s psychosocial quality of life was affected by divorce. Psychosocial quality of life improved when parents had divorced by mutual agreement, the mother had a university education, when more time had elapsed since separation and was poorer in boys and in children of older mothers. And, quality of life can be improved as more time elapses after the divorce, this is because may be it can be interpreted as a process of accepting the family modification. And lastly, poor oral hygiene was observed in children who spent more time in leisure activities and that lifestyle factors could make an impact on oral hygiene and periodontal health. A need exists for addressing obesity, oral health and nutrition, jointly in health promotion strategies, to improve well-being of children and also to empower good life-style factors (Anand et al., 2014).
Sunday, January 19, 2020
Japanese Occupation in South-east Asia Essays -- Pacific War Japanese
Japanese Occupation in South-east Asia Table of Contents Chapter 1: Introduction Background                                                  Pg. 3 Thesis                                                       Pg. 3 Research questions                                             Pg. 3 Rationale                                                  Pg. 3 Methodology                                                  Pg. 4 Chapter 2: Literature Review                                   Pg. 5 Primary sources                                             Pg. 5 Secondary sources                                             Pg. 6 Chapter 3: Research Methodology                                   Pg. 8 Procedure                                                  Pg. 8 Types of sources                                             Pg. 8 Compiling and presenting the data                                   Pg. 8 Chapter 4: Results and findings                                   Pg. 10 Background information                                        Pg. 10 Conflict between Japan and United States                              Pg. 11 The Japanese Occupation (1942 – 1945)                              Pg. 11 Chapter 5: Discussion and interpretation                              Pg. 24 The causes or motives of Japanese Occupation                         Pg. 24 The effect of Japanese Occupation                                   Pg. 25 Propaganda                                                  Pg. 26 Chapter 6: Conclusion                                        Pg. 28 Acknowledgements                                             Pg. 29 Chapter 1: Introduction Background:      The Pacific War opened on 7 December 1941, and Japanese troops started invading other Asian countries. Singapore, which was a colony of Britain at that time, fell to the Japanese on 15 February 1942. Since then, the people in South-east Asia had great sufferings. During the Japanese Occupation, many people suffered and some even died. Finally, the Japanese surrendered after the bombing of Nagasaki and Hiroshima, and the nightmare was finally over. Thesis:      War does more harm than good. Research questions: †¢Ã‚     What are the main causes & motives of the Japanese Occupation †¢Ã‚     What are the main effects of Japanese Occupation to the people at that time? †¢Ã‚     What are the instruments... ...lusion The people under the Occupation suffered, and the people in Japan also suffered after the bombing of Nagasaki and Hiroshima. We learned that war does not do any party good, as we can see from the end of the war. We should learn our lessons from this event, and from the causes we can learn how to prevent it. Therefore, we should place our attention on the recent US and Iraq war, and stand on the side of peacefulness. We should also educate ourselves to differentiate between propaganda and the reality. Lastly, I had proven my theory, â€Å"War does more harm than good.†Acknowledgements http://www.iwm.org.uk/upload/package/25/pearl_harbour/hb_rise_of_japan.htm http://www.iwm.org.uk/upload/package/25/pearl_harbour/hb_ww1.htm http://www.iwm.org.uk/upload/package/25/pearl_harbour/hb_us_lead.htm http://www.iwm.org.uk/upload/package/25/pearl_harbour/hb_j_agg.htm http://www.iwm.org.uk/upload/package/25/pearl_harbour/hb_count.htm http://www.japan-guide.com/e/e2130.html http://www.japan-guide.com/e/e2129.html http://www.diggerhistory.info/pages-posters/jap.htm http://www.s1942.org.sg/ Understanding our past, Singapore from Colony to Nation - Federal Publications
Saturday, January 11, 2020
GFP protein
Green Florescent Protein, abbreviated as GFP, is a protein composed of 238 amino acids that is commonly found in mnemiopsis, comb Jelly. It has a major wavelength at 396 nm and a minor one at 475 nm. GFP is what gives mnemiopsis their bright green florescent glow. ultraviolet light, or blue light, is necessary to see the florescent glow of this protein. GFP is an irregular protein because It Is highly resistant to denaturation by temperature and PH. It can survive In temperatures up to 98 degrees and has a pH of 12. 2 due to Its complex exterior, called the beta barrel. At an pH higher than 12. It denatures. It also has an Isoelectric point at 5. 3. The peripheral beta barrel cannot be digested or broken apart by protease because of the strong bonds holding It together. The beta barrel protects the chromophore, which Is the substance which gives GFP Its green glow. When CFP Is extracted from the plasmid of an E. Coll or from a Jellyfish, It contains an array different contaminants ma king it difficult for scientist to do experiments with GFP. A procedure in purifying GFP from a crude cell extract to nearly 100% GFP so that it can be analyzed and used in scientific experiments and research is necessary.The goal is to ptimize each protocol used to purify crude GFP. Methods Ammonium Sulfate Precipitation To purify the crude samples of GFP, the ion exchange method separates substances inside the test tube by similar charge. A sample of crude GFP of 7. 5 mL in a plastic tube was used for the experiment. Knowing that 43. 6 grams of ammonium sulfate in a 100 mL solution yields a 70% percent saturated solution, the proportion 43. 6g 11 00 mL=x/7. 5 mL was used to determine that 3. 27 grams of ammonium sulfate needs to be added to the experimental sample. After adding the ammonium sulfate, the solution was stirred gently to prevent frothing.Once most of the solution is transferred, the tube was placed on a triple beam balance along with another tube that went through the same process. The centrifuge was set at 15,000 rpms for 15 minutes so that the hydrophobic materials will separate and become the supernatant while the GFP pellet will remain behind. Once the 15 minutes elapsed, a new pipette was used to remove the supernatant, leaving behind the pellet of GFP and hydrophilic contaminates. To remove the hydrophilic substances, 5 mL of 4 molar ammonium sulfate and 15 mL of 10 mL tris at a p of 8 was added Into the oak ridge entrifuge test tube.The solution Is then put Into the centrifuge at 15,000 rpm for 15 minutes again. Once 15 minutes has passed, the supernatant, containing the GFP, was removed by a pipette and put In a microfuge. Hydrophobic Interaction Hgure yaropnoDlc Interactlon set up One molar ammonium sulfate was added to the column to wash the sample. Adding 1 molar ammonium sulfate washes the sample because a high salt concentration increases hydrophobicity of the GFP and the buffer, causing most of the GFP to be at the very top of the column. Substances that are hydrophilic get flushed out of the olumn while the more hydrophobic substances stay in the container.After the column has been eluted with 1 molar ammonium sulfate, the tris buffer is added to the ammonium sulfate to dilute it into . 5 molar ammonium sulfate. The volume of 1 molar ammonium sulfate inside the oak ridge centrifuge test tube is the volume of the tris buffer that will be added. After the column chromatography has been flushed with . 5 molar ammonium sulfate, more hydrophobic substances will be flushed out since the hydrophobicity of the tris buffer and the GFP has decreased. This causes the GFP to spread out in the column. Finally the amount of . 5 molar ammonium sulfate is diluted with tris buffer to . 5 molar ammonium which should cause most of the GFP to be flushed out of the column along with other substances that are very hydrophobic. While this experiment is going on the liquid that comes out of the column is collected in multiple test tubes. These test tubes contain GFP and other contaminants. The solutions are than read by a spectrophotometer. Each test tube will be tested by the spectrophotometer so that a graph can be made. Anion Exchange Figure 2: Siphon Bridge set up for Anion Exchange Figure 3: Centricon Test Tube In order to use anion exchange, the starting condition of the sample needs to be in a low salt solution.However after the GFP had gone through hydrophobic interaction, it was in a high salt solution. Before facing this dilemma, the fractions were pooled by centricon which decreases the overall sample volume by removing some buffer and salt solution. This greatly increases the GFP concentration in the samples. The fractions are placed in the centricon and then into a centrifuge for 25 minutes at 3,000 rpm to be separated by size. The large proteins stay in the entricon while buffer and salt solution goes into the plunger. To reduce the concentration of salt in the GFP sample, the sample is diluted 10 folds.Since the amount of GFP that was restored was 18 mL, 162 mL of tris buffer needed to be added. The diluted GFP is then put in the chromatography container, containing positively charged DEAE which is attracted to the GFP at a low salt concentration. After the GFP has been completely filled, the column is connected to a beaker that contains a low salt concentration. the low salt concentration beaker is connected to a high salt concentration beaker. As one drop of low salt solution goes into the chromatography column, one drop of high salt solution goes into the low salt solution.Gradually the salt concentration increases in the low salt beaker and in the column chromatography, causing GFP to spread down the container. The eluted GFP dripped out of the column chromatography to be collected in test tubes. I nree pnase partltlonlng Figure 4: Precipitate of GFP. T-butanol is one top while contaminates are on bottom GFP then went through three-phase partitioning, also known as TT P. The fractions taken after an anion exchange was 15 millilieter. Ten ml of 4 M ammonium sulfate was added to this volume to increase the salt concentration of the solution to 1. M, which is about 40% salt saturation. Twenty-five milliliters of t-butanol was added then added which was the same amount of ammonium sulfate and GFP in the container. The container was then placed in the centrifuge for ten minutes at 4600 RPM, causing the mixture to split into three layers; butanol on top, GFP in solution on the bottom, and precipitated contaminants in-between. The top layer of butanol and disk of precipitate were taken out. The volume of GFP solution was again matched in utanol and the container went into the centrifuge again. An aspirator was used to extract the GFP into a microfuge. . 6M ammonium sulfate was then added to the microfuge and the container was placed in a micro centrifuge for one minute at 13,000 RPM. Butanol and other contaminants that had not been take out previously f ormed a disc, was then taken out with an aspirator and the remaining GFP was then left in the microfuge. HPLC Figure 5: HPLC basic layout After the sample went through three phase partitioning, it was put through the High Performance Liquid Chromatography for a final purification. First liquid was put into the HPLC to clean out any previous GFP inside the loop of the HPLC and the column of the HPLC.Then, GFP in the microfuge was sucked into an injector to be put into the HPLC. Pushing the top of the injector slowly, GFP entered into a loop inside the HPLC. Once the GFP was placed in the loop, a knob was turned clockwise to the word lock. The GFP was then sent to the column where it was purified further by size through the minuscule beads. About 6,000 pounds of pressure per square inch was produced by the HPLC to push the GFP through the beads. While this was occurring, a pectrophotometer connected to the HPLC read the wavelengths of substances.Near the 396 nm wavelength, GFP was col lected in a microfuge tube. A UV light was held near tne exlt 0T Results e HPLC to measure tne amount ng sample. Graph 1: Results of the sample after HIC at a wavelength of 395 nm Graph 2: Results of the sample after HIC at a wavelength of 280 nm Graph 3: Results of the sample after HIC of the entire spectrum Seventeen test tubes were received after the HIC purification process. A blank consisting of tris buffer and ammonium sulfate was sampled in the spectrophotometer against liquid from each of the seventeen test tubes.Graph one represents the sample after HIC at a wavelength of 395 nm while graph two Results shows the results after HIC at a wavelength of 280 nm. After HIC, the fractions 12 to 16 were chosen for their purity and recovery of GFP. Graph one show the amount of GFP in each fraction number while graph two shows the total amount of protein in each fraction number. Graph three shows the spectrum of the entire sample. Graph 4: Results after Anion Exchange at a 397 nm wave length Graph 5: Results after Anion Exchange at a 280 nm wavelength Graphing 6 Thirteen test tubes were collected from the Anion Exchange purification process.This time the samples were blanked against tris buffer at 8. 0 pH and 0. 5 molar sodium chloride. Graph four shows results of the Anion Exchange at a 397 nm wavelength and graph five shows the results after Anion Exchange at a 280 nm wavelength. Once again, the graph at a 297 nm wavelength shows the amount of GFP while the graph at a 280 nm wavelength shows the amount of total protein. Graph six represents the results of the entire spectrum. The GFP peak was a lot more visible. Step Iotal sample (mL Abs (280) Total Protein Abs (397) GFP Ratio Crude sample 120 1600 . 25 At-ns042- 20 1 . 61 . 9 118 HIC 18 . 28 . 173 . 618 15 . 126 . 130 1. 03 3 Phase Partitioning . 01 n/a . 75 . 243 . 257 1. 06 Table 1: This is the overall data table. The second column shows the total volume at the start of each purification step. The following two columns are the peaks of the graphs at those wavelengths. The last column represents the ratio of GFP to the total Protein. The most desirable ratio is 1. 25. Dlscusslon The first method in purifying the crude GFP was using the ammonium sulfate precipitate. When ammonium sulfate is placed in water, it dissociates into ammonium (NH4+) and sulfate ions (S042-).Water, composed of two hydrogen ions and one oxygen ion, is a polar molecule because the oxygen has a high electronegativity. Oxygen has a greater affinity making the oxygen portion of water negative and the hydrogen portion of the water positive. The dissociated positively charged ammonium ion is allured to the negatively charged oxygen while the negatively charged sulfate ions are attracted to the dissociated positively charged hydrogen. The attraction between the ammonium sulfate and the water was so strong that the GFP and other proteins were left unoccupied, causing them to precipitate.When GFP in the 70% salt solution was placed into the centrifuge, substances such as DNA and RNA was removed because they became part of the supernatant. At a 70% salt concentration, only hydrophilic substances stay in solution while the more hydrophobic substances precipitate. When the GFP in a 25% solution of salt was placed in the centrifuge, the GFP and other substances went back into solution because there not enough water was occupied by the salt. Before the GFP is placed in the centrifuge, it must be balanced with another centrifuge with the same weight and the two containers must be placed across from one another.This is vital because the centrifuge needs to be balanced when it is rotating at an incredibly fast speed. Failure to have balanced centrifuge containers can result in a broken centrifuge and loud sounds. Also when mixing the GFP with salt, it is important not the shake the container or frothing will occur, making it difficult to transfer the solution in to an oak ridge centrifuge tube. The second p urification procedure that GFP underwent was hydrophobic interactions. During this purification, GFP binded to the non-polar Phenyl Sepharose beads because of its non-polar and hydrophobic traits.However the water in tris buffer is strong enough to separate the attraction between GFP and the Phenyl Sepharose. Therefore a high salt concentration is necessary to occupy the water so that the GFP and the Phenyl Sepharose to be attracted together. At a high salt concentration, GFP with bind easily to the Phenyl Sepharose since very little water molecules would interfere with the attraction and at a low salt concentration, GFP would not bind easily to the Phenyl Sepharose because tnere wlll De a lot 0T unoccuplea water molecules tnat wlll De aDle to InterTere wltn the GFP and Phenyl Sepharose attraction.Before the experiment, ten millimolar tris buffer at a pH of 8 was used to clean the column in order to keep the pH stable and to wash away the salt, ammonium sulfate, in the column. Remov ing the salt is vital because the buffer that once surrounds the salt will be allured to the hydrophobic benzene and to the hydrophobic patches on the GFP. Since the hydrophobic patches of the GFP are already filled, they will be flushed out, leaving mostly beads of benzene and the 10 millimolar tris buffer at a pH of 8. Once the column has been clean, it needs to be equilibrated so that the salt concentration is the same through the olumn.The step gradient used, started ata 1 molar ammonium sulfate concentration and was halved until a . 25 molar concentration to separate substances by hydrophobicity. The third purification procedure was anion exchange. In this procedure, GFP and other contaminants are separated by charge. The beads in the containers are different from the beads from the hydrophobic interaction because on they have a different chemical called DEAE which makes them positively charged. GFP has both protons and electrons on it which is why it was not easily attracted t o the DEAE, which is why the GFP is put in a basic solution.Ata high pH, the amount of negatively charged hydroxide increases and these hydroxides are allured by the protons on the GFP. The protons are than neutralized, making GFP a negatively charged molecule. The isoelectric point of GFP is at a pH of 5. 3. Ata pH higher than 5. 3, it is negatively charged and when it is at a pH lower than 5. 3, it is positively charged. Once the column chromatography is filled with GFP and connected to a beaker of low salt which connected to a beaker of high salt, anion exchange occurs. As the salt concentration increases, the GFP slowly spreads down the column and eventually out f the column into test tubes.Between the HIC and the Ion exchange chromatography, the sample the fractions were pooled and put in a centricon causing the GFP concentration in the samples to increase. This occurred because the ultrafilter only allowed particles smaller than protein to go in to the pusher. The large protei ns stay in the centricon while buffer and salt solution goes into the plunger. The sample of GFP was also diluted 10 folds because the sample needs to be in a low salt solution to use anion exchange and after the GFP had gone through hydrophobic interaction, it was in a high salt solution.The anion exchange method creates a continuous salt gradient because as one drop of low salt solution goes into the column chromatography, causing GFP to spread down the container. The follow procedure was the three phase partitioning purification. T-butanol and 1. 6 molar ammonium sulfate were essential for this procedure. T-butanol has a low density causing in to stay above the GFP solution. In addition it has an attraction for water and other hydrophobic substances causing 5 mL of water to be drawn out of the GFP sample and precipitated substances to float between the t-butanol and the GFP sample.Fresh t-butanol is necessary after removing the old t-butanol with the contaminants because at that point, the salt concentration had increased since water was drawn out. was aDle to De preclpltatea Decause 0T tne nlgn salt concentration. The final procedure for purifying GFP was using the HPLC which separated substances by size. The beads used in the HPLC column are miniscule and porous. The pours on the beads give substances of the same size more opportunities to leave the HPLC at the same time. Since the beads are so small, high pressure is needed to push the GFP sample through the beads.Naturally, smaller substances will exit the HPLC first while larger materials will exist last. In all scientific experiments room for error is unavoidable. During the HIC, IEX, three phase partitioning, and the HPLC, amounts of GFP were lost due to the GFP sticking to a container, a pipette, and even spills. During the HIC some of the GFP was lost due the overflowing the test tubes with liquid exiting the column. During the HPLC some GFP was lost because not all GFP dripping out of the HPLC wen t in to the microphage. Other errors include letting the column dry because the liquid was not dded to the beaker about the column.During the spectrophotometer runs, the blank was no inserted correctly causing the reading of the GFP to be incorrect. In addition, the order in which the GFP samples were suppose to be placed in the spectrophotometer was messed up. Judging from the overall purification table, table 1, the purification was quite successful. Originally, the ratio was only . 25, but by the end of all the purification procedures, it obtained a ratio 1. 06. A 1. 25 ratio is most desirable and through the purification, the ratio was nearly reached. The anion exchange, three phase artitioning, and the HPLC purification were the most impacting procedures.The anion exchange greatly increased the purity of the crude sample compared to the HIC purification. The three phase partitioning and HPLC purified the GFP even more. Some improvements to the protocols would be to start with t he anion exchange purification so that overall, the salt solution would go from a low salt concentration to a higher salt concentration. This also eliminates the need to dilute the solution. In addition, an automatic machine could be used to shift the test tubes that collect the iquid exiting the columns to prevent overflowing test tubes and the risk losing GFP.GFP is unique because of its florescent glow. This glow can be used as a marker or an indicator. If a glowing marker could be placed on infectious cells such as tumor cells or cancerous cells, it would revolutionize the treatment of these diseases because doctors will be able to track where the harmful cells are. In addition, if it is possible to trigger the florescence of GFP with UV light, it can eventually be used in light bulbs to produce light. GFP light bulbs would last for an incredibly long time ince they are very resistant to denaturing.In addition, in vehicles, GFP can be mixed in the motor oil, transmission oil, po wer steering oil, air conditioning oil, and other oils so that if a leak occurs in a car, it can easily be spotted by shinning UV light on the car. The purification of GFP can lead to endless new innovations in electrical engineering, automotive repair, and curing deadly diseases.
Friday, January 3, 2020
Standard vs. Tipping Bucket Rain Gauges
A rain gauge is a weather instrument that gathers and measures the amount of liquid precipitation that falls from the sky. How a Tipping-Bucket Gauge Works A tipping bucket rain gauge has several components that allow it to accurately measure rainfall. As rain falls, it lands in the funnel of the tipping bucket rain gauge. The rain travels down the funnel and drips into one of two very carefully calibrated ‘buckets’ balanced on a pivot (like a see-saw). The top bucket is held in place by a magnet until it has filled to the calibrated amount (usually approximately 0.001 inches of rain). When the bucket has filled to this amount, the magnet will release its hold, causing the bucket to tip. The water then empties down a drainage hole and raises the other to sit underneath the funnel. When the bucket tips, it triggers a reed switch (or sensor), sending a message to the display or weather station. The display counts the number of times the switch is triggered. Because it knows how much rain is needed to fill the bucket, the display can calculate the rainfall. Rainfall is measured in inches; 1 of rain would fill a container with straight edges to a level of 1. Getting the Best Results From Your Rain Gauge To get the most accurate results from a tipping bucket rain gauge, you need to properly install the rain gauge. The rain gauge must be positioned on a flat surface – if the surface isn’t flat, the see-saw may tip before the bucket has filled to the calibrated level, or not tip at all. If the bucket doesnt tip at the calibrated level, the rainfall calculated will not be correct. Use a spirit level to determine whether a surface is flat, and then fix the gauge to the flat surface to ensure you are getting an accurate reading.The rain gauge must be positioned on a surface that does not vibrate – surfaces such as a porch or fence can move and vibrate. The tipping bucket is very sensitive and any vibrations could cause the gauge to tip even if it is not raining.The instrument must not be positioned near trees – being positioned near trees could allow leaves or pollen to fall inside the funnel and block it, causing an inaccurate reading.It must not be positioned in a sheltered area – being positioned in a sheltered location (such us beside your house or a fence) co uld significantly increase or decrease the amount of rain depending on the wind direction, and cause an inaccurate reading. The gauge should be positioned at least twice as far away from the object as the object’s height (e.g. if the fence is 6 feet high, the gauge should be positioned at least 12 feet away).Your weather equipment must not be located near any magnetic, steel, or iron objects – magnetic, steel, or iron objects can affect the amount of time the magnet will hold the bucket or whether it will hold it all, causing an inaccurate reading. Will a Rain Gauge Measure Snow? If it snows where you live, most rain gauges will not be able to measure the snow fall; snow will block the opening of the collection funnel. However, special snow gauges are available to measure this. Following these recommendations should ensure your get an accurate result from your tipping bucket rain gauge.
Subscribe to:
Posts (Atom)