1.
Srudy Of Gamma-Aminobutyric Acid A Receptor Delta Subnuit Gene Mutations Involved In Generalized Epilepsy With Febrile Seizures Plus (GEFS+) Patients in Punjab
by Iram Javed | Dr. Muhammad Wasim | Dr. Abu Saeed Hashmi | Dr. Asif Nadeem.
Material type: ; Format:
print
Publisher: 2012Dissertation note: World health organization (WHO) reports that neurological disorders affect one billion people worldwide, including 50 million affected by epilepsy. Epilepsy is a common neurological disorder characterized by recurrent, periodic, spontaneous and unprovoked seizures. Generalized epilepsy with febrile seizure plus (GEFS+) is an autosomal dominant disorder and a heterogeneous familial condition in which family members express febrile seizures initially, and then show multiple phenotypes of myoclonic epilepsy including partial or absence seizures and generalized tonic conic seizures. Molecular genetics techniques have identified various GEFS+ associated mutations in many genes i.e. sodium channel genes (SCN2A, SCN1A, and SCN1B) and some GABA receptor genes (GABRG2 and GABRD). GABAA receptors are the principal intermediaries of fast inhibitory neurotransmission in the eNS and have been frequently reported to playa significant role in a number of seizures. GABRD gene encodes the delta (8) subunit and is usually located in extrasynaptic GABAA receptors. The present study was aimed to investigate coding regions of GABRD gene for analyzing the mutations involved in epilepsy. Blood samples of unrelated true representative ofGEFS+ were collected from psychiatry departments of different hospitals of Lahore. DNA were extracted with the standard protocol and amplifications of the GABRD regions were done with specially designed primers. Later on, sequencing of target fragments was carried out. Sequences were analyzed through BioEdit software and then aligned with the help of custalW2 software. Out of 14 GEFS+ patients, only 3 were identified with a novel heterozygous transition mutation in intron 5. Further study, with much larger sample number, is required to revise the effects of this polymorphism and accurately identifying the associated factors. There is a need to explore the other gene mutations causing epilepsy in local population of Punjab and Pakistan that will ultimately help to develop genetic counseling strategies, gene therapies and prenatal diagnostic procedures for the population of Pakistan.
Availability: Items available for loan: UVAS Library [Call number: 1394,T] (1).
2.
Effect Of Date Palm Pollen On The Plasma And Intra-Testicular Testosterone Levels Of Male Albino Rats
by Yasir Arfat | Dr. Muhammad Tayyab | Dr. Abu Saeed Hashmi | Dr. Ali Raza.
Material type: ; Format:
print
Publisher: 2012Dissertation note: Considerable evidence exists for the efficacy and safety of short courses of low
dose testosterone therapy for treating infertility and delayed puberty. This treatment is
associated with high levels of patient satisfaction. There is not yet sufficient evidence for
the routine use of other therapies. Experimentally, date extract had been shown to
increase sperm count and increase stimulating concentration of testosterone count in
guinea pigs and to enhance spermatogenesis, follicle stimulating hormone (FSH) and
luteinizing hormone (LH) in rats. Intratesticular testosterone (ITT) is thought to play a
key role in the control of spermatogenesis but is rarely measured.
The present study is therefore designed to examine the effect of date palm pollen
(DPP) (Phoenix dactylifera) on the plasma and intra-testicular testosterone levels using
male albino rat as an experimental animal with the hope that the result of this study may
pave the way for treating male infertility and delayed puberty.
Adult male albino rats were divided into two groups (control and experimental).
Experimental group were given date palm pollen (DPP) suspension in a single oral dose
of 120 mg/kg of body weight for 35 days. Where as the control were given equal amount
of distilled water. Blood samples of control and experimental groups were taken for
measurement of serum testosterone levels at day 0, 12, 24 and finally at day 36.Aanimals
were sacrificed. Testes were removed for gross and biological studies. Intra-testicular
testosterone levels were measured at the end of experimental studies.
There were no statistically significant differences in the variable of control group.
Experimental group who received DPP suspension for 35 days showed statistically significant increase in body weight, weight of paired testes, serum and intra- testicular testosterone levels as compared to control group.
Availability: Items available for loan: UVAS Library [Call number: 1411,T] (1).
3.
Bioconversion Of Wheatbran To Glucose By Gluoamylase From Aspergillus Fumigatus
by Hassan Ali | Dr. Muhammad Tayyab | Dr. Abu Saeed Hashmi | Dr. Asif Nadeem.
Material type: ; Format:
print
; Literary form:
drama
Publisher: 2012Dissertation note: Background:
Glucose is produced by hydrolysis of starch. Many crops like maize, rice and wheat can be used as the source of starch. Wheat bran is an agricultural waste byproduct which can be converted to glucose using glucoamylase. Wheat bran is very cheap source for carbohydrates. It is mainly composed of carbohydrates; hemicelluloses, cellulose and starch. Glucoamylase is an enzyme that yields glucose from the nonreducing chain of amylose and amylopectin by hydrolyzing ? -1,3, ?-1, 4 and ?-1,6 linkages of starch. Glucoamylases are produced by plants, animals and microorganism. Microbes, including bacteria, yeast and fungi are major source for the production of glucoamylases. Aspergillus fumigatus is found in soil and in decaying organic matter and it has an essential role in carbon and nitrogen recycling.
Hypothesis: A. fumgiatus might be a good source for the production of glucoamylase through submerged fermentation conditions.
Parameters/Methodlogy: Aspergillus fumigatus was identified macro and microscopically. Enzyme production was measured by DNS method. The effects of different sources of carbon, phosphorous and nitrogen on glucoamylase production were also examined. In order to get the optimum production of glucoamylase, the effect of temperature, pH and incubation period was analysed separately.
Methodology: Initially the A. fumigatus was isolated and conditions were optimized for the growth and production of glucoamylase. Production of enzyme was examined by DNS method. The effects of various carbon, nitrogen and phosphorous sources were examined on the production of glucoamylase. From the present study it was concluded that maximum production of glucoamylase can be obtained from A. fumigatus using wheat bran as the substrate at pH of 4.8, temperature of 40oC with an incubation time of three days.The use of wheat bran as substrate wheat bran for the production of glucoamylase will reduce the cost for the production of glucoamylase.
Availability: Items available for loan: UVAS Library [Call number: 1509,T] (1).
4.
Bioconversion Of Industrial Wastes To 6-Aminopencillanic Acid With Escherichia Coli.
by Hasan Javed | Ms. Shagufta Saeed | Dr. Abu Saeed Hashmi | Dr. Muhammad.
Material type: ; Format:
print
Publisher: 2013Dissertation note: 6-aminopenicillanic acid is ?-lactam nucleus produced by penicillin acylaseupon hydrolysis of penicillin. 6-APA is main component of semi-synthetic penicillins. Penicillin acylase is most valuable enzyme and is produced by many microbes such as Escherichia coli. Different media and method were used for the isolation, identification an characterization of E. coli. Total 30 strains of E. coli were isolated from fecal matter of equine species and tested for the penicillin acylase activity. About 13 isolates gave the enzyme activity.
For the production of cell mass, different low cost media was used to cut down the price of production. Corn steep liquor, molasses, milk whey and wheat bran was tested for the growth of E. coli. These industrial wastes can minimize the production cost of 6-APA which has a high demand for the production of semi-synthetic penicillins. Corn steep liquor showed better growth of E. coli and can be used as the cheap source of carbon and nitrogen.Phenylacetic acid was also used in the growth medium and it was used as the inducer for enzyme. Without phenylacetic acid in medium, enzyme production decreases.
Corn steep liquor is the best sources for production of cells which is 0.520 mg mL-1 Molasses also better for fermentation and highest value is 0.336 mg mL-1. Milk whey media needs further studies for the better production of cells with using different concentrations.it gave best production 0.112 mg mL-1 Wheat bran is not proper source for cell production and does no showed E. Coli growth. All the strains showed growth in corn steep liquor, milk whey and molasses but not in wheat bran. Among all the strains horse sample (Ho-9) showed better cell production in all the media used.
Availability: Items available for loan: UVAS Library [Call number: 1571,T] (1).
5.
Identification Of Pesticide Residues In Different Vegetable Collected From Market Of Lahore, Pakistan.
by Anam Munawar | Dr. Muhammad Imran | Dr. Abu Saeed Hashmi | Dr. Muhammad.
Material type: ; Format:
print
Publisher: 2013Dissertation note: Pesticides are the chemicals which are used to kill or repel the unwanted objects such as pests. Different types of pesticides are present which undergo a different mechanism and kill the pests. Four different types are being used in Pakistan such as organophosphate, organochlorine, pyrehtroid and carbamates. Use of organophosphate and organochlorine become less due the presence of residues. Use of pesticides is increased for a number of purposes such as to increase the rate of production, to decrease the damage of crops and to increase the saving time of different vegetables.
Vegetables are the main source of income of Pakistan, and vegetables are common in our use. Vegetables contain different nutritional elements of our diets. That's why vegetables play an important role in the nutritious diet of a person. The spray of different chemicals on vegetables not only decreases the nutritional elements but also increase the risk of different diseases.
As pesticides leave their residues in vegetables, different techniques can be used to detect the residues and their maximum residue limit, at which limit these pesticides are harmful for humans. Pesticides can also act on unintended individual such as human beings and cause different acute and chronic diseases.
Different vegetables were selected for analyses that are common in use and available in every season. Pesticides which were selected are that which are common in Pakistan and from different pesticide classes.
In present study vegetables of different areas of Lahore were collected and analyzed through HPTLC and GC/MS. HPTLC was used to analyze and calculate the concentration and GC/MS was used for the confirmation of results, and it was concluded that which vegetable contain the high concentration of pesticides. It was studied that which vegetable absorb large amount of pesticides. Potato, tomato, egg plant, okra and cucumber of different markets of Lahore contain high concentration of pesticides as compared to the other vegetables.
Availability: Items available for loan: UVAS Library [Call number: 1510,T] (1).
6.
Biochemical Identification Of Various Causes Of Anemia In Females From District Pakpattan
by Hafiz. Muhammad Toqeer | Dr. Muhammad Tayyab | Dr. Abu Saeed Hashmi | Mr. Muhammad.
Material type: ; Format:
print
Publisher: 2013Dissertation note: Anemia is estimated to be affecting almost 600 millions people all over the globe and is regarded as deficiency in Hemoglobin concentration. The decreased amount of hemoglobin in blood could not been able to fulfill the oxygen demand of tissues in body. Keeping in view the above situation, a study was planned to investigate the various types of anemia in dist. Pakpattan.
One hundred blood samples were collected from females randomly selected from various parts of district Pakpattan. The samples were divided into two groups on the basis of age. Group A contains the patients with age between 14 to 26 years where as Group B consist of patients with age 27 to 40 years. Samples were processed in-order to estimate Complete Blood Count, serum iron level, serum ferritin levels, vitamin B12 assay and HPLC based estimation of various variants of hemoglobin.
The results demonstrated that 62% of the total female population of dist. Pakpattan was found to be anemic. Among Group A, 66.66% were anemic due to iron deficiency and 33.33% were due to chronic disease. Group B contained 59.09% anemic, out of these patients, 57.69% were anemic due to iron deficiency, 38.46% due to chronic disease and 2.27% due to deficiency of Vitamin B12. Iron deficiency was found to be the major cause of anemia that is followed by anemia due to chronic disease and Vitamin B12 deficiency. The intensity of anemia was 5% higher in young age females (Group A) as compared to the elder age females (Group B).
This work provided the information about the prevalence of various types of anemia in the population of dist. Pakpattan. The data will be helpful for developing strategy for the control of anemia in future. Further study with a large number of samples, is required throughout the country for the establishment of a data base that will be a good step to control various types of anemia.
Availability: Items available for loan: UVAS Library [Call number: 1611,T] (1).
7.
Isolation, Purification And Characterization Of Xylanase From Aspergillus Flavus (Wild Stin) Using Agriculture Waste as Substrate
by Hadia Rehman | Ms. Asma Waris | Dr. Abu Saeed Hashmi | Dr. Muhammad.
Material type: ; Format:
print
; Literary form:
drama
Publisher: 2013Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1634,T] (1).
8.
Identification Of Pesticides Residues In Defferent Samples Of Milk
by Neelam Shahzadi | Dr. Muhammad Imran | Dr. Abu Saeed Hashmi | Dr. Muhammad.
Material type: ; Format:
print
; Literary form:
drama
Publisher: 2013Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1646,T] (1).
9.
Comparison Of Locally Available Synthetic And Non-Synthetic Powders For Latent Fingerprint Development
by Arman Khan | Dr. Muhammad Wasim | Dr. Abu Saeed Hashmi | Dr. Wasi.
Material type: ; Format:
print
Publisher: 2013Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1718,T] (1).
10.
Estimation Of Caffeine In Decaffeinated Coffee And Tea Available In Pakistan
by Muhammad Abbas Sadiq | Dr. Muhammad Wasim | Dr. Abu Saeed Hashmi | Ms. Sehrish.
Material type: ; Format:
print
; Literary form:
drama
Publisher: 2013Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1719,T] (1).
11.
Estimation Of Cyanide In Different Speciis Of Apple Seed
by Zohra Bhatti | Dr. Muhammad Wasim | Dr. Abu Saeed Hashmi | Dr. Ali Raza.
Material type: ; Format:
print
Publisher: 2013Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1748,T] (1).
12.
Method Development And Estimation Of P-Phenylenediamine In Biological Sample.
by Muhammad Adnan Jamil | Dr. Muhammad Wasim | Dr. Abu Saeed Hashmi | Ms. Sehrish | Faculyt of Biosciences.
Material type: ; Format:
print
; Literary form:
drama
Publisher: 2013Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1754,T] (1).
13.
Biochemical Evaluation Of Armoracia Rusticana And Raphanus Sativus On Alloxan Induced Diabetic Rats
by Nadia Rana (2012-VA-540) | Ms. Asma Waris | Dr. Abu Saeed Hashmi | Dr. Muhammad Wasim.
Material type: ; Literary form:
not fiction
Publisher: 2014Dissertation note: Diabetes mellitus (DM) is a metabolic disorder characterized by hyperglycemia in which the body does not produce or properly utilize insulin. It the reason of interruption in protein, carbohydrate and lipid metabolism and caused the complications such as nephropathy, microangiopathy and retinopathy. It is the most widespread endocrine disorder, affects more than 176 million people worldwide (WHO 2004).
Diabetes mellitus is generally classified into three types; type I, type II diabetics and gestational diabetes (Velho and Foguel, 2002). Type I diabetes mellitus is commonly occur among young people, it is also known as juvenile-onset diabetes or insulin dependent diabetes mellitus. Type I is the result of absolute deficiency of insulin that is commonly caused by the chronic autoimmune disease that results from complex interaction of both genetic and environmental factors (Pietropolo 2001). Type II diabetes mellitus is mostly occur in adults aged 40 years or above, it is commonly known as non-insulin diabetes mellitus characterized by too much hepatic glucose production, reduced insulin secretion from beta cells of pancreas, and peripheral tissues such as muscle adipose and liver become resistant to insulin (Ahmad 2006).
Association of hyperglycemia with long term damage, dysfunction as well as ultimate organs failure, mainly the heart, blood vessels, eyes, kidney and nerves has previously been recognized (Hung et al. 2005).
Dyslipidemia is another main reason of mortality and morbidity that results in development of cardiovascular complications (Reasner 2008). It is a main risk factor of diabetes and mostly result from prolonged hyperglycemia and insulin resistance in both (type I and type II) diabetic patients is called ‘diabetic dyslipidemia’ (Mooradian 2009). Hyperlipidemia and an increase in blood cholesterol and triglyceride are results from decrease in lipolysis which is caused by deficiency of insulin, eventually increases the risk of heart attack and atherosclerosis (Avramoglu et al. 2006). The risk of heart disease, stroke, kidney disease, retinopathy, neuropathy, ulceration and gangrene of extremities is increased with association of diabetes mellitus (Rotshteyn and Zito, 2004).
According to current statistics, diabetes mellitus is worse or greater in developing countries than the developed countries worldwide (Oputa 2002). So there is a great need to discover, design and test new drugs having dual therapeutic properties to control and cure both closely related critical diseases, diabetes and dyslipidemia and their mutually linked chronic complications (Bhandari et al. 2002).
In order to design and develop the drugs for the treatment, one of the best strategies is experimental animal models to understand pathophysiology of any disease (Rees and Alcolado, 2008; Chatzigeorgiou et al. 2009). For studying and testing anti-hyperglycemic agent, several animal models have been developed for the past few decades (Srinivasan and Ramarao, 2007). Chemical induction of experimental diabetes by alloxan is one of the most effective methods (Etuk 2010).
Alloxan is a widely used diabetogenic agent that induced the type I diabetes in animals but it also represent the end stage type II diabetes milletus: as there is severe deficiency of insulin in plasma, the end stage type II diabetes mellitus also adopts the characteristics of T1DM (Viana et al. 2004). Alloxan exerts its action by generating reactive oxygen species (ROS) along with cytosolic calcium raised in islet B of pancreas, when administered parenterally (Szkudelski 2001). Diabetic dyslipidemia is also acquired by the untreated alloxan induced diabetic animals (Alnoory et al. 2013).
Currently herbal remedies are in great demand due to side effects associated with therapeutic synthetic drugs (Mahmood et al. 2011). There are large numbers of plants that have shown effective hypoglycemic activity after laboratory testing, more than 1200 plants species are used in the treatment of diabetes mellitus worldwide (Eddouks et al. 2005).
It is believed that antioxidants present in the diet help to reduce certain diseases, vegetables are rich in these compounds (Astley 2003; Bazzano et al. 2002). There are large number of herbs, spices and other plant materials that have shown hypoglycemic and antioxidant properties, and are less harmful than synthetic drugs (Eidi et al. 2006). For the development of new pharmaceutical lead along with dietary supplement to already existing therapies, medicinal plants provide a valuable source of oral hypoglycemic compounds (Bailey and Day, 1989).
Raphanus sativus (radish) belong to the family Brassicaceae and it is an edible root vegetable (Lewis-Jones et al. 1982). Radishes contain high quantity of calcium, magnesium potassium, copper, ascorbic acid, folic acid, vitamin B6, and riboflavin and low amount of saturated fat and are very low Cholesterol (Nunes et al. 2011). Roots, seeds and leaves are the different parts of radishes (Raphanus sativus) that are used for medicinal purposes (Nadkarni et al. 1976). Radish roots are beneficial to protect the cell membranes against lipid peroxidation and also inhibit the changes in membrane caused by fat rich diet (Sipos et al. 2002).
Radishes (Raphanus sativus) have good hypoglycemic potential coupled with antidiabetic efficiency (Shukla et al. 2011). Due to hyperlipidemia the probabilities of cardiovascular disease increases in diabetic patient. Raphanus sativus (radish) is a traditional plant which is used to lower plasma lipid. It has the capability to lower the plasma triglyceride, cholesterol, and phospholipids in normal rats (Taniguchi et al. 2006).
Radishes are recommended as an alternative treatment for various diseases including hyperlipidemia, coronary heart diseases and cancer due to its high medicinal and nutritional value (Cetin et al. 2010). Phosphatase, catalase, sucrase, amylase, alcohol dehydrogenase and pyruvic carboxylase are the main enzymes that found in the radish roots (Singh et al. 2013). It is beneficially used in curing poor digestion and liver dysfunction (Lugasi et al. 2005), antioxidant activities (Wang et al. 2010), anti tumorigenic (Kim et al. 2011), anti-diabetic (Shukla et al. 2010). The leaves of radish are good source of protein (Singh and Singh, 2013).
Armoracia rusticana (Horseradish) belongs to the Brassicaceae family; it is a hardy perennial plant, mustard and cabbage are also including in this family. The roots of horseradish are rich in vitamin C and B1, iron, potassium, calcium and magnesium, phytoncide and essential oils; Allyl isothiocyanate a (volatile aglycone) which is released by a glycoside is identical with the essence of mustard plant (Istudor 1998). Root of horseradish smells pungent due to the allyl sulfide, a substance present in garlic and onion.
Armoracia rusticana is a source of many compounds that have been broadly studied for various health benefits (Lin et al. 2000). It contains several substances that have beneficial effects on peripheral blood flow. Its utilization normalizes the blood pressure and prevents the risk of thrombosis and sulfurous substances also improve the elasticity of cerebral and coronary blood vessels (Cirimbei et al. 2013). It has antibacterial properties due to allyl isothiocyanate present in volatile oils, especially mustard oil (Rosemary 1976).
The main component of the horseradish and the other vegetables from Brasicaceae family is sinigrin, degraded by the myrosinase enzyme complex to the allyl isothiocyanate (Wang et al. 2010). The enzyme horseradish peroxidase, is a heme-containing enzyme found in the plant that utilizes hydrogen peroxide to oxidise a extensive variety of organic and inorganic compounds, widely used in molecular biology and biochemistry (Bladha and Olssonb, 2011).
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14.
DNA Based Characterization Of Arginase Gene From Geobacillus Sp. SBS-4s
by Raabia Bibi (2012-VA-537) | Dr. Muhammad Tayyab | Dr. Abu Saeed Hashmi | Dr. Muhammad Imran.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Geobacillus is a group gram-positive, rod-shaped, aerobic, endospore-forming and obligate thermophilic bacteria, isolated from the diverse habitats, hot springs, thermal environments, terrestrial soils, deep sea sediments (Zeigler, 2014), petroleum and soil of desserts (Claus and Berkeley 1986). It grows at a wide range of temperature from 45 to 75°C and pH ranging from 6.2 to 7.8 (Nazina et al. 2001). These bacteria survives at higher temperature where most of other living species fail to survive (Claus and Berkeley 1986). Geobacillus have achieved a significant population with a worldwide distribution, probably in large part due to adaptive features of their spores (Zeigler, 2014). These can be found singly or in short chains and motile by means of peritrichous flagella and is capable of secreting a wide variety of extracellular and intracellular enzymes i.e amylase, lipase, carboxypeptidase, cellulase, xylanase, protease and galactosidase (Fogarth et al. 1974; Obeidat et al. 2012).
Geobacillus sp. SBS-4S was isolated from hot spring located in Gilgit, Northern areas of Pakistan. It was found to be an aerobic, gram-positive and rod-shaped bacteria having ability to hydrolyze a variety of sugars, carboxylic acids and hydrocarbons at elevated temperatures from 45 to 75°C. SBS-4S was found to be involved in the production of various intra and extra cellular enzymes (Tayyab et al. 2011).
Arginase is the enzyme responsible for the degradation of arginine resulting in the production of urea and ornithine (Kaur et al. 2009). It is accomplished by the cleaving of the guanidinium group from arginine which yields urea (Turras et al. 2008). Arginase present in many mammals (Homo sapiens), Bacilli (cyanobacteria), protozoa (Entamoeba histolytica), yeast (Saccharomyces cerevisiae), fungi (Neurospora crassa) and plants (Lathyrus sativus) etc (Kaur et al. 2009). The crystal structure of arginases have been determined by X ray crystallographic studies. This is a manganese dependent enzyme. The enzyme shows its activity through the metal ion. Metal ion is actively responsible for the incorporation of water molecules essential for the activity of the enzyme. A second proposed mechanism, based on electron paramagnetic resonance (EPR) studies postulates direct coordination of the substrate to manganese and disruption of the aqua bridge. Arginases are homo-oligomers, with a typical subunit mass of 32 to 36 kDa (Bewley et al. 1999).
There are two types of arginases, arginase-I and arginase-II, located in the cytoplasm and mitochondria, respectively. The principal ureagenic enzyme activity arginase-I is most abundant in normal mammalian liver and acts in coordination with the other enzymes of the urea cycle to sequester and eliminate excess nitrogen from the body. The second form arginase-II can be found in many organs, with the highest levels found in kidney and prostate where as lower levels in macrophages and lactating mammary glands (Iyer et al. 2002).
Important role of arginase in controlling the cellular levels of arginine and ornithine, which are required for various critical metabolic processes, including protein synthesis and the production of creatine, polyamines, proline and nitric oxide (NO). Type II arginase is found in a variety of different tissues and have a key role in the regulation of urea cycle arginine metabolism by regulating levels of arginine in the cell (Bewley et al. 1999). The enzyme arginase plays key role in the pathogenesis of pulmonary disorders such as asthma through dysregulation of L-arginine metabolism and modulation of nitric oxide (NO) homeostasis and it also play role in the development of chronic airway remodeling through formation of ornithine with downstream production of polyamines and L-proline, which are involved in processes of cellular proliferation and collagen deposition (Benson et al. 2011). Arginase involved in tissue repair processes by the synthesis of L-ornithine, which is the precursor of polyamines and proline that are involved in cell proliferation and collagen synthesis (Maarsingh et al. 2009).
Genetically engineered arginase as fusion protein with prolonged half-life and increased efficacy are used to treat different tumor lines that inhibit cell proliferation and impaired cellular migration in vitro and in vivo (Li et al. 2013). This is a arginine-degrading and ornithine producing enzyme and is used to treat arginine-dependent cancers (Yu et al. 2013). Chemically modified arginase-II has been employed for the treatment of taper liver tumor and L5178Y murine leukemia (Kaur et al. 2009). The enzyme was cloned and expressed in E. coli and subsequently conjugated to polyethylene glycol to increase the circulating half-life and decrease the immunogenicity of the recombinant mycoplasma enzyme. The human hepatocellular carcinoma, melanoma cell lines and tissue samples do not express argininosuccinate synthetase (ASS), making them auxotrophic for arginine and thus reasonable candidates for arginine deprivation (Yang et al. 2010).
Arginase is induced in murine myeloid cells mainly by T-helper 2 cells cytokines and inflammatory agents and participates in a variety of inflammatory diseases by down-regulation of nitric oxide synthesis, induction of fibrosis and tissue regeneration. In humans, arginase I is constitutively expressed in polymorphonuclear neutrophils and is liberated during inflammation. Myeloid cell arginase-mediated L-arginine depletion profoundly suppresses T cell immune responses and this is a fundamental mechanism of inflammation-associated immunosuppression. Pharmacological interference with L-arginine metabolism is a novel promising strategy in the treatment of cancer, autoimmunity or unwanted immune deviation (Munder, 2009).
Arginase has very important role in nitrogen fixation and fruit ripening (Yu et al. 2013). Putrescine (1,4-butanediamine) is the product obtained from arginine with the highest market value and it is used as an intermediate in a large number of industries, including the pharmaceutical industry, agrochemical industry and textile industry (Turras et al. 2008).
Arginine is a semi-essential amino acid and is the precursor for the formation of nitric oxide (NO) by nitric oxide synthases (Getz and Reardon, 2006). One of the major functions of arginine within the body is as an intermediate in the urea cycle. In the cytosol of hepatocytes, arginase-I removes the guanidine group from arginine to produce urea and ornithine. Urea is then transported from the hepatocyte into the bloodstream and ornithine is used to regenerate arginine within the hepatocyte. Arginine deficiency causes several disorder like, hyper cholesterolemia, hypertension, diabetes mellitus, kidney failure, hyper homo-cysteinemia, smoking, and aging (Alvares et al. 2012). Arginine is used to modulate the cellular immune response during infection. The generation of nitric oxide from arginine is responsible for efficient immune response (Das et al. 2010).
Arginine is synthesised in humans and other mammals from citrulline in two steps through the urea cycle enzymes, argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL). ASS catalyses the conversion of citrulline and aspartic acid to argininosuccinate, which is then converted to arginine and fumaric acid by ASL (Yang et al. 2010).
Ararinase play important role in conversion of arginine to 1,4–butanediamine (a building block for nylon-4,6), through two main transformations: the hydrolysis of arginine to ornithine and urea; and the decarboxylation of ornithine to 1,4–butanediamine and carbon dioxide. Both steps can be catalyzed chemically or enzymatically (Turras et al. 2008).
The present study deals with the characterization of arginase gene.
Availability: Items available for loan: UVAS Library [Call number: 2244-T] (1).
15.
Expression And Mutational Analysis Of Breast Cancer Susceptibility Gene 1 (Brca1) And Cyclooxygenase-2 (Cox-2) Gene In Feline And Canine Tumours
by Haleema Sadia (2007-VA-567) | Dr. Muhammad Wasim | Prof. Dr. TahirYaqub | Dr. Abu Saeed Hashmi.
Material type: ; Literary form:
not fiction
Publisher: 2014Dissertation note: Cancer is the first cause of death in cats and dogs while in human it is the second most cause of death (Jemal et al. 2008). According to an estimation, cancer related deaths in the world are 13% and 70% of these deaths are in poor countries (World Health Organization 2012). Such natural cases of cancers in cats and dogs especially, in dogs offer an opportunity to use the dogs for comparative cancer studies and as an animal model for anticancer drug development (Pawaiya 2008). Inu a series of more than 2000 autopsies, it was found that almost forty five percent dogs that lived for ten or more years expired because of cancer (Bronson 1982). Dogs are affected by skin cancer 35 times more often than humans. They are also affected 4 times more often by mammary gland cancer, 8 times more often by bone cancer, and twice more often by leukemia, than humans (Cullen et al. 2002). The regulation of cell proliferation, genome stability and programmed cell death are important for systemic homeostasis.
1.1Historical perspective on cancer causation
Hippocratic and Galenic medicine attributed the spread of black bile (one of the four humours) in the tissue as the cause of the cancer (Diamandopolus 1996) is an idea survived intact through the Middle Ages and Renaissance. With the discovery of the lymphatic system by Gasparro Aselli in 1662, the black bile theory was superseded by the idea that cancer was an inflammatory reaction to extravasated lymph; a theory modified 150 years later by John Hunter who introduced the notion that contaminated coagulating lymph was the origin of the cancer (Kenneth 2003). A German pathologist Johannes Muller first time demonstrate that cancer is made up of cells (1838) but he also gave an idea that cancer cells were originated from a bud called Blastema instead of normal cells (Kardinal and Yarbro 1979). Following Schleiden and Schwann's cell theory of tissues,it was Rudolf Virchow (Muller’s student) who in 1855 demonstrated that every cell was derived from another cell (omnis cellula e cellula), including cancer cells (Mazzarello 1999; Porter 1999). In 1867 Wilhelm Waldeyer supported the theory of the normal cell for the origin of cancer and he believed that metastasis resulted from transportation of cancer cells by blood or lymph (Porter 1999).
Around the turn of the twentieth century the beginning of tumour transplantation experiments led to the new view of the cancer cell as an autonomous cell. The first successful tumour transplants were described in 1876 by the Russian veterinarian Mstislav Aleksandrovich Novinski (Novinski 1876). He reported in his thesis entitled “On the Question of the Inoculation of Malignant Neoplasms” the first successful serial passage of tumours through transplantation in dogs. Novinski's transplantation experiments were based on the inoculation of canine transmissible venereal tumour (CTVT) in puppies. Novinski stated that successful tumour transplantation depends on the inoculation of a living element of the tumour and that the transplantation of the element of a cancerous tumour to healthy tissue acts as an infecting agent. In 1888 Wehr repeated Novinski's transplantation experiments in dogs with similar results (Shimkin 1955). It is interesting to note that the dogs used for transplantation of CTVT did not come from a single breed and were therefore not highly inbred. The allo-transplantation of tumours seemed less surprising in the late 19th Century than it does today with our modern knowledge of histo-incompatibility.
The successful results obtained with CTVT served as model for tumour transmission in other animals. Hanau in 1898 inoculated two rats with vulvar epidermoid carcinoma and observed growth of the tumour in the recipients (Shimkin 1955). In 1901 Leo Loeb supported the transplant ability of tumours in rats (Witkowski 1983; Brent 1997). In 1903 a Danish veterinarian Carl O. Jensen determined the successful growth of transplanted tumours in mice by heredity (Brent 1997). The discovery that the tumour could be successfully transplanted into (Witkowski 1983; Brent 1997) other mice, led the scientists to use rodent system to supply tumours for experiments. The observation that a single tumour could be expanded through many generations exceeding the life span of the laboratory mouse led Leo Loeb to the "cancer immortality" concept (Witkowski 1983).
The earliest observations reported by John Hill in 1759 and by Percival Pott in 1775 on the association of a specific tumour to a specific profession or work, led to the idea that some chemicals can cause cancer (Greaves 2000). In 1918 Yamagiwa and Ichikawa induced cancer by applying coal tar to rabbit skin(Greaves 2000; Luch 2005). After the discovery of the X rays by Wilhelm Conrad Roentgen in 1895, Frieben published data in 1902 indicating that cancer rates were increased among persons working with X-rays (Cassileth 1983; Greaves 2000)
1.2 Tumour Progression
The first detailed characterization of the dynamic nature of cancer was described by Leslie Foulds (Foulds 1949). Foulds showed that tumours progress (evolve) through different stages, characterized by the acquisition of different phenotypic traits such as increased growth rate, hormone dependence, invasiveness, formation of metastasis (Foulds 1949; Fould 1954; Foulds 1957). With the progress of molecular biology the phenotypic view had been replaced with the somatic mutation theory, where cancer evolved through the accumulation of different mutations in several genes (Greaves 2000). The accumulation of mutations in somatic cells implicated the presence of different cells bearing different mutations and also the presence of natural selection, which selected the cells with advantageous mutations. One of the questions arising from the somatic mutation theory was whether a tumour had a single or a multiple origin.
This observation was supported by a karyotype analysis in chronic myelogenous leukaemia (CML) by Peter Nowell and David Hungerford in 1960 (Nowell 2002). They described the presence of an unusually short chromosome 22 in all CML tumour cells analyzed, and the absence in the normal cells from the same patients. This observation suggested that this mutation was a somatic mutation that occurred in one cell in the bone marrow, which gave it a selective advantage to expand as a clone. Nowell postulated that a tumour develops by a Darwinian evolutionary process, where cells with mutations conferring a growth advantage are selected and expanded (Nowell 1976; Greaves 2002). In 1954 Peter Armitage and Richard Doll analyzed human cancer incidence over the age, and showed that chances of cancer increased in older people (Armitage and Doll 1954).
The concept that cancer might be contagious also recurs throughout the past 300 years.In the 17th and 18th centuries, physicians Daniel Sennert and Zacutus Lusitanus supported the hypothesis that cancer was contagious. In fact in 1779 a hospital in Paris was directed to move the cancer patients from the city (Cassileth 1983; Kenneth 2003).
1.2.1 Exogenous and endogenous factors
In 1844 the Italian physician Domenico Antonio Rigoni-Stern noted that cancer of the cervix was frequent among married ladies, rare among unmarried ladies and absent in Italians nuns. In contrast, breast cancer was more frequent among nuns (Greaves 2000). These observations led to the hypothesis that cervical cancer was sexually transmitted, and we now know that the cause is a papilloma virus (Hausen 2002).In 1908 Wilhelm E and Olaf B, transferred the leukemia in chicken by tissue filterates (Wyke 2003). In 1911, Peyton Rous demonstrated that viruses were the cause of solid tumours (Sarcoma) in chickens but it took many decades before his data were accepted (Dulbecco 1976). The notion that viruses can cause cancer was a discovery that brought back the fear that cancer was a contagious disease.
There are many exogenous and endogenous risk factors that affect the tumor suppressor genes and oncogenes (Todorova 2006). Tumour viruses (Bishop 1980), chemical carcinogens (Loeb et al. 2000), natural chemicals, (Ames et al. 1990), herbicides (Glickman et al. 2004), physical carcinogens like radiation (Upton 1978) are exogenous factors while inherited genetic defects, immune system (Rosenthal 1998) and hormonal factors (Rodney 2001) are among endogenous risk factors.
Although tumour cells are generally described as independent evolving units, recent results suggest that tumour cells are able to stimulate stromal cells to produce growth factors that increase tumour proliferation (heterotypic stimulation) (Kinzler and Vogestein 1998; Skibe and Fuseing 1998; Iyengar et al. 2003). It has been demonstrated that cells involved in the immune response to tumours may produce factors such as inflammatory chemokines that may also promote the tumour proliferation (Pollard 2004; Wyckoff et al. 2004)
1.2.2 Two hit hypothesis
Retinoblastoma is a tumour that becomes manifested early in life. Retinoblastoma can be inherited or sporadic. According to the two hit hypothesis in the inherited form a single mutation in the Retinoblastoma (Rb) gene is present in the germ line which gives the genetic predisposition to develop cancer, but a second mutation in the normal Rb allele which occurs in the retinoblast must be acquired to develop cancer (Knudson 2001). In the sporadic form the two mutations in the Rb alleles occur in the somatic cells. Although the epidemiological and molecular observations have consolidated the multistage theory of cancer, the number of mutations and in which sequential order they have to be acquired to develop cancer is still an open question (Hanahan and Weinberg 2001; Hahn and Weinberg 2002b).
1.2.3 Oncogenes
Early experiments involving transforming retroviruses and the transfer of genes from tumour cells into established rodent cells allowed the identification of several cancer causing genes called oncogenes. The result of these experiments suggested that cancer could be induced by the mutation of one proto-oncogene. However, the rodent cells used as recipient in the gene transfer experiments were not normal, but were immortalized, thus acquiring the ability to proliferate indefinitely. When the normal rodent cells were used, the transfer of a single oncogene failed to induce transformation, while the transfer of two oncogenes resulted in transformation. Human cells require more mutations than rodent cells and that there are differences also between cell types within the same species (Rangarajan et al. 2004).
1.3 Cancer Hallmarks
Despite the enormous variety of tumours affecting different types of tissues in animals and humans, research over the past 50 years has revealed that all malignant cancers share the same essential alterations (Hanahan D and Weinberg RA 2000).
These hallmarks include:
Immortalization
Evasion from programmed cell death (apoptosis)
Independence from growth stimulation
Resistance to growth inhibition
Angiogenesis
Invasion and metastasis
Genetic instability.
These hallmarks are briefly described below.
1.3.1 Immortalization
Telomeres contain DNA sequence repeats and protein. The repeat sequence consists of hexameric motifs such as GGGTTA in humans, extended for 10 –20 kilobases. The 3’ end has a 100-400 nucleotide over-hang (Mathon and LIoyd 2001). Telomeric DNA is generated by an enzyme called Telomerase Reverse Transcriptase (TERT) which has two subunits, RNA and catalytic protein subunit. This RNA binds the telomeres DNA ends thus acting as template for telomere elongation. The chromosome ends are protected by several proteins: TRF-1, TRF-2, and POT–1 (Mathon and LIoyd 2001; Hahn and Weinberg 2002a). Several experiments have shown that senescence is activated when the telomeres are shortened down to 5 kb and that senescence is triggered by the shortest telomere present in the cell (Hemann et al. 2001).
Many reports have suggested that the replicative senescence is not activated by the erosion of the double strand repetitive sequence, but by the degradation of the 3’end single strand overhang, resulting in loss of protective capping (Stewart et al. 2003). Telomere length is maintained by the activation of telomerase or by an alternative mechanism called alternative lengthening of telomeres (ALT), where the telomeres are regenerated through recombination-based inter chromosomal exchange of sequence information (Bryan et al.1997; Dunham et al. 2000). In the normal cell telomerase is transiently expressed, since it can be detected only in S phase, but in neoplastic cells its expression is increased and is detectable throughout the cell cycle (Mathon and Lloyd 2001). In tumour cells the senescence and crisis barriers are avoided by the activation of telomerase regenerating the telomeres and by the inactivation of tumour suppressor and pro-apoptotic genes (Hanahan and Weinberg 2000; Hahn and Weinberg 2000b).
1.3.2 Apoptosis.
The sensors detect the intra- and extra-cellular signals. The intracellular signals include DNA damage, hypoxia and oncogene overexpression (Evan and Littlewood 1998). The extracellular signals monitor the cell-cell and cell-matrix homeostasis (Aoshiba et al. 1997; Prince et al. 2002; Alberts et al. 2002a). The signals detected by the sensor are mainly conveyed to the mitochondria, where a series of cytoplasmatic proteins of the Bcl2 family control the release of cytochrome C from the mitochondria (Alberts et al. 2002a). The release of cytochrome C activates an array of intracellular proteases called caspases causing protein and DNA degradation (Hanahan and Weinberg 2000). The caspases can be directly activated by extracellular proteins such as FAS ligand, which binds to the death receptor FAS (Houston and O’ Connell 2004).
Once the caspase cascade is triggered it cannot be inactivated (Alberts et al. 2002a). It has been reported that the tumour suppressor p53 can trigger the caspase cascade by the overexpression of the Bax protein, a member of the Bcl2 family, which in turn increases cytochrome C release thus inducing apoptosis (Hanahan and Weinberg 2000). In CTVT it is likely that expression of c-myc is up-regulated, due to insertion of a LINE-1 element as discussed later. Ectopic c-mycexpression can promote tumour growth and survival, as seen, for instance, in immunoglobulin gene c-myc chromosome rearrangements in Burkitt's lymphoma (Hemann et al. 2005).
1.3.3. Independence from growth stimulation
1.3.3.1. Growth factors
Thus the proliferation of a cell is dictated by the needs of the cells around it (Hanahan and Weinberg 2000). In contrast, a tumour cell escapes from the external dependence to become an autonomous evolving unit, by producing its own growth signals.
1.3.3.2 Growth factor receptors
Another mechanism selected by tumour cells is the overexpressions of growth factor receptors, which induce the tumour cells to become sensitive to concentrations of growth factor that normally, do not trigger proliferation (Hanahan and Weinberg 2000). Proliferation can also be induced by a mechanism independent of the growth factor, for example the alteration of the cytoplasmic tail of growth factor receptor causes self-activation of the receptor, which therefore becomes independent from the external microenvironment (Alberts et al 2002b).
1.3.4 Resistance of growth inhibition
Like growth signals, the anti-proliferative signals derive from soluble factors or surface proteins that are produced by neighbouring cells, or are induced by components of the extracellular matrix (Hanahan and Weinberg 2000; Alberts et al. 2002d). These external inhibitory signals activate different intracellular pathways that regulate the cell cycle (Alberts et al. 2002c).
The Rb protein and its related proteins, p107 and p130 play a key role in controlling this transition (Weinberg 1995). The association of Rb with the transcription factor E2F inhibits the transcription of genes involved in the G1-S progression (Alberts et al. 2002c). The hyper-phosphorylation of the Rb protein induces the dissociation with E2F, therefore allowing progression to S phase (Alberts et al. 2002c). Normally complexes of cyclin and cyclin dependent kinase (CDK) induce the phosphorylation of the Rb protein (Alberts et al. 2002c). Many tumours can avoid the antigrowth signals by altering Rb activity or the proteins involved in Rb phosphorylation (Mittnacht 2005).
1.3.5 Angiogenesis
Although the majority of the new vessels in adult tissues are derived by sprouting from existing vessels, many evidences indicate that progenitor endothelial cells are derived from the bone morrow contributing to the vessel growth (Zhang et al. 2000; Contreras et al. 2003; Nishimura and Asahara 2005; Religa et al. 2005). Although endothelial cells are highly proliferative in response to several angiogenic factors, they have long half-lives up to several years (Carmeliet 2003). In order to adapt the vascular system to the tissue's requirements, several mechanisms regulate the process of angiogenesis (Carmeliet 2003). A key molecule involved in the angiogenesis process is the vascular endothelial growth factor (VEGF) (Carmeliet 2003). In addition it has been demonstrated that tumours can activate or inactivate pro- and anti-angiogenic factors respectively present in the extracellular matrix by producing several proteases (Gately et al. 1997; Harlozinska 2005).
1.3.6 Metastasis
In cancer during tumour progression, some tumour cells acquire the ability to migrate and form new colonies at secondary sites and these cells then make new tumour cells (Hanahan and Weinberg 2000). It has been estimated that 90 % of mortality associated with cancer is due to metastasis (Sporn 1996). Results show that few cells in the primary tumour acquire the ability to grow in the secondary sites and that the tendency to metastasise is acquired in the early steps of tumour progression (Van’t Veer and Weigelt 2003).
Progressive alteration of normal tissue homeostasis by tumour and stromal cells, allow tumour cells to move throughout degraded matrix, and to invade surrounding tissues (Hanahan and Weinberg 2000). Tumour cells are also aided to migrate by soluble factors (chemotaxis) and bound adhesion molecules (haptotaxis) (Nguyen 2004). In order to invade new organs, circulating tumour cells need to stop and exit the systemic circulation. In an unspecific manner, the extravasation may be due to the fact that large arteries progressively narrow in to arterioles and then capillaries and tumour cells can be trapped in this small vessel, thus allowing the migration in the new organ (Nguyen 2004). Although the exact mechanism behind the tumour homing is not completely understood, recent results suggest that the selective homing of cancer cells may be due to three mechanisms: 1) presence in the target tissue of specific growth factors or appropriate extra-cellular matrix that favour the selective tumour growth, 2) presence in the target organ vessel endothelium of specific adhesive proteins that interact with the tumour cells, favouring the tumour invasion, 3) production of a chemotaxis soluble factor by the target tissue that attract the tumour cells ( Fidler 2003).
1.3.7 Genetic instability
Over the past 25 years numerous genetic alterations have been described in human and animal tumours. These genetic alterations can affect the DNA sequence and the chromosomes (Lengauer et al. 1998). The mutations of DNA include: substitution, deletion, translocation and insertion and they can affect one or more nucleotides. The necessity to transmit genetic information faithfully between generations demands genetic stability (Eisen and Hanawalt 1999)
In normal conditions the genome is affected by spontaneous mutations caused by physiological DNA instability and by imprecision of the DNA polymerase proofreading activity during the DNA replication (Alberts et al. 2002e). In eukaryotic cells, several enzymes have been described with DNA polymerization activity, and five are the most important DNA polymerases involved in DNA replication and repair, alpha, beta, gamma delta and epsilon. To date the only polymerase involved in mitochondrial DNA replication is polymerase gamma. In vitro studies on the fidelity of DNA duplication has shown that the nucleotide mis incorporation rate varies among polymerases, with one in 5000 bases for beta and one in 10 000 000 for delta and epsilon polymerases (Umar and Kunkel 1996; Loeb and Loab 2000). To avoid non-complementary nucleotide incorporation, polymerase delta, gamma and epsilon contain a proofreading activity (Kunkel and Alexander 1986). Normally DNA replication is carried out by delta polymerase, but recent reports show that in some tumours this priority is shifted in favour of less accurate polymerases, thus increasing the mutation rate (Loeb and Loeb 2000). Environmental agents such as ultraviolet light, ionizing radiations and toxic substances in the dietary uptake can induce mutations (Loeb and Loeb 2000).
1.3.7a Single Base Excision Repair
When a mutation effects on a single nucleotide then base excision repair take place. BER employs enzymes called DNA glycosylases, which are specific in removing a specific mutated base (Krokan et al 2000).
1.3.7b Nucleotide excision repair
The nucleotide excision repair (NER) system is able to repair DNA damage induced by UV. In contrast to BER, the NER system recognizes altered nucleotides by scanning the DNA for a conformational alteration (bulky lesion) (Wood 1996).
1.3.7c Mismatch repair
The mismatch repair (MMR) pathway includes a series of proteins that are involved in correcting errors that escape the DNA polymerase proofreading activity during DNA replication. They are also involved in suppressing recombination between non-identical sequences both in mitosis and meiosis (Kolodner and Marsischky 1999). Unlike BER and NER, MMR does not act on damaged or mutated sequences, but it targets only the newly synthesized DNA strand.
Inactivation of the MMR system produces microsatellite instability (MSI) (Atkin 2001).
1.3.7d. Homologous recombination
Homologous recombination repairs double strand breaks by using an intact and homologous DNA molecule as a template. In eukaryotes several proteins are involved in the homologous recombination process (Kanaar et al. 1998; Haber 2000).
1.3.7e. Non-Homologous End Joining
Non-Homologous End Joining (NHEJ) is the more important repairing mechanism when there is break in DNA double strand and it is very important mechanism in mammals (Khanna and Jackson 2001). During the NHEJ process small deletions are generated. Given that majority of the mammalians genome is composed of non-coding regions, the probability that in normal situations the NHEJ process induces mutation in genes is low (Alberts et al 2002e). However, if there are multiple break points NHEJ increases the occurrence of illegitimate recombination
(Rothkamm et al 2001).
1.3.7f Chromosome Instability (CIN)
The cell reproduces by a series of events that allow DNA replication and cell division in a process known as the cell cycle. In order to check the correct order of events that take place in the cell cycle, a complex cell-cycle control system has evolved (Alberts et al 2002c). This system checks normal cell cycle progression by a series of stage-specific sensors known as checkpoints that are able to induce the arrest of the uncompleted stage until it is completed.
The two fundamental processes in the cell cycle are the duplication and the division of the chromosomes, which take place during the Synthesis (S) and Mitosis (M) phase respectively. To prevent the possibility that two daughter cells have non-identical genomes, there are two checkpoints known as DNA replication and DNA damage checkpoints before mitosis, and one known as spindle-attachment checkpoint during mitosis (Alberts et al 2002c).
Chromosome instability (CIN) is also associated with structural alteration of chromosomes, which include reciprocal and non-reciprocal translocations, amplifications, deletions and insertions (Cairns 2005). Structural chromosome instability, resulting from DNA breaks and rearrangements, is due to alteration of cell cycle checkpoints, DNA damage response and telomere integrity (Gollin 2005). Structural alterations may results in altered gene expression or produce fusion or chimeric proteins with dysregulated or new properties (Greaves and Wiemels 2003). Studies have shown that a large proportion of human tumours with chromosome instability have a high rate of loss of heterozygosity (Rajagopalan and Lengauer 2004). Therefore it has been argued that chromosome instability could accelerate the rate of inactivation or activation of tumour suppressor genes or oncogenes respectively (Rajagopalan and engauer 2004).
CIN-associated genes can be classified on the basis of the mutations (Michor et al. 2005). Class I genes of CIN, e.g Mitotic Arrest Deficient gene (MAD-2 ) boost up CIN in case one allele is mutated or deleted. Class II genes of CIN e.g. Human Budding Uninhibited by Benzimidazoles (hBUB-1) gene boost up CIN if mutation is in one allele in a dominant negative fashion. Both Class I and Class II genes are required at the spindle assembly checkpoint (Amon 1999; Hoyt 2001). Class III genes of CIN e.g. Breast cancer gene BRCA1 and another Breast cancer gene BRCA2 boost up CIN if both alleles are mutated. BRCA genes have very important role at checkpoint and it is involved in DNA repairing and recombination (Yarden et al. 2002).
1.4 Evolutionary Dynamics of Tumour Development
According to clonal evolution theory, cancer is the result of somatic mutations selected during tumour evolution (Nowell 1976). It has been argued that tumour cells cannot acquire the mutations needed for tumour progression at a physiological mutation rate, but that the tumour cell must acquire an increased mutation rate (Cairns 1998; Loeb and Loeb 2000).
In order to induce cancer the mutations must affect a variety of genes that restrain somatic conflict (Frank and Nowak 2004). These genes are known as cancer related genes and can be subdivided in three categories: Gatekeeper, Caretaker, and Landscaper (Michor et al 2004). Gatekeeper mutations increase the cellular proliferation rate by the alteration of oncogenes, tumour suppressor genes and apoptotic genes (Michor et al 2004). Caretaker mutations increase genome instability by inactivating genes involved in maintaining genome integrity (Lengauer et al 1998). Landscaper mutations increase tumour proliferation by affecting genes involved in regulating the external cellular microenvironment (Bissel and Radisky 2001).
While mutations affecting oncogenes behave in a dominant way, because only one mutated allele can induce a tumour phenotype, mutations affecting tumour suppressor genes can be neutral if the normal allele compensates the mutant allele, disadvantageous if the mutant allele triggers apoptosis, and advantageous if the mutated allele is inactivated and the second allele is insufficient to balance the wild type allele (Michor et al. 2004). In small compartments the inactivation of the two alleles of a tumour suppressor gene, is unlikely, unless the mutation rate is increased by genetic instability (Nowak et al. 2005). Loss of heterozygosity increases with chromosome instability (Michor et al. 2004).
1.5 Tumours of Feline and Canine included in this study
1.5.1Mammary Tumours
Mammary gland tumours are most frequent in dogs (Moulton 1990) while in cats it is third in prevalence, after haemopoietic and skin tumours (Misdorp et al. 1999). The average age of peak prevalence of tumours in cats is approximately 9.3 years (Roccabianca et al. 2006). Mammary tumours can also affect male cats and dogs, with the average age for them being 12.8 years (Rutterman et al. 2000). Siamese has twice the risk in comparison to other breeds of cat (Weijer et al. 1972). Same predisposition was observed with our data, that all five cases collected in this study were belonging to Siamese breed. Mammary tumours are more prevalent in Pakistan and all the cats and dogs were between 5-11 years old. This suggests that there are more chances of mammary tumours in older cats and dogs. Mammary tumours included in this study were 23% all 22 tumours studied.
1.5.2 Canine Transmissible Venereal Tumours (CTVT)
Canine transmissible venereal tumours first reported by Blaine in 1810 (Blaine, 1810) is a transmissible cancer in dogs. Studies found that CTVT was transmitted by transplantation of living cells (Novinski 1876), confirming it as a transmissible cancer. CTVT is of clonal origin, originating from a founder dog 11,000 years ago (Katzir et al. 1985; Murgia et al. 2006; Rebbeck et al. 2009; Murchison et al. 2014). It is one of only two transmissible cancers known (Murchison 2008) and is spread by allogeneic transfer of cells between dogs, usually during coitus. It manifests as a tumour, associated with the external genitalia of both male and female dogs, although tumours can also arise in the mouth, nose or skin. It is purported to be of histiocytic origin (Mozos et al. 1996; Mukaratirwa and Gruys 2003), and usually remains rather localised, except for rare cases of metastatic spread.
Recorded cases of metastasis include involvement of the lymph nodes (Higgins 1966), skin (Dass 1986) and eye (Barron et al. 1963), among others. Experimental transplants of CTVT tumours into subcutaneous sites in experimental dogs are characterized by progressive and regressive phases. This is seen as a rapid volume increase, followed by tumour shrinkage, and eventually complete regression accompanied by serum-transferable immunity to reinfection (DeMonbreun 1934). In this project we collected 6 samples for BRCA1 and COX-2 studies in different tumours while 16 more samples for Department of Veterinary Medicine, University of Cambridge, UK. Although the prevalent rate of CTVT is in second number, many attentions were paid to collect CTVTs. For BRCA1 and COX-2 studies the 28% were CTVT out of 22 different tumours.
1.5.3 Perianal adenomas/Adenocarcinomas
There are many glands present around the anus of dogs. These are sebaceous and non-secretary glands, while anal sac glands are positioned at 4 and 8 o clock to the anus and secrete their secretions into the lumen of theanal track (Yang Hai-Jie et al. 2008). Perianal adenomas are more frequent than adenocarcinomas (malignant form). In this study 3 tumours were collected which were 14% of total canine tumours collected. These tumours are mostly common in medium to older age dogs.
1.5.4 Granuloma
Granuloma is also called as lick granuloma in dogs it is a type of skin cancer It typically results from the dog’s urge to lick the lower portion of one of her or his legs. This study reported 9% of total tumours included in this study.
1.5.5 Oral Tumours (Squamous cell Carcinoma)
Oral tumours are 4th common cancers in canines. Male dogs have 2.4 times greater risk of developing oral tumours than female dogs (Dorn et al. 1968). This study reported 9% oral tumours in a period of 2 years.
1.5.6 Lymphoma
Lymphoma is the second most prevalent intra –ocular tumours of dogs. Basic cause of lymphoma in dogs is unknown but genetic (chromosomal segregation), environmental and infectious factors such as retroviruses play vital role in developments of this cancer (Fighera et al. 2002). This study reported 9% Lymphomas of total collected tumours.
1.6 Rationale behind selection of genes
1.6.1 BRCA1 gene
BRCA1 gene is tumour suppressor gene, it is involved in repairing the DNA double strands breaks and in case of failure it leads the cells towards apoptosis (Starita. 2003). BRCA1 forms BRCA1 Genome Surveillance Complex (BASC) when it combines with different types of tumour suppressor genes, DNA damage sensors and signal transducers (Wang et al. 2000). It is involved in Ubiqutination, transcription regulation (Friedenson 2007; Friedenson 2008). In humans BRCA1 was first identified at chromosome 17 (Hall et al. 1990) and it was isolated in 1994 (Miki et al. 1994). It is present at 17q21 with a length of 100 Kb. In canine it is located on chromosome 9. BRCA1 has 22 exons in canines and felines; it encodes a protein of 1882 amino acids in canine and 1871 amino acids in feline. Many scientists from different research showed that women who have famililal mutations in the BRCA1 or BRCA2 (BRCA1/2) genes have increased risk of breast cancer (Struewing et al. 1997).
Fig 1: BRCA1 mechanism in DNA repairing. http://www.publichealthunited.org/leading-by-example-angelina-jolie-and-the-brca1-gene-mutation/
1.6.2 Cyclooxygenase-2 Enzyme (Prostaglandins, COX-2).
Cyclooxygenase-2 enzyme (Cox-2) is also called as Prostaglandins Endoperoxide synthase (PTGS). It is involved in the synthesis of prostaglandins which act as biological mediators in many body functions. It was first isolated from prostate gland that’s why it is called as Prostanglandin. Cyclooxygenase enzymes have two types, cyclooxygenas-1 and cyclooxygenase-2. Cyclooxygenase-1 is constitutively produced in the cell while cyclooxygenas-2 is inducible and it is constitutively produced only in kidneys, seminal vesicles and central nervous system. Its high expression has been recorded in many different types of tumours, it has been involved in anti-apoptosis, cell proliferation, tumour angiogenesis, cell invasion and immune suppression activities. In canine COX-2 is present on chromosome 7 having 604 amino acids and 10 axons. This correlation of cyclooxygenase-2 in cancer development suggests using new therapeutics against it. Studies have shown cycoloxygenase-2 high expression in number of different tumours (León-A 2008), such as intestinal, pancreatic, ovarian, prostatic, nasal cavity, oral cavity and mammary tumours of dogs (McEntee et al. 2002; Mohammed et al. 2004; Borzacchiello et al. 2007; Eplattenier et al. 2007; Mullins et al. 2004; Pireset al. 2010; Dore et al. 2003).
Fig 2:COX-2 mechanism of actionhttps://www.google.com.pk/search?q=cox+2+mechanism+of+action.
1.6.3 DLADQA1 (MHCII gene) (Additional work performed at Department of Veterinary Medicine, University of Cambridge, UK).
The Major Histocompatibility Complex (MHC) is a cell-surface protein mediating immune recognition through its interactions with T cells (Fig 3). There are three classes of MHC molecules in mammals - the classical MHC-I and II, and non-classical MHC-III Table 1). MHC-I interacts with CD8+ cytotoxic T cells, whilst MHC-II binds to CD4+ helper T cells. MHC molecules mediate antigen presentation to T cells. MHC-I typically presents self- peptides, whilst MHC-II presents foreign peptides. MHC molecules are extremely variable and polymorphic across the population, with a huge number of alleles at each MHC locus.
This allows MHC molecules themselves to behave as antigens in transplant rejection, with the graft MHC peptide recognized as non-self by the recipient, and thus rejected. It would be expected that CTVT, an allergenic graft, should be rejected for two reasons: host MHC will present tumour antigens as foreign non-self to the host immune system, and tumour MHC will present a mismatch to the host immune system as a foreign antigen itself (Fig 3). This project focuses on the DLADQA1 locus (Wagner et al. 2002), a classical MHC-II gene on dog chromosome 12. There is high level of MHC allelic variability in any population (Niskanen et al. 2013).
Fig 3:MHC is involved in graft rejection.
This rejection (represented by the red arrow) occurs according to two principles. Firstly, host T cells may recognize the host MHC presenting a foreign peptide that should activate an immune response. Secondly, host T cells would also be able to recognize the tumour MHC presenting any peptide as foreign, since it is not self-MHC. It is thus surprising that CTVT is able to persist as an allogeneic graft. MHC expression was previously characterised molecularly by Murgia et al through RT-PCR of a MHC-I (DLA88) and MHC-II (DLADRB1) gene (Murgia et al. 2006). They found that there was downregulation of expression of both these MHC genes. DLA88 showed low levels of tumour-specific expression, whilst there was no detectable tumour expression of DLADRB1. Murgia et al. also performed MHC genotyping for a number of CTVT samples and confirmed that all CTVTs shared the same haplotype (Murgia et al. 2006). They identified two clusters at the DLADQA1 locus, with some CTVTs appearing to be haploid the locus, whilst others remained diploid. This is in contrast to evidence that suggests the DLADQA1 locus had undergone a copy-neutral loss of heterozygosity (LOH) (Murchison et al. 2014).
1.6.4 Technologies used in this research work.
Different technologies are being used in cancer research such as PCR, flow cytometry, immunohistochemistry (IHC), in- situ hybridization (FISH, CSH) and microarray for diagnosis (Pawanet al. 2010). Here, I used Real time PCR for gene expressional analysis of BRCA1 and COX-2 and DLADQA1 (MHCII). Histopathology (Hematoxyline and Eosin staining) was performed for the diagnosis of tumours. CTVT diagnostics qPCR was also performed to measure the allele’s quantity of LINE-myc gene and CDKN2A gene. Conventional PCR measures at End-Point, while Real-Time PCR collects data during the PCR shows the data and quality of data during exponential growth phase also it has increase dynamic range of detection, it is very sensitive and no need for post PCR processing. Immunohistochemistry was performed to find out the expression of MHCII antigens in CTVTs. The serum protein electrophoresis and serum biochemistry was also measured. Western blotting was performed to detect antibodies in CTVTs (protein expression). It is a very good technique to measure the gene expression at protein level in fluidic material of cells. We performed capillary electrophoresis to find the mutations/SNPs in our genes of interest (BRCA1, COX-2 and DLADQA1). Genetic analyzer was used to find the sequence variations in our genes of interests. Other methods used for sequence variation studies, like SSCP, DGCG and HPLC miss the mutations (Rassi 2009). So the sequencing by capillary electrophoresis was the best option for this study.
Availability: Items available for loan: UVAS Library [Call number: 2250-T] (1).
16.
Mutational Analysis Of Hepatitis C Virus Ns4b Gene Encoding Protein
by Faiza Nisar Bukhari (2013-VA-12) | Dr. Muhammad Imran | Dr. M.Yasir Zahoor | Dr. Abu Saeed Hashmi.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Hepatitis C virus (HCV) infection has an estimated global prevalence of 2%–3% with approximately 270 million infected people worldwide of which 10 million belongs to Pakistan. HCV is a positive-sense single-stranded RNA virus from the family of Flaviviridea. Its genome encodes 10 proteins including a 261 amino acids protein NS4B. NS4B is a non-structural protein that performs hyperphosphorylation of NS5A, transactivates interleukin 8 promotor, suppresses HCV translation and modulates the ER stress response. Mutation in NS4B encoding gene may be targeted to develop an effective vaccine against HCV before virus invasion in host immune system.
Serum was collected from 20 patients who are infected with HCV. RNA was isolated from these samples to reverse-transcribe it into cDNA. This cDNA were PCR-amplified and amplicons were detected by UV light after their separation through agrose gel electrophoresis followed by seguencing of NS4B encoding gene.
Variations in HCV NS4B gene was analyzed by using Mega 6 software and sequence 2 blast analysis NCBI tool. Protein modeling was performed with phyre 2, DIANNA tool and I-Tasser online server and quality of model and antigenicity was checked by Vaxajen V 2.0 and Epijen softwares. Conservative analysis and epitope mapping were performed by using Immune Epitope Database (IEDB).
A systematic approach was employed for the prediction of potential epitopes in NS4B protein. Vaxijen V 2.0 was used to determine overall antigenicity of NS4B using cut off value of 0.4. Values above this threshold level show probable antigens in the protein. The topology of NS4B was determined by TMHMM server with the help of membrane topology data. Regions outside membrane and transmembrane were eradicated for epitopes prediction. T cell epitopes propred was used with proteosome cleavage site filter of 4% threshold. From the following data analysis 12 to 15 possible T-cell epitopes can be predicted; only these T cell epitopes were included in conservation analysis.
The present study provides information about mutational changes in HCV protein NS4B and thus preliminary data for the development of vaccine against HCV. This can also lead to future prospects for diagnosis, treatment and prognosis of HCV.
Hepatitis C virus (HCV) infection has an estimated global prevalence of 2%–3% with approximately 270 million infected people worldwide of which 10 million belongs to Pakistan. HCV is a positive-sense single-stranded RNA virus from the family of Flaviviridea. Its genome encodes 10 proteins including a 261 amino acids protein NS4B. NS4B is a non-structural protein that performs hyperphosphorylation of NS5A, transactivates interleukin 8 promotor, suppresses HCV translation and modulates the ER stress response. Mutation in NS4B encoding gene may be targeted to develop an effective vaccine against HCV before virus invasion in host immune system.
Serum was collected from 20 patients who are infected with HCV. RNA was isolated from these samples to reverse-transcribe it into cDNA. This cDNA were PCR-amplified and amplicons were detected by UV light after their separation through agrose gel electrophoresis followed by seguencing of NS4B encoding gene.
Variations in HCV NS4B gene was analyzed by using Mega 6 software and sequence 2 blast analysis NCBI tool. Protein modeling was performed with phyre 2, DIANNA tool and I-Tasser online server and quality of model and antigenicity was checked by Vaxajen V 2.0 and Epijen softwares. Conservative analysis and epitope mapping were performed by using Immune Epitope Database (IEDB).
A systematic approach was employed for the prediction of potential epitopes in NS4B protein. Vaxijen V 2.0 was used to determine overall antigenicity of NS4B using cut off value of 0.4. Values above this threshold level show probable antigens in the protein. The topology of NS4B was determined by TMHMM server with the help of membrane topology data. Regions outside membrane and transmembrane were eradicated for epitopes prediction. T cell epitopes propred was used with proteosome cleavage site filter of 4% threshold. From the following data analysis 12 to 15 possible T-cell epitopes can be predicted; only these T cell epitopes were included in conservation analysis.
The present study provides information about mutational changes in HCV protein NS4B and thus preliminary data for the development of vaccine against HCV. This can also lead to future prospects for diagnosis, treatment and prognosis of HCV.
Availability: Items available for loan: UVAS Library [Call number: 2339-T] (1).
17.
Production Of Single Cell Protein By Arachniotus Ruber Using Remnants Of Carrot As Substrate And Its Biological Evaluation In Broiler Chicks
by Lutfullah Siddiqui (2012-Va-601) | Ms. Shagufta Saeed | Dr. Abu Saeed Hashmi | Dr. Muhammad Wasim.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: CD Error. Summary could not opened. Availability: Items available for loan: UVAS Library [Call number: 2371-T] (1).
18.
Mutational Analysis Of Hcv Gene Encoding E1 Glycoprotein
by Muhammad Saad Junjua (2013-VA-893) | Dr. Muhammad Imran | Dr. Wasim Shehzad | Dr. Abu Saeed Hashmi.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Hepatitis C virus (HCV) is a positive single stranded RNA virus that belongs to Flaviviridae family and causes liver related issues like hepatocellular carcinoma, cirrhosis and chronic liver disease. HCV is affecting people worldwide; more than 170 million peoples have been affected so far and the number is increasing day by day. Its prevalence in Pakistan is about 3% to 6%. There is lot of variation in its genome and it is classified into 6 major genotypes and these genotypes are further classified into many subtypes. Size of HCV is about 9500 bps which only encodes single polyprotein. This 3000 to 3300 amino acids polyprotein is processed by cellular and viral proteases to generate 10 polypeptides consisting of 4 structural (Core, E1, E2 and P7) and 6 non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A, NS5B). The most immunogenic gene from all the genes is E1. It involves in the interaction with the host cell and easily escapes from the immune system of host due to the presence of hypervariable regions in E1 gene.
To isolate the E1 gene, RNA extraction was done using the kit method. RNA was converted to cDNA which was then amplified in two rounds of PCR using nested primers from HCV core region. After confirming the presence of HCV RNA in serum samples, PCR amplification of HCV E1 gene was carried out using gene specific nested primers. Amplified E1 gene products were Sanger sequenced and aligned with standard sequence to find out genetic variations. E1 gene sequences were converted to protein sequences for which secondary protein structures were made and analyzed. No noticeable change was seen in these secondary protein structures. The protein sequences were also analyzed for the presence of B-cell and T-cell epitopes; two T-cell epitopes (QAFTFRPRR, FLVGQAFTF) were found which may inform the development of a proper vaccine against HCV. Availability: Items available for loan: UVAS Library [Call number: 2399-T] (1).
19.
Physical, Chemical and Biological Treatment of Rice Husk to Improve Its Nutrative Value
by Rahat Naseer (2003-VA-196) | Dr. Abu Saeed Hashmi | Dr. Muhammad Tayyab | Prof. Dr. Habib ur Rehman.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Thesis submitted without CD. Availability: Items available for loan: UVAS Library [Call number: 2450-T] (1).
20.
Biochemical And Homology Analysis Of Jak2 Gene In Canines And Hominidae
by Marya Saadullah Khan (2014-VA-324) | Ms. Huma Mujahid | Dr. Abu Saeed Hashmi | Dr. Muhammad Yasir Zahoor.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: Cancers are considered to be the most lethal of all diseases known out of which myeloproliferative neoplasms comprise of a very little percentage.The frequency of these disorders is known in human beings and a lot of work has been done on humans. But there is a lot of scope for research on this area in canines. As dogs were found to have strong homology with human beings, we compared canine cJAK2 exon 13 sequence with the humanhJAK2 exon 13 and found 96 % homology. Mutations in JAK2 gene are well known to cause three types of disorders i.e. polycythemia vera caused by a well-known point mutation in exon 14 causing substitution of valine for phenylalanine in JH2 domain of the protein.Essential thrombocythemia and idiopathic myelofibrosis may also be caused by this mutation but similar clinical conditions arise without the presence of this mutation. Studies have revealed that other point mutations such as deletion, addition or substitution are also responsible for these disorders.
JAK2 is an intracellular protein which performs phosphorylation of STAT molecules upon their activation. Although the whole protein in its good state is important for its function but the two domains JH1 and JH2 are vital. JH1 domain acts as a tyrosine kinase enzyme and its activity is controlled by JH2 domain also known as pseudo tyrosine kinase domain. Any mutation in these domains leads to protein conformation defect and thus prevents its performance. Besides V617F mutation, other mutations are being discovered in this part of gene. Researchers have found mutations in exon 12, 13 and 15 that have been found to be involved in development of myeloproliferative neoplasms in different cases of patients.
Blood picture do not reveal any direct clue except for increased erythrocytes alone or along with other cells like increased platelets. Therefore blood indices are not reliable parameter to indicate the type of mutation involved in these disorders. Also LDH and EPO levels are not correlated with the disorder. Although EPO test must be done to exclude the possibility of secondary PV and erythropoiesis.
Availability: Items available for loan: UVAS Library [Call number: 2544-T] (1).
21.
Comparative Genomic Study of Motor Neuron Disease in Horses and Human
by Shakeela Daud (2011-VA-534) | Dr. Muhammad Wasim | Prof. Dr. Tahir Yaqub | Dr. Abu Saeed Hashmi.
Material type: ; Literary form:
not fiction
Publisher: 2017Dissertation note: cd not submitted Availability: Items available for loan: UVAS Library [Call number: 2810-T] (1).
22.
Evaluation Of Bioactive Peptides/ Proteins/ Alkaloids From Extracts Of Croton Tiglium, Lawsonia Inermis And Eruca Sativa Against Mastitis Causing Bacterial Strains
by Rubia Saeed (2011-VA-377) | Dr. Muhammad Tayyab | Dr. Abu Saeed Hashmi | Dr. Muhammad Nawaz.
Material type: ; Literary form:
not fiction
Publisher: 2017Dissertation note: Mastitis is considered as one of the most prevalent disease in dairy animals of Pakistan. Bacteria which are found in most mastitis cases are S. aureus, S. agalactiae and E. coli. Infections caused by these bacteria are being treated by various antibiotics but due to their development of resistance towards these drugs, there is need to explore some alternatives like medicinal plant extracts for the treatment of mastitis. Croton tiglium, Eruca sativa and Lawsonia inermis have been reported to have antimicrobial activity, thus the extracts of these medicinal plants will be explored to their antimicrobial activity against mastitis causing bacterial strains.
Present study purpose was to evaluate the bioactive proteins/alkaloids/peptides from extract of C. tiglium, E. sativa and L. inermis against mastitis causing bacterial strains. For this purpose, the leaves and seeds samples of selected medicinal plants (C. tiglium, E. sativa and L. inermis) were collected from Bagh-e-Jinnah and were identified from Department of Botany, University of the Punjab, Lahore. The ethanolic and aqueous extracts were prepared to evaluate the antimicrobial activity against mastitis causing bacterial strains. For this purpose, dust free leaves and seeds of selected plants were cut into small pieces, homogenized in ethanol/buffer and centrifuged. The resulting supernatant was then collected to check its antimicrobial activity against S. agalactiae, S. aureus and E.coli. Antimicrobial activity was analyzed by well diffusion method. Regarding the antimicrobial activity assay, the overnight grown cultures of the selected microbial strains was spread on the LB agar plates and the extracts was applied to wells incubated was done at 37°C for overnight. Inhibition zone was measured. Then the extracts having maximum activity were purified by GC.MS and the nature of extract was examined. All experiments were performed in triplicates so mean and average of the values was taken. Availability: Items available for loan: UVAS Library [Call number: 2853-T] (1).
23.
Production, Purification & Characterization Of Recombinant Thermostable Phytase And Its Biological Evaluation In Broiler Chicks
by Furqan Sabir (2007-VA-524) | Dr. Muhammad Tayyab | Dr. Abu Saeed Hashmi | Dr. Ali Raza Awan.
Material type: ; Literary form:
not fiction
Publisher: 2017Dissertation note: Phytate is the principle storage form of phosphorus in plants particularly in cereal grains and legumes. Mono-gastric animals doesn’t have ability to utilize phytate as phosphorus source. The animals release the undigested phytate from body with manure that cause environmental pollution. Phytases are responsible for the hydrolysis of phytate, resulting in availability of free phosphorus for the animal. The present study deals with the production and characterization of recombinant thermostable phytase and its biological evaluation in the broiler chicks. The PCR resulted in the amplification of 1.8 kb phytase gene using the genomic DNA of Thermotoga naphthophila as template. The purified PCR product was ligated in pTZ57R/T and the ligated material was utilized for the transformation of E.coli DH5α cells. The positive clones were selected on the basis of blue white screening. The restriction digestion of plasmid DNA from positive clones using NdeI and Hind III resulted in the release insert from the vector. The purified phytase gene after restriction digestion was ligated into pET21a already restricted with the same restriction enzymes and the expression was analyzed using E.coli BL21 CodonPlus (DEL) cells. SDS-PAGE demonstrated the intra-cellular production of recombinant phytase. The conditions were optimized for the optimal production of recombinant phytase (PHYTN). The maximal production of PHYTN was recorded when the BL21 CodonPlus cells having recombinant pET21a having phytase gene were induced with 1.4 mM IPTG and 6 hours post induction incubation period. The recombinant protein was purified using various chromatographic techniques and the purified protein was utilized for characterization. PHYTN showed optimal activity at 80 °C and pH 6 in sodium acetate buffer. The enzyme was found metal dependent and presence of Fe3+ or Cu2+ showed enhancing effect on PHYTN activity. Thermostability studies demonstrated that PHYTN retains 90% residual
SUMMARY
71
activity when the protein was incubated at 80 °C for 1h in the presence of 1.5 mM Fe3+. The kinetic studies of PHYTN demonstrated km and Vmax values of 50 mM and 2500 μmole/min respectively when sodium phytate was used as substrate. The characterized PHYTN was used for poultry trials to check the efficacy of the enzyme in poultry birds.
The results depicted that PHYTN put significant effect on the bird weight gain, feed intake and feed efficiency ratio. Presence of 1000 IU/kg of PHYTN resulted in the weight gain in 3rd, 4th and 5th week of trials from 504.766 to 533.535 g, 767.933 to 823.733 g and 999.833 to 1120.277 g respectively when compared with the control. The study demonstrated that this recombinant thermostable phytase is suitable for poultry feed industry and its domestic production will contribute the economic availability of PHYTN for the poultry feed industry. Availability: Items available for loan: UVAS Library [Call number: 2870-T] (1).
24.
Effect Of Camel Milk Lactoferrin Against Carbon Tetrachloride Induced Hepatic Toxicity In Sprague Dawely Rats
by Nasreen Asghar (2014-VA-546) | Dr. Muhammad Nasir | Dr. Sanaullah Iqbal | Prof. Dr. Abu Saeed Hashmi.
Material type: ; Literary form:
not fiction
Publisher: 2017Dissertation note: Liver is a vital organ involved in regulation of several significant metabolic functions and is responsible for maintaining homeostasis of the body as well as detoxification of chemicals, drugs and other metabolites Chronic Hepatitis and mortality due to liver cirrhosis and hepatocellular carcinoma is common in Pakistan. Camel milk Lactoferrin has antiviral, anti-inflammatory, anti-carcinogenic properties. Liver cirrhosis is a serious and irreversible disease. it is common in Pakistan population which leads to mortality (Khan AA, 1995). (Anderson and Smith, 2001).These problems can be controlled by taking special measures. Nutraceutical foods like camel milk have many beneficial uses in this regard. Camel milk lactoferrin might be used for the cure of hepatic fibrosis induced by CCI4 in Sprague Dawely rats. Seventy five (75) male Sprague –Dawley rats were purchased from National Institute of Health Islamabad kept in animal house of UVAS Lahore and randomly divided into 5 groups under completely randomize design (CRD). In group (2-5) carbon tetrachloride (CC14) was subcutaneously injected with a mixture of 40% CCI4 (a mixture of pure CC14 and sterile olive oil) at 200 uL/100g body weight as single dose, 48 hrs before the starting of treatment or 0 day. After 48 hrs, rats were considered hepatic injured except the (+ve) control group. Among all groups, four groups –ve control, 30mg/kg/b.wt, 60mg/kg/b.wt and 90mg/kg/b.wt were supplied with standard diet plus Lactoferrin (in different concentrations and doses) orally, while control group were provided only standard diet throughout the efficacy study (30 days). Daily feed and water intake was monitored and cages were cleaned regularly. Body weight was recorded before decapitation throughout the experimental period. The efficacy studies
data was analyzed through analysis of variance (ANOVA).Statistical significance was defined as P≤0.05.Means were compared for significance difference using Duncan‟s Multiple Range test (DMRt) and Least significance difference test (LSDt).
Organ to body weight ratio and decreasing tendency in (-ve) control group was observed, while increasing tendency for body weight was observed in camel milk lactoferrin treatments groups during the study. In the present investigations, higher amount of transaminases (AST,ALT) and cholestatic liver enzyme (ALP) were observed however, their concentrations were significantly decreased significantly in camel milk lactoferrin treatments as compared to rising trend in (-ve) control group.
Hypercholesterolemia is due to of oxidative stress induced by CCl4 and characterized by elevated levels of cholesterol .In the present research, it is concluded that camel milk lactoferrin treatments were effective in improving lipid profile.it is also obvious from the current results that camel milk lactoferrin treatments improved the hemoglobin (Hb) level although the effect was found non-significant .Likewise , results of present study also suggested that different treatments, time interval and their interaction had non-significant effects on white blood cells count in rats.
Histopathology results of present study given exposed that CCl4 consequences prominent hepatic steatosis, hepatic cord rupture and necrosis .Post treatment of camel milk lactoferrin reduced the severity of CCl4 –induced liver intoxication. Fatty change and necrosis were improved in the histological sections of camel milk post-treated rats.(-ve) control group treated with CCl4 and basal diet showed severe hepatotoxicity, severe inflammation of hepatocytes and portal vein congestion, while group C and D treated
with CCl4 ,basal diet and 30mg/kg/b.wt ,60 mg/kg/b.wt camel milk lactoferrin respectively showed moderate improvement in hepatotoxicity .group E treated with CCl4 and 90 mg/kg/b.wt lactoferrin showed mild hepatotoxicity in rats. Availability: Items available for loan: UVAS Library [Call number: 2943-T] (1).
