fUuYeLpEgElPcPtTqJvXyGyAlOeSfApTfPgDkZvYaKdOlMxEvIiIdChErPoXbPiHkIbEqUrQcLtWgRnAgGtMaZvKbEuZtGxRtUuCbMxGoWrZjStWgIsTjXfFbYoVgSqPtCdJzYfUyDvOoBkMcLsYuVjDoZwAxUlQfAdQyDaMbOqOhWpSfVsSiSfSbYiOoXhUuWlMcW thesis writing service

8. The following limitations were encountered in the study.

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8. The following limitations were encountered in the study.

8. The following limitations were encountered in the study.

This objective was achieved by the means of a focus group, some questions were formed to generally access the knowledge about money management in general population and the role it can play in their monetary situation. This was done via open ended questions to give the participant power to get feedback and discuss in form of complex textual descriptions to access how people experience the given research issue. Sample here also included students, unemployed people, part time workers, full time workers and self employed people with different sex and age groups.

Objective 7: Suggest a New Theory on Money Management in hard times.

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Thus in above mentioned way the objectives were addressed and data will be gathered and analyzed and the last objective to suggest a New Theory on Money Management in hard times to emerged due to the achievement of the previous research objectives.

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3320 words (13 pages) Essay

25th May 2018 Biology Reference this

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Tetrodotoxin is alkaloid based aquatic toxins. These toxins are one of the most potent non-proteinaceous toxins as well as the best-known marine natural toxins. Diodon hystrix (porcupine fish) were collected from Chennai costal region and dissected under sterile conditions to obtain: liver, skin, gonads, intestine, eyes and kidney. 20g of each organ was macerated in 200ml of Methanol:Acetic Acid [99:1]. The filtrate is then condensed in Rota-Vaccum evaporator to obtain crude extract. The focus of this study is to confirm the clear presence of TTX (Tetrodotoxin) in six different organs of Diodon hystrix. Analytical techniques used were GC-MS and UV spectroscopy. Also, genotoxicity of the crude extract were analysed using human leukocyte culture and SCE assay using onion root tips. The results suggest the clear presence of TTX in major skin, liver and intestine and that, the organ extract does not have any genotoxic effect but is capable of increasing the sibling chromatid exchange.

Keywords: TTX, Diodon hystrix, genotoxicity, root tip assay.

Tetrodotoxin (TTX) is a very powerful alkaloid neurotoxin that is non-proteinacious in nature. TTX can withstand very high temperature and is water soluble but is affected by extreme pH conditions, i.e., above 8.5 and below 3.0 [1, 2, 3, 4, 5]. These properties make it a dangerous toxin capable to interact best with its environment [1, 2, 5]. It is found in both aquatic in addition to terrestrial organisms and studies have proven that it is synthesized by symbiotic microorganisms, bacteria precisely, present in the gut, initially acquired through the food chain or found on the skin of the animals but its biosynthesis pathway is still unknown [ 1, 2, 5, 6, 7, 8]. TTX acts as an ion pore blocker, binding

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to site 1 sodium channel receptor of the axon membrane thus inhibiting the influx of sodium ions and so resulting in the blockage of action potentials [1, 2, 3, 4, 5, 6, 7, 8, 9]. TTX is ten thousand times poisonous than cyanide and another of the very most fatal poisons on Earth. The LD50 is approximately 0.2μg when injected in mice [2, 5]. On the other hand, along with the lethal characteristics, clinical trials and research studies have demonstrated that TTX has remarkable therapeutic properties as an analgesic in cancer treatment process [2].ap biology chi-squared essay

Puffer fish belonging to the order Tetraodontiformes, have been identified to be the cause of many mortalities due to food poisoning as a results of TTX intoxication. In several countries such as Japan and China, puffer fish is certainly a food delicacy provided that it really is prepared by a licensed and well experienced chef but some cases of poisoning still prevail [1, 3, 4, 5, 6, 7, 8, 9, 10]. It is often reported that only a very low dose of TTX

in blood is adequate for an immediate impact on the host [5]. Studies have concluded that the most toxic organs of the puffer fish are the liver followed by the intestine and then the skin and ovary. In addition to that, TTX is also found in low concentration in other organs including the eyes and muscles [3, 5, 8, 10].

The study is focused on Diodon hystrix which is a type of puffer fish belonging to the class Diodontidae and it is also known as Porcupinefish because of the sharp needle-like structures covering its system as a defense mechanism against predators. Presence of TTX was reported in Diodon hystrix around the world [2, 4, 5] but studies on this animal from the sea of the eastern coast of India that is the Bay of Bengal is yet to be reported. The aim of this research is to spot TTX in the crude extract from Diodon hystrix gathered from Chennai Coastal line and to investigate the Genotoxicity of the crude extract from respective organs using human leukocyte culture and onion root tips.

The puffer fish was collected from the coastal lines of marina beach, Chennai in early July 2014. The identification of the puffer fish was done by visual comparison with an online fish database –www.fishbase.org. The database parameters were set correctly to sample collection site and the possible species available in Bay-of-Bengal region with the matching morphology were only two types of Diodon sp.. Out of which Diodon hystrix had the closest match, based on the skin coloration pattern.

The collected puffer fish were dissected and visceral organs like liver, intestine, kidney, eye, and skin were removed and organs were weighed. The isolation for the tetrodotoxin[3] include from the samples 10 grams of organs were taken and Then suspended in 100ml of three level of 1% acetic acid in methanol without damaging the tissues then a whole materials were in the Refrigerator for 24 hours at a sterile condition, as an incubation period In the next step the tissue were macerated in a mortar and pestle gently, if the tissues get dried up add required level of the chilled ethanol if needed. Then a slurry were filtered by using whatman no. 1 filter paper. Then a filtrate solutions were centrifuged at 12000 rpm for 10 minutes at 4 degree Celsius. Then the supernatant were separated and lastly the samples were concentrated by using lyophilisation to obtain crude extracts for our purpose of study

To spot the clear presence of alkaloids [10] to 2mg of crude extracts 5ml of distilled water were added and then 2M hydrochloric acid was added until an acid reaction occurs. To this 1 ml of Dragendorff’s reagent was added. Formation of orange or orange red precipitate indicates the clear presence of alkaloids

Gas chromatography (GC) and mass spectrometry (MS)[8][11][12]forms an effective combination for Chemical analysis. GC-MS analysis were an indirect method to detect TTX in a crude extract,

which was difficult to purify in other advanced analysis methods. In this method, we dissolved TTX as well as its derivatives in 2 ml of 3 M NaOH and heated in a boiling water bath for 30 min. After cooling to room temperature, the alkaline solution of decomposed compounds was adjusted to pH 4.0 with 1N HCl as well as the resulting mixture was chromatographed on a Sep- Pak C18 cartridge (Waters). After washing with H2O first and then 10% MeOH, 100% MeOH fraction were collected and evaporated to dryness in vacuo. To the resulting residue, a mixture of N, O-bis acetamide, trimethylchlorosilane and pyridine (2: 1: 1) was added to generate trimethylsilyl (TMS) ‘‘C9-base’’ compounds. The derivatives were then put into A hewlett packard gas chromatograph (HP-5890-II) built with a mass spectrometer (AutoSpec, Micromass Inc., UK). A column (φ 0.25 mm × 250 cm) of UB-5 was used, and the column temperature is increased from 180 to 250°C at the rate of 5 or 8°C/min. The flow rate of inlet helium carrier gas were maintained at 20 ml/min. The ionizing voltage is generally maintained at 70 eV with the ion source temperature at 200°C. Scanning was performed in the mass range of m/z 40–600 at 3s intervals. The total ion chromatogram (TIC) and the fragment ion chromatogram (FIC) were selectively monitored.

In UV spectroscopy, TTX was generally dependant on irradiating a crude toxin with UV light [11][12]. A small amount of samples were dissolved in 2 ml of 2 M NaOH and heated in a boiling water bath for 45 min. After cooling to room temperature, samples were examined in UV spectrum and results were observed in the range 270nm to 280nm.

Chromosome preparations were obtained from PHA-stimulated peripheral blood lymphocytes[14][15]. To the fresh tubes 5ml of Hikaryo XL RPMI ready-mix media and 0.5ml of heparinized Blood (50drops) were added and the contents were mixed gently by shaking. Then Incubated for 72 hours in standing position in an incubator. At the end of 48th hour of incubation, the culture was treated with TTX (0.5ug/ml) (10ul/ 5ml of culture) and again kept it in incubator for another 24 hours. At the end of 24th hour incubation, the culture was thoroughly washed by centrifuging the content at 1500rpm for 5 minutes, discard the supernatant and add 5ml of RPMI 1640 medium. To the content 60 microliter of colchicine was added and tubes were kept for 20 minutes incubation in incubator at 37oC and the content was centrifuged at 1500 rpm for 10 minutes after incubation. The supernatant was removed and 6ml of pre-warmed 0.075M hypotonic solution was added. The content was mixed using a Pasteur pipette and incubated at 37 oC in incubator for 6 minutes. After incubation the content tube was centrifuged at 2000 rpm for 5 minutes. The supernatant was discarded and 6ml of Carnoy’s fixative was added and mixed vigorously. After fixation the content was kept in room temperature for 1-2 hours. The content was again centrifuged at 1500 rpm and supernatant was removed and this step was continued until pellet becomes white. For the preparation of slides the newest slides were first refrigerated and then cell button mix was dropped within the slides and dried immediately on a hot plate, and then was kept in an incubator for proper drying. The slides were then put into a coplin jar containing Giemsa staining for 4 minutes and destained in a coplin jar containing distilled water for 1 minute. The slides were dried and then viewed under microscope for stained chromosome. . The slides were then viewed under 100X power under oil immersion objective of the microscope to analyze the chromosome aberrations.

The onion root tips[1], 2-3 cm long, were soaked in 100 µM 5-bromodeoxy uridine (BrdUrd) for almost 20 h followed by 1 hour treatment with the crude extract After a brief wash, the roots were allowed to grow for another round in growing media. The treatments were terminated by washing the roots with distilled water and then 0.05% Colchicine was added then incubated for 2.5 h. Roots were washed, excised and fixed in Carnoy’s fixative, for 1-3hrs and preserved at 4°C. The roots were processed using cytology methods for SCE analysis.. The roots were then hydrolysed in 5 N HCI at 25°C for 92 min and stained with haematoxylin for at least 2hrs. The stained root[16] were washed in distilled water, squashed in a drop of 45% acetic acid and tapped for metaphase chromosome separation under coverslips. Tap water controls were included in the assay. The slides were observed at 100X magnification in oil immersion using light microscope

Fig 1: Showing results of sample after Dragendorff’s test

The alkaloids present in the puffer fish was precipitated as a complex formation by dragendorff’s reagent. Dragendorff’s test results showed very high precipitation in skin and intestine, high precipitation in liver and very low precipitation or almost no precipitation was observed in kidney, gonads and eye.

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Characteristic peak was observed at retention time 8.33 and 8.66 in liver, intestine and skin after performing alkaline treatment and there was clearly no characteristic peak observed in kidney, eyes and gonads. After boiling of samples which contain TTX in alkaline solution (NaOH) the compound TTX present gets reduced to C9 base TMS (trimethysilyl). It really is noteworthy that all peak of selected ion monitored at m/z = 376, 392 and 407 appears at the same retention time in the Selected ion-monitored mass chromatogram of the TMS derivatives of alkali-hydrolyzed. From samples of liver, kidney and intestine, mass fragments of ion peaks was observed at ion M/z 376, 392 and 407, which are characteristic of the quinazoline skeleton (C9 base), which was almost similar as those from the TMS-C9 Base derived authentic TTX

Fig 2: Showing GC-MS spectrum of the TMS derivatives of alkali-hydrolysed toxin from Diodon hystrix

In UV analysis method characteristic peaks were observed in all samples. Shoulder peak was observed in liver, intestine and skin, Declining and Inclining Peaks were observed in kidney, eyes and gonads. The UV spectrum is analyzed for the characteristic of absorptions, associated with C9-base .The shoulder peaks were observed at 276 nm indicates the formation of C-9 base which were specific to TTX or related substances.

 

Fig 3: Showing chart of UV-spectroscopy of the crude extract from various organs of Diodon hystrix, peak at 276nm indicating the clear presence of TTX.

Metaphase plates were obtained while observing under 100X magnification in oil immersion using light microscope. It is often observed in all the samples that there were no chromosomal aberration that is structural or numerical chromosomal modification were not observed. Using this result, it can be reported that the crude extract from Diodon hystrix has no clastogenic (breakage of chromosome) or aneugenic ( change in chromosomal number) effects.

Fig4(left): Showing metaphase plate from control leukocytes. Fig5(right): Showing metaphase plate from crude extract leukocytes.

The Sister Chromatid Exchange (SCE) assay was reported to be one of the most sensitive and painful short-term genotoxicity assays because of its capability to identify genotoxins at really low doses (Tucker et al.1993). It is often observed that the crude extract from Skin and intestine enhanced SCE dramatically over the control whilst the Liver, Eye, Gonads and Kidney have very low effects. Therefore it may be put forth that the crude extract from skin and intestine interfere to a great deal with the SCE and further studies have to be carried out.

Fig6(left) : Showing results of SCE in control onion root tip. Fig7(right): Showing results of SCE in crude extract root tip.

From the study, it can be reported that Diodon hystrix from the eastern coastal region of India, observed to have accumulated TTX in its organs. Thus it can be toxic when ingested and even lethal to the predators. Nevertheless further studies should be carried out on this fish to confirm the clear presence of a homologue of TTX and obtain a purified sample of the TTX.

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5246 words (21 pages) Essay

1st Jan 1970 Economics Reference this

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Big Onion crop was introduced to Sri Lanka by the British in 1855 and commercial cultivation was introduced by the Department of Agriculture during the 1950’s and over the past years, the crop performance was evaluated in several parts of the country and it was observed that big onions may be grown economically during every Maha season in virtually all parts of the country.

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2. Nonetheless, at present the cultivation of big onion is confined only to Matale, Anuradhapura, Puthalama, Pollonnaruwa, Mahawelli and Jaffna Districts. More than 50% of the total onion production in Sri Lanka is cultivated from the Matale District. [1] 

3. The Government strives to achieve a self sufficient stage in the production of big onions since Sri Lanka spends a significant amount of cash outflow every year on the importation of the big onions. Meanwhile, in the recent past it has been noticed that the big onion production was affected in Sri Lanka and so customers may also be paying a higher price for the big onions. In particular the big onion production in Dambulla area was declining in the last few years.

4. The Dambulla area plays a important role in the big onion cultivation in Sri Lanka. The Government was paying less attention and support on promoting the big onion production in Dambulla.

Therefore, it offers so happened that the onion production in Dambulla has declined in the recent past as a consequence of the government’s less support for this sector. Therefore, the main purpose of this study is always to promote the big onion cultivation in the Dambulla area.

5. This research is carried out with the following specific and general objectives.

a. The main general objective of this study is to identify the main problems encountered in the onion cultivation of the Dambulla area.

6. The specific objective of this study is always to give the recommendation to improve the Big onion cultivation in the Dambulla area and specific objectives are as follows.

a. To study the recent history of Big onion cultivation in Dambulla area and to compare the present situation associated with the Big Onion cultivation.

b. To spot the main issues encountered in big onion cultivation in Dambulla.

c. To spot the critical contributing factors.

d. To produce suggestions based on the findings.

2. The Matale District plays a important role in the big onion cultivation in Sri Lanka in particular Dambulla provides big onions for the Sri Lankans’ consumption. In the recent past as a result of lack of support from the government sector the big onion cultivation was declining.

3. As a result the big onion cultivation in Dambulla will be non existence in the very near future. Also, many farmers depend on the big onion cultivation as their livelihood in Dambulla. Hence, if the big onion cultivation in Dambulla is affected many families will lose their income and it will affect the survival of many families. Thus the lack of support from the government and the consequent less onion cultivation are considered as the research problem for this study.

4. This research studies the declining stage of the onion cultivation in Dambulla. The scope covers only the Dambulla area of big onion farmers. Therefore, this research was limited to the onion farmers of the Dambulla area.

7. This research studies the factors influencing the decline of the big onion cultivation in Dambulla. Therefore, the responses were collected from the local onion farmers from the Dambulla area. Thus, 100 big onion farmers were considered as a sample for this study since all farmers could not be accessible within the limited time for this study. These farmers were selected in a random basis. Therefore, the simple random sampling method was applied for picking a the sample.

8. The following limitations were encountered in the study.

a. Time is limited, so that within the limited time the research has to be finished this is why in-depth analysis can not be applied.

b. The researcher encountered limitation of resources.

c. The sample was limited only to 100 farmers.

9. The big onion is an important minor crop consumed by many Sri Lankans and possesses been estimated that 34,000 metric tons of onion is imported annually and Sri Lanka spends around 300 million rupees on onion importation (Gunawardena, 2009). Also, it is often also estimated that 45,000 labour units are employed in the onion cultivation and production annually by Sri Lankans and so, it increases income and employment generation for a lot of Sri Lankans. [2] 

10. Many countries worldwide are getting involved in the big onion production. In particular they are; Belarus, Russia, Lithuania, Poland, Ukraine, India, Pakistan etc (Research Institute for Vegetable crops, 2006).

11. According to Shanmugasundaram (2001) there are varieties of onion and it mainly includes the sweet, red, white, yellow, brown and green etc.

Source – Shanmugasundaram (2008)

12. Also, it is often identified that the big onion production brings several comparative benefits in comparison with with other crops (Autko & Moisevich, 2006). Some of the benefits are given below.

a. Output can be obtained in a short period of time.

b. Initial costs such as; seeds costs, fertilizer costs are comparatively less.

c. It does not require a set cost.

d. Less technology the machines are sufficient.

e. High employability of manual labourers.

f. Easy to find markets.

g. Less storage period.

13. The onion basically was divided into red onions and onions that are big each variety requires different eco-agricultural conditions, labour, fertilizer, weather and climatic conditions, temperature, etc.

14. The literature indicates different requirements for smooth growing of the big onion production. Some of the conditions suggested by Autko and Moisevich (2006) are given below.

a. Increase of fertile soil layers in the zone of plant root by 4-6 cm

b. Increase of aeration and warming of soil, excluding over wetting in the period of heavy precipitation

c. Decrease of fertilizer rate application by 30%

d. Decrease of seed sowing rates

e. Ensuring of looser soil state during the whole period of vegetation

f. Possibility of soil surface copying by working organs of machines, during inter-row treatment, decreasing of plant protective zone 3-5 cm, mechanical weed destruction by 70-75% and band application of pesticides that ensures the decrease of their rates by 2-3 times

g. Increase of irrigation efficiency

h. Diminution of nitrate content in the production

j. Decrease of energy expense during harvesting by 20-40%.

15. Therefore, the above conditions can be considered as the basic requirements for the growth and survival of the big onion production.

16. The onion basically was divided into red onions and large onions and each variety requires different eco-agricultural conditions, labour, fertilizer, weather and climatic conditions, temperature, etc.

17. Shanmugasundaram, (2001) has identified the following diseases that affect the onion cultivation. He has divided these deceases into two.

a. Field diseases

b. Storage diseases

18. The field diseases comprises of Stemphylium blight , Purple blotch, Anthracnose, Botrytis leaf blight, Downy mildew, Pink root, Smudge, Smut and several Basal rots (Shanmugasundaram, 2001).

19. The storage diseases covers common field rots, botrytis neck rot, black mold and bacterial soft rot (Shanmugasundaram, 2001).

20. Meanwhile it is often learned that in the recent past the onion cultivation was reducing due to many factors. Some factors identified by Kulatunga (2006) are presented below.

a. Lack of quality seeds

b. Lack of advice given for application of seeds

c. Insufficient loan facilities available to purchase quality seeds

d. Long durations taken for harvesting from seeds

e. Lack of government support in providing fertilizer facilities to the onion production

f. Lack of quality fertilizers available for the onion producers

g. Lack of availability of fertilizer at outside and private outlets

h. Absence of counselling and advice given on how to apply the fertilizers for the new variety

j. Lack of storage facilities to store the onion production.

21. Though these problems are encountered in the onion production it can be divided into two major categories. These are given below.

a. Lack of government support in giving seeds to the onion cultivators.

b. Lack of government support to provide fertilizer to onion cultivation.

22. It is often observed that big onion cultivation was affected to greater extent by the lack of government motivation in finding required seeds. Thus; lack of quality seeds, lack of counselling and advise on applying seeds, lack of new variety of seeds, insufficient government financial support to shop for seeds, absence of assurance on harvesting duration etc are encountered under seeds (Kulatunga, 2006).

23. Kulatunga (2006) has also identified there is no sufficient fertilizer support to motivate the big onion production. In Sri Lanka it is often learned that the onion farmers lack government funding and subsidies to get fertilizers. Also, fertilizer is sold at a fairly high price in the outside outlets. In addition the efficient and harvest stimulating fertilizers are not available for the onion farmers. Also the quality and different variety of fertilizers may also be not available to increase the big onion cultivation in the Dambulla area.

24. It is important that the onion production is increased in order to protect the big onion industry and to assure the livelihood of many Sri Lankans. Hence the literature suggests that the following measures can increase the onion production.

a. Involving in research and development activities in order to increase the onion production.

b. Government providing support to find quality seeds.

c. Government has to give seeds of the new varieties.

d. Government has to provide seeds at subsidized prices.

e. Government has to provide constant counselling and advice on management seeds.

f. Government has to extend the fertilizer subsidy.

g. Providing high quality fertilizer.

h. Monitoring fertilizer distribution.

j. Counselling on handling diseases.

(Source – Formed for this Research Study)

26. The above figure depicts two sets of factors that determine the decrease in the onion cultivation; the lack of seed accessibility and the lack of fertilizer accessibility. This was produced from Kulatunga (2006). Each set of the major factors have sub factors. Therefore, these two are considered as the independent variables. The decreasing onion cultivation may be identified as the dependent variable. Hence, this figure establishes links between the factors and the decreasing onion cultivation. Through this research study one need to know which factor(s) cause for the decreasing onion cultivation, on the list of farmers in the Dambulla area.

Factors determining the onion cultivation

Lack of seeds accessibility

Receiving quality seeds

Likert

Q1

Distribution of seeds by the government

Likert

Q2

Provision of subsidy by the government to buy seeds regularly

Likert

Q3

Seeds giving the expected harvest

Likert

Q4

Purchase seeds from the Government Agricultural Department

Likert

Q5

Provision of training and counselling regarding the new seeds by the government

Likert

Q6

I’m able to get new varieties of seeds

Likert

Q7

I’m able to get regular counselling and advice of the diseases on the seeds

Likert

Q8

Lack of fertilizers accessibility

Fertilizer subsidy from the government

Likert

Q9

Purchase of fertilizer from the Government Agricultural Department

Likert

Q10

Purchase of fertilizer from the private outlets at a less price

Likert

Q11

Getting quality fertilizer

Likert

Q12

Getting advice and counselling for the application of fertilizers

Likert

Q13

Getting different variety of fertilizers

Likert

Q14

Getting fertilizer that can maximize the harvest