101.
Production, Purification And Characterization Of Laccase From White Rot Fungus
by Afrah Shafique (2012-VA-577) | Ms. Faiza Masood | Dr. Abu Saeed Hashmi | Dr. Tanveer Hussain.
Material type: ; Literary form:
not fiction
Publisher: 2014Dissertation note: Laccase (oxidoreductase, EC 1.10.3.2) are blue copper dependent oxidases and the mainligninolytic enzyme produced by white rot fungus. Laccase catalyze the oxidation of large snumbers of phenolic compounds (Kunamneni et al. 2007; Poonkuzhali et al. 2011). These enzymes have a molecular weight 60-90 kDa and consist of 15–30% carbohydrate. Laccases are the earliest and maximum investigated enzymatic systems. Laccase was initially found by Yoshilda in 1883 in the sap of Japanese laquer tree named as Rhusvernicifera. After a while in 1896, Bertrand and Laborde determined that laccase is a fungal enzyme.(Shraddha et al. 2007; Giardina et al. 2010).
Laccases are present extensively in nature, originating from plants, bacteria and fungi (Poonkuzhali and Palvannan 2011). In fungi, laccases are widely distributed in ascomycetes, deuteromycetes and basidiomycetes. The laccase producing fungus include Trametes versicolor, Pleurotus ostreatus, Polyporus, Trametespubescens, Cerrenaunicolour,PhanerochaetechrysosporiumandFunaliatrogiietc (Dwivediet al. 2011). Laccases occur morein fungi, than in the higher plants. Laccases are also present in few bacteria such as S.lavendulae, S.cyaneus, and M.mediterranea(Viswanath et al. 2008; Arias et al. 2003). In vegetables laccases have been recognized in turnips, apples, pears, cabbages, potatoes, beets, asparagus and various other vegetables (Jhadav et al. 2007).
Enzymes are produced by every living organism, however enzyme produced by microbes have various benefits over the enzyme originated from plants and animal origins.Laccases by nature
are important because of its huge diversity of catalytic activities, economical in production and comparatively more stable than other enzymes.The field of biotechnology proposes expanding possibilities for the production of several enzymes from microorganisms. New methods and techniques have been advanced by using enzyme as biocatalysts to produce big added value products like growing food requirements,good quality chemicals and medicines. Moreover enzymes are also utilized for environmental actions and for diagnostic and analytical motives. (Buchholz et al. 2005). Microbial enzymes are used as cost effective and environmentally sensitive substitutes for chemical processing in several industries and bioremediation. Therefore the commercial demand for microbial enzymes is increasing (Radhika et al. 2013).
Fungal laccases have boundless biotechnological functions across the globe like the decolouration and detoxification of industrial effluent, bleaching of pulp, phenolicselimination from wines, in preparation of biosensors in detergents blockindye transfer- functions (Yaver et al. 2001).It is also used in the formation of anticancer drugs, and included in few cosmetics to lessen their toxicity (Couto and Herrera 2006).In recent years, laccase have been skillfully practiced to the field of nanobiotechnology due to its capacity to mobilize electron transfer reactions without further addition of cofactor(Shraddha et al. 2007).
Laccase is ample in several white- rot fungi that are involved in lignin metabolism (Bourbonnais et al. 1997, Leontievskyet al. 1997). Fungal laccases have immense redox potential (up to +800 mV) than bacteria or plant laccases. The action of these laccases seems to be appropriate in nature and also has significant applications in the field biotechnology. These laccases are associated with the deterioration of lignin and also in the elimination of conceivably lethal phenols appear during the breakdown of lignin (Thurston et al. 1994).
The white rot fungus is corporeal in preference to morphological and composes of those fungi that are adequate of degrading lignin, which is a heterogenous polyphenolic compound in huge amount within the lignocellulose wastes(Eaton and Hale. 1993).Theircapability to deteriorate cellulose, hemicellulose, these are the polysaccharides forming the essential part of lingo cellulose is the basic metabolic processbetween the fungi and happen under the span of environmental conditions.The degeneration of lignindoesn’tprovide net energy so it is degraded during the secondary metabolism in order to gain polysaccharides present in lignin and carbohydrate complexes, supplying energy to which the organisms don’t have access(Jeffrics. 1990).The white rot fungi varyingly secrete one or more three extracellular enzyme namely manganese peroxidase, lignin peroxidase and laccase that are fundamental for degradation of lignin, ant they are generally mentioned as lignin modifying enzymes LMEs (Pickard et al. 1999).
Laccase is the subjects of demanding research in the recent years, because of their several properties like extensive substrate relevance, doesn’t required the inclusion of cofactors because they use oxygen as cofactor which is frequently present in the environment (Eugenio et al. 2009). Maximum number of laccases produced by various organisms is excreted as extracellular enzymes and this makes the purification process quite accessible. Laccase commonly display appreciable extent of stability. Due to these properties laccases are ideally applicable in diverse biological processes such as the treatment of industrial effluent, biopulping and biobleaching (Eggert et al. 2006).
The huge potential of laccase requires advancement in its production and, with huge activities and low cost (Herrera et al. 2007). The use of lignocellulosic agricultural waste as substrates is a tradition for the production of enzyme like laccase because it is ligninolytic in nature (Niladeviet al.2011). It is highly crucial to optimize the fermentation parameters for the adequate production of laccase (Revankaret al. 2007). .
The advantages of agro-industrial leftovers for cultivation media is of immense concern as agriculture waste cut down the expenditure of enzyme production and enhance the understanding on energy protection and recycling (Mansuret al. 2003).These agriculture wastes are comparatively economical and also contain ample nutrients such as lignin, cellulose andhemicellulose. These nutrients serve as inducer to energize the production of enzyme (Vassil et al. 2000).Due to these properties these agricultural waste can be used as substrate for the production of ligninolytic enzymes during the process of fermentation.
Laccase can be produced at varying rates by using a wide range of organisms grown on different substrates and by using several methods of fermentation, such as solid state, semisolid state, and submerged (Rodriguez et al. 1999; Boran et al. 2011). However, for effective laccase production, it is very important to use efficient laccase-producing organisms, suitable fermentation methods, and cheap and widespread sources. Accordingly, one of the most suitable approaches for the production of this enzyme is to use the most efficient agricultural wastes for increasing the production of the ligninolytic enzymes (Elisashviliet al. 2008).
Pakistan is an agricultural country and each year manufactures tons of agricultural by products. These agricultural wastes are accessible in markets at a very reasonable price and can be utilized as substrates in fermentation technique (Minussi et al. 2007). Agricultural waste products like rice husk, wheat bran, corn cob, millet husk and cereal huskhave been utilized by various scientists for laccase production (Osma et al. 2011; jhadav et al. 2009).The chemical properties of these agricultural wastes make them important and economical fermentation medium for biotechnological purposes(Giardina et al. 2010).The cellulose and hemicellulose constituents of lignocellulose wastes are widely used by several organisms but lignin, which is the maximum contrary material to microbial degradation, is transformed conveniently by only few organism of thw white rot fungus (Dwivedi et al. 2011).Lignin serves as a barrier that protects cellulose and hemicellulose from enzymatic attack, however, white rot fungi can attack this barrier in order to obtain energy from cellulose. These fungi produce different extracellular ligninolytic enzymes such as laccase, manganese peroxidase, and lignin peroxidase (Couto et al. 2006).
Fermentation is a biological approach that is used for the transformation of complicated substrates into basic composites by different microorganisms like bacteria and fungi. In the procedure of this metabolic breakdown the microorganisms also release various added compounds like carbon dioxide and alcohol asidefrom the conventional products of fermentation. These added compounds are known as secondary metabolites (Pandey et al. 1999). These Secondary metabolites span from enzymes, antibiotics, peptides and growth factors (Balakrishnan and Pandey. 1996; Machado et al. 2004; Robinson et al. 2001). They are also known as bioactive compounds becausethey carry biological activity(Demain et al. 1999).
Submerged fermentation is a type of fermentation in which components are present in a liquid media like broths and syrup. The co-active composites are poured into the fermentation broth. In this media the substrates are employed quiet immediately, due to this reason the nutrients in the media are either fortified or regained continuously. This type of fermentation approach is optimum for microorganisms such as bacteria, fungi because they depend upon on immense moisture content. The increased benefit of this approach is that the purification of the desired products or enzymes is quiet effortless. Submerged fermentation is especially used in the abstraction of secondary metabolites that are utilized in liquid form (Subramaniyam et al. 2012).
Furthermore 75% of the commercial enzymes are made by using submerged fermentation, it also supports the usage of genetically modified organisms to a large expanse then solid state fermentation. Submerged fermentation is also used on large extent because it doesn’t require equipment concerning solid state. On the contrary solid state fermentation is a mechanism operated in absence of free flowing water by utilizing solid support in form of natural substance ( Poonkuzhali et al . 2011). .
The major purpose of conducting this research is to design optimized fermentation process which produces effective amount of enzyme by using agricultural wastes. The use of agricultural wastes as substrates is economical and increase awareness on energy conservation .The enzyme can be used further for bioremediation because it not substrate specific and can act on broad range of substrates.
Availability: Items available for loan: UVAS Library [Call number: 2213,T] (1).
102.
Bioconversion Of Molasses To Glucose Oxidase Through Solid State Fermentation With Aspergillus Niger
by Wajeeha Zafar (2012-VA-574) | Dr.Abu Saeed Hashmi | Dr. Muhammad Tayyab | Dr. Muhammad Wasim.
Material type: ; Literary form:
not fiction
Publisher: 2014Dissertation note: Enzymes can be defined as soluble colloidal organic catalysts which are produced by living cells and are capableof acting independently of the cells. Glucose oxidase belongs to oxidoreductaseand is also called as glucose dehydrogenase. The glucose oxidase enzyme (GOX) oxidizes glucose to gluconic acid. In cells, it aids in breaking the sugar down into its metabolites. Glucose oxidasehas found several commercial applications including glucose removal from dried egg; improvement of color, flavor, and shelf life of food materials; oxygen removal from fruit juices, canned beverages. It has also been used in an automatic glucose assay kit in conjunction with catalase and chiefly in biosensorsfor the detection and estimation of glucose in industrial solutions and in body fluids such as blood and urine. It is often extracted from Aspergillusniger. GOX is a dimeric protein. The active site where glucose binds is in a deep pocket. This enzyme acts outside of cells, is covered with carbohydrate chains (Raba and Horacio 1995).
Aspergillus niger is the potential source for the production of glucose oxidase and is preferreddue to its high production ration of extracellular enzyme. The ability of Aspergillus niger toutilize a wide range of waste products as nutrition source makes it more economical source of the enzyme (Rajesh et al .2002).
The glucose oxidase fromA. nigerisalso an intracellularenzyme present in the mycelium of the organism. Aspergillus nigeris a filamentous fungus belonging to phylum Ascomycota. It Produces microscopic conidia on conidiophores that are produced asexually. Hyphae possess septa and are hyaline. They are supported at their base by foot cells from which conidiophores originate. It possesses long, double-walled, smooth and colorless to brown conidiophores.It is commonly foundin mesophilic environments such as soil, plants and enclosed air environments. It is capable of surviving in various environments, it is not only a xerophilic fungus, but is also a thermo tolerant organism. It is because of this property that it exhibits a high tolerance to freezing temperature(Schuster 2002).
Glucose oxidase was first isolated from mycelia ofA. nigerandPenicilliumglaucumby Müller.
A large number of microbes including bacteria and filamentous fungi have been used for the production of glucose oxidase. Glucose oxidase is produced at large scale using A. nigerand P. amagasakiense. Many bacteria are also involved in the production of this enzyme; some of these are Zymomonasmobilis, Micrococcus and Enterobacte(Yogananth et al. 2012).
Glucose oxidase (GOX) from Aspergillus niger is a well-characterised glycoprotein consisting of two identical 80-kDa subunits with two FAD co-enzymes bound. Both the DNA sequence and protein structure at 1.9 A have been determined that these identical subunits size vary from 70 to 80 KDa. It catalyzes the oxidation of D-glucose (C6H12O6) to D -gluconolactone (C6H10O6) and hydrogen peroxide. It is produced naturally in some fungi and insects where its catalytic product, hydrogen peroxide, acts as an anti-bacterial and anti-fungal agent (Ikram et al . 2014).
Glucose oxidase has a molecular weight of 160,000 a.m.u. (Tsugeet al .1975) and consists of two identical polypeptide chain subunits having nearly equal molecular weights linked by disulphide bonds (O'Malley and Weaver 1972) and it is highly specific for β-D-glucose (Bentley 1963). Each subunit of the glucose oxidase contains one mole of Fe and one mole of FAD (Flavin adenine dinucleotides) and it contains 74% protein, 16% natural sugar and 2% amino sugars (Tsugeet al. 1975). The Glucose oxidase enzyme in its purest form is pale-yellow powder. The molecular weight of GOX ranges from approximately 130 kDa to 175 KDa (Kalisz et al. 1997).
Gluconic acid, the oxidation product of glucose, is a mild neithercaustic nor corrosive, non-toxic and readily biodegradable organic acid of great interest for many applications. As a multifunctional carbonic acid belonging to the bulk chemicals and due to its physiological and chemical characteristics, gluconic acid itself, its salts (e.g. alkali metal salts, in especially sodium gluconate) and the gluconolactone form have found extensively versatile uses in the chemical, pharmaceutical, food, construction and other industries (Anastassiadis and Morgunov 2007 ).
This enzyme is present in all aerobic organisms and normally functions in conjunction with catalase (Coxon and Schaffer 1971). This enzyme is also used as an antioxidant (Berg et al. 1992). It is mainly available from microbial sources and is normally produced by aerobic fermentation of Aspergillus nigerand Penicillium species (Fiedurak1996; Lu et al. 1996; Plush et al .1996; Rando et al. 1997). It has high specificity for D-glucose (Kuly and Cenas 1983).
This enzymeis also widely used to produce gluconicacidthatGOXtogether with Horse Reddish peroxidase has a range of applications in the food industry for glucose determination. GOX is being used in the textile industry producing hydrogen peroxide for bleaching process. This enzyme is also used to determine capillary glucose in screening of gestational diabetes (Mesiggi et al. 1988). This enzyme is utilized to extend the shelf life of fish(Field et al.1986) andproduction of calcium gluconate, gluconic acid and its derivatives (Khurshid2009).
Solid state fermentation [SSF] has been recently considered as the most cheapest and more environmentally friendly relative to submergedliquid fermentation [SLF] in the production of value added industrial based products such as enzymes, bio fuels.Advantages of Solid State Fermentation over Submerged Fermentation isHigher volumetric productivity, usually simpler with lower energy requirements, Might be easier to meet aeration requirements, Resembles the natural habitat of some fungi and bacteria and Easier downstream processing (Mienda et al. 2011).
Molasses is a dark brown, almost black, moist granular sugar. Its distinctive molasses taste is due to its high content of minerals. Nutritively, it has high iron content (Draycott and Philip 2008).
Aspergillus niger is a fungus, one of the most common species of the genus Aspergillus.The genus Aspergillus isimportant economically, ecologically and medically (Nizamuddinet al.2008).
Glucose oxidase enzymewas producedthrough the microbial fermentation. For that purpose solid state fermentation wasdevelopedwithA.niger. Solid state fermentation was applied to utilize agricultural residue such as Molasses as substrate.
The current research work was focused on production ofextracellular Glucose Oxidase (GOX) fromAspergillusniger using industrial waste such as molasses as substrate by Solid state ( static ) fermentation.
Availability: Items available for loan: UVAS Library [Call number: 2219-T] (1).
103.
Talaash-e-Baharaan
by Jamila Hashmi.
Edition: 1st ed.Material type: Publisher: Lahore: Sang e Meel Publications; 2011Availability: Items available for loan: UVAS Library [Call number: 891.4393 Hashmi 30526 1st 2011 Novel] (1).
104.
Delignification Of Rice Husk By Organic Solvent Treatment To Increase It’s In Vitro Digestibility
by Awais Alam (2012-VA-604) | Dr. AbuSaeed Hashmi | Miss Huma Mujahid | Dr. Asif Nadeem.
Material type: ; Literary form:
not fiction
Publisher: 2014Dissertation note: The major constituent of plant cell wall is lignocellulose. Plant biomass mostly consist of cellulose, hemicellulose and lignin alongside little measures of pectin, protein, extractives (dissolvable nonstructural materials, for example, sugars, nitrogenous material, chlorophyll, waxes) and ash. Lignocellulosic biomass is the most abundant organic material in nature. There is an expected yearly overall production of 10–50 billion dry tons representing about 50% of the worldwide biomass yield (Parveen et al. 2009).
Numerous physicochemical, structural and compositional variables decrease the digestibility of cellulose present in lignocellulosic material. So a treatment is required to increase the digestibility of lignocellulose biomass by exposing the cellulose present in plant fibers. Different techniques have been utilized for treatment, including chemical treatment, ammonia fiber explosion, biological treatment and steam explosion to modify the cellulosic structure to increase the availability of cellulose for digestion (Haoran et al. 2013). At that point, acids, bases and enzymes might be utilized to break down the cellulose into its respective sugars. Cellulolytic enzymesare broadly used to break down cellulose into its constituent sugars.
Among various agricultural wastes a broadly available waste is Rice husk (RH) which is rich in lignocellulosic material. Internationally, roughly 600 million tons of rice paddy is delivered every year. By and large 20% of the rice paddy is husk, giving a yearly aggregate generation of 120 million tons (Abbas et al. 2010). Pakistan is a rice producing country a great part of the husk produced from processing of rice is either blazed or dumped as waste. Rice husk yield in Pakistan is more than 1780 thousand tons every year (Asif et al. 2013).
Rice husk produced during rice refining, makes disposal issue because of less business interest. Additionally, handling and transportation of RH is hazardous because of its low density. Rice husk ash (RHA) is an incredible environmental risk bringing about harm to land and encompassing range here it is dumped. Thus, business utilization of rice husk and its ash is the option answer for disposal problem (Dilip et al. 2014).
RH are essentially made up of lignocellulose (60wt. %) and silica (11wt. %). The greater part of past investigations concentrated on the preparation of silica or other silicon based materials from RH, while the lignocellulose in RH was mostly glazed and then wasted. Thus, a methodology for comprehensive usage of RH has been produced to expand its digestibility by the breakdown of lignocellulosic mass. (Ajay et al. 2012)
Numerous techniques have been adopted for treating lignocellulosic feedstocks. However just a few of them appear to be encouraging. These treatment techniques include dilute acid treatment, steam blast (CO2 blast), pH controlled water treatment, ammonia fiber expension, ammonia recycle percolation (ARP) and lime treatment. Some survey articles have been appeared for microbial biomass treatment. But the present study gave presentations on organosolv treatment process. Despite the fact that organosolv treatment is more expensive at present than the leading treatment forms, it can give some significant side products. It appears that organosolv treatment is more practical for biorefinery of lignocellulosic biomass which considers the usage of every bit of biomass parts. An essential streamlining and usage of side products may lead the organosolv treatment to be a guaranteeing one for bio refining lignocellulosic feedstock in future. Organosolv treatment yields three different parts: dry lignin, a watery hemicellulose stream and a moderately pure cellulose division (Xuebing et al. 2009).
Availability: Items available for loan: UVAS Library [Call number: 2230-T] (1).
105.
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.
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106.
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).
107.
Polymorphism Of The Slc11a1 Gene Associated With Resistance To Bovine Tuberculosis.
by Qamar Raza Qadri (2009-VA-569) | Dr. Asif Nadeem | Dr. Tahir Yaqoob | Dr. Abu Saeed Hashmi.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Bovine Tuberculosis (bTB), caused by Mycobacterium Tuberculosis is a health threat to
livestock. Information on genetic resistance or susceptibility because of polymorphisms of
candidate genes could be used in making selection decisions. Solute carrier family 11 (protoncoupled
divalent metal ion transporters), member 1 gene (SLC11A1), is a known candidate gene
which is associated with natural resistance to infection by Mycobaterium spp in buffalo.
Polymorphism in this gene can be studied for breeding disease resistance animals. Blood samples
were collected from Nili Ravi buffalo breed of Pakistan. DNA was extracted by organic method.
Primers were designed using Primer3 software. Amplification of gene was done by Polymerase
Chain Reaction. PCR products were sequenced bi-directionally on ABI 3130XL Genetic
analyzer. Sequence alignment was performed for polymorphism identification. The analysis of
identified polymorphism has been done by CHROMAS software. Sequences were aligned by
BLAST tool of NCBI. The results of analysis showed that no polymorphisms were identified in
exonic region of gene. This might be due to less sample size. Genetics play important role in
fighting against pathogens. Identifying the genes involved can lead to marker-assisted selection
strategies. Availability: Items available for loan: UVAS Library [Call number: 2332-T] (1).
108.
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).
109.
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).
110.
Mutation Anlysis Of DTNBP1 Gene In Pakistani Patients With Schizophrenia Disorder
by Hafiza Sidrah Yasin (2013-VA-11) | Dr. Asif Nadeem | Dr. Maryam Javed | Dr. Abu Saeed Hashmi.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Schizophrenia (SCZ) disorder is a mental complex, heterogeneous and chronic neurodegenerative disorder with a cumulative prevalence of 1%. SCZ is an idiopathic disorder of the cortex and hippocampus. Environmental as well as genetic factors contribute to its complex pathogenesis. A functional repeat polymorphism in the Dystrobrevin Binding Protein 1 (DTNBP1) gene promoter conveys susceptibility for SCZ disorder. The DTNBP1 has been implicated in rare autosomal dominant forms of SCZ disorder because of mutations associated with severe disease progression and a typical physical signs and symptoms, indicative of neurodegeneration. Mutation in DTNBP1 gene has association with change in dysbindin protein which leads to change in abnormal neurotransmitter trafficking which leads to decrease in neuronal size, brain atrophy and reduced glutamate release in schizophrenia disorder. A systematic approach was applied to proceed the present study in order to identify the single nucleotides polymorphisms in schizophrenic patients. Blood samples (n=40) were collected from schizophrenia disorder patients. DNA was extracted by organic method. Primers were designed using Primer3 software. The amplification of gene was done by Polymerase Chain Reaction. PCR products were sequenced bi-directionally on ABI 3130XL Genetic analyzer. Sequence alignment was performed for polymorphism identification. The analysis of identified polymorphism was done by CHROMAS software. Sequence was aligned by Blast tool of NCBI. Difference between allele and genotype frequency of studied gene was evaluated and analyzed by using “SNPator”. The present study provides information about the susceptibility and genetic basis of the individual towards this disease and identified polymorphisms provides the opportunity to diagnose the disease earlier on the basis of particular SNPs in Pakistani patients. Availability: Items available for loan: UVAS Library [Call number: 2382-T] (1).
111.
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).
112.
Aazan
by Habib Ur Rehman Hashmi.
Edition: 1stMaterial type: ; Literary form:
not fiction
Publisher: Multan, Pakistan: Maktaba Qasmia; 2008Availability: Items available for loan: UVAS Library [Call number: 297.3 Habib 23622 1st 2008 Islam] (1).
113.
Dasht-e-Soos
by Jameela Hashmi.
Edition: 1stMaterial type: ; Literary form:
not fiction
Publisher: Lahore: Sang-e-Meel; 2010Availability: Items available for loan: UVAS Library [Call number: 891.4393 Jameela 20984 1st 2010 Urdu.Literature] (1).
114.
Bioconversion Of Agricultural Waste To Alginate By Azotobacter Vinelandii Using Fermentation
by Shagufta Saeed (2008-VA-742) | Dr.AbuSaeed Hashmi | Prof. Dr.Ikram-ul-Haq | Dr. Muhammed Tayyab | Dr. Ali Raza Awan.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Alginate is an exopolysaccharide composed of varying ratios of β-D mannuronic acid and its C5 epimer α-L-guluronic acid linked together by β-1,4 - glycosidic bond. It has wide range of industrial applications particularly in food sector as a viscosifier, stabilizer, thickener, emulsifier, gelling and water binding agent. Commercial alginate is extracted from brown algae but due to variation in composition of biopolymer isolated from species of different locations, there is growing interest in bacterial alginate.
At present two strains of bacteria are reported to produce alginate, Pseudomonas and Azotobacter. Hence present study was designed to produce alginate by Azotobacter vinelandii utilizing cheap substrates to save the foreign exchange. To achieve the goal, different physio-chemical parameters were optimized to have hyper-production of alginate through submerged fermentation. Different agricultural wastes like wheat bran, rice polishing and molasses were utilized as substrates through fermentation with Azotobacter vinelandii.On fermentation of 7.5% (w/v) wheat bran by A.vinelandii, maximum alginate production (5.21 g/L) was observed at 48 hours of incubation time with 6% (v/v) inoculum size, pH 7.0, 300C and agitation speed of 200 rpm. Addition of different optimum levels of ionic salts i.e. 1.5% CaCl2 and 2% MgSO4. 7H2O in the growth medium gave significantly (P< 0.05) higher quantity of alginate (6.08 g/L) where as addition of KH2PO4 and NaCl reduced the yield of alginate. Among different nitrogen sources tested, 2% corn steep liquor resulted significantly (P<0.05) higher yield of alginate (7.46 g/L).
The bacterial strain was improved by exposure to physical (UV irradiation) and chemical mutagens (Nitrous acid and ethidium bromide) to obtain more than 90% killing. The survivors were screened for hyper-production of alginate against the wild strain of A.vinelandii using pre-optimized conditions. The highest alginate production (13.8 g/L) was obtained by the ethidium bromide treated strain (EtBr-02). The mutant strain was used for optimization of fermentation parameters. The maximum concentration of alginate (15.61 g/L) was obtained by utilizing 10% (w/v) wheat bran, 8% (v/v) inoculum at 48 hours of incubation, pH 7.0, 300C and an agitation speed of 200 rpm. Inclusion of 2.5% cornsteep liquor raised the alginate concentration to 15.8 g/L.
Batch fermenter studies were carried out in 2 L fermenter with working volume of 1.5 L using the mutant strain A.vinelandii, EtBr-02. Optimization of process parameters like agitation, aeration and pH in the fermenter showed that maximum alginate (16.8 g/L) was achieved at 300 rpm, 2.5 vvm aeration and controlled pH condition at 32 hours of incubation time.
The alginate produced was identified by FTIR spectrum after precipitation. The purity of alginate was estimated by HPLC against the standard alginic acid from Sigma-Aldrich and was found to be 98% pure. The alginate produced was used at 3% concentration for immobilization of yeast cells. Immobilized and free cells were compared for ethanol production using 10% sucrose as the carbon source in fermentation medium. The maximum amount of ethanol obtained was from free cells i.e. 38 g/L whereas immobilized cells produced 32.5 g/L ethanol. The advantage of immobilization is that beads can be reused in eight sequential fermentation cycles of 10 h each. Thus a cheap and practical bioprocess of alginate production was developed, that can be exploited commercially to save foreign exchange.
Availability: Items available for loan: UVAS Library [Call number: 2460-T] (1).
115.
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).
116.
Bio-Conversion of Molasses to Phytase Through Solid State Fermentation With Aspergillus Niger
by Faseeha Nasim (2012-VA-633) | Dr. Abu Saeed Hashmi | Ms. Faiza Masood | Prof. Dr. Saima.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: CD Corrupt. Availability: Items available for loan: UVAS Library [Call number: 2484-T] (1).
117.
Nucleotide Sequence Variation In Heat Shock Protein 70-1 Gene Of Capra Aegagrus Blythi
by Fehmeeda Fatima (2014-VA-775) | Dr. Asif Nadeem | Dr. Maryam Javed | Dr. Abu Saeed Hashmi.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: Heat shock protein 70 (HSP 70) plays a vital role in survival of an organism by providing
cytoprotection against various kinds of stresses. Among all the HSPs present in the cell, the
ubiquitous HSP 70 proteins are the most abundant and temperature sensitive. Considering the
importance of HSP70-1 gene in conferring thermotolerance, present study has been designed to
characterize this gene in Sindh ibex which is a wild goat species of Pakistan. The
characterization of HSP70 gene might be helpful for deriving phylogenetic relationship among
different species and identifying new functions among the related species. Blood/meat samples
(n=25) were collected from Kirthar national park, Sindh. Standard DNA extraction method was
used for DNA extraction. PCR primers were designed by Primer3 software and amplification of
gene was done by Polymerase Chain Reaction. PCR product was sequenced bi-directionally by
Big DyeTM Terminator on ABI 3130XL Genetic Analyzer. Multiple sequence alignment was
performed for polymorphism identification. Genetic diversity was calculated by using DnaSP
v.5.0. Phylogenetic analysis using the MEGA v.6.0 software package was performed and
neighbor joining and UPGM trees were constructed. The results indicated that Sindh ibex
HSP70.1 gene was highly similar to of domestic goat, sheep, cattle, buffalo, camel and horse
which indicates their origin from a common ancestor. The results of this data might be helpful in
designing effective conservation strategies for Sindh ibex. Availability: Items available for loan: UVAS Library [Call number: 2524-T] (1).
118.
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).
119.
Molecular Exploration Of Zinc Finger Bed-Type Containing 6 Gene For Growth Trait In Beetal Goat
by Kanwal Rashid (2014-VA-496) | Dr. Maryam Javed | Dr. Asif Nadeem | Dr. Abu Saeed Hashmi.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: Zinc finger, BED-type containing 6 (ZBED6), is a novel transcription factor.It acts as a repressor of IGF2 transcription in skeletal muscle myogenesis and development. it is mainly involved in organism development, signaling, cell to cell interaction, hepatic fibrosis, clathrin mediated endocytosis and tight junction signaling cascades. Chromatin immunoprecipitation (ChIP) sequencing using C2C12 cells identified about 2,500 ZBED6 binding sites in the genome, and the deduced consensus motif gave a perfect match with the established binding site in Igf2. Silencing of Zbed6 in myoblast cells affect Igf2 expression, cell proliferation, wound healing, and myotube formation. Genes associated with ZBED6 binding sites showed a highly significant enrichment for certain Gene Ontology classifications, including development and transcriptional regulation.Functional and signaling assays of BED6 gene indicate its probable role in controlling growth traits in Goat. Blood samples (n = 40) were collected. Inorganic method of DNA extraction used. Primers for PCR amplification will be designed using Primer3 software. PCR products will be sequenced bi-directionally on ABI 3130XL Genetic analyzer. The results of sequencing were analyzed using CHROMAS software. Sequence alignment tools (blast 2)were used for SNPs identification. Difference between allele and genotype frequency of studied gene evaluated by chi square test, likelihood test and analysis was done by POPGENE and one way ANOVA.Novel Variations identified which have probable implementation in selection of superior goats with higher tendencies towards weight gain. Availability: Items available for loan: UVAS Library [Call number: 2554-T] (1).
120.
Sequence Analysis Of Mitochondrial Atpase 8/6 Gene Variants In Sindh Ibex (Capra Aegagrus Blythi)
by Javeria Zafar (2014-VA-222) | Dr. Asif Nadeem | Dr. Maryam Javed | Prof. Dr. Abu Saeed Hashmi.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: ATPase 8/6 gene plays a vital role in survival of an organism by generating energy in the form of ATP synthase. Considering the importance of ATPase8/6 gene in energy generating, present study has been designed to characterize this gene in Sindh ibex. The characterization of ATPase8/6 gene might be helpful for deriving phylogenetic relationship among different species and identifying new functions among the related species. Tissue/blood samples (n=15) were collected from Kirthar National Park, Sindh. Standard DNA extraction method was used for DNA extraction. PCR primers were designed by Primer3 software and amplification of gene was done by Polymerase Chain Reaction. PCR product was sequenced bi-directionally by Big Dye TM Terminator on ABI 3130XL Genetic Analyzer. Multiple sequence alignment was performed for polymorphism identification. Genetic diversity was calculated by using DNAsp. Phylogenetic analysis using the MEGA6 software package and an equally weighted maximum parsimony analysis was performed using the close-neighbor-interchange algorithm. The results indicated that Sindh ibex ATPase8/6 gene was highly similar to Capra caucasica. The results of this data might be helpful in designing effective conservation strategies of different species of wild animal. Availability: Items available for loan: UVAS Library [Call number: 2587-T] (1).
121.
Tuhfa tul Razafi Milad e Mustafa (PBUH)
by Raza Muhammad Shah Hashmi.
Edition: 1stMaterial type: Publisher: Lahore: Al Madina; ndAvailability: Items available for loan: UVAS Library [Call number: 297.1 Raza 22320 1st nd Islam] (1).
122.
Assessment Of Afflatoxins Contamination In Peanuts
by Zanib Hashmi (2009-VA-512) | Dr. Naureen Naeem | Dr. Sanaullah Iqbal | Prof. Dr. Aftab Ahmad Anjum.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: Peanut is the most important agricultural crop of Pakistan. Peanut is a dicotyledonous, herbaceous, pubescent, rigid or low growing plant and the only species cultivated is (Arachishypogaea L.). Peanut is rich in protein, fat and carbohydrates, some percentage of Ca, K, P, Mg and vitamin E is also present. Peanut is an excellent source of edible oil as it contains about 50 to 53 percent good quality oil used in ghee, margarine and salad. There is high risk of contamination of peanuts with aflatoxins(AFB1, AFB2, AFG1 and AFG2) because of fungal attack during the drying of peanut pods. Out of all these aflatoxins AFB1 is most important. Aflatoxins are toxic, carcinogenic secondary metabolites of Aspergillusflavus, Aspergillusparaciticus and Aspergillusnomius. Aflatoxins can cause illness to human results in Aflatoxicosis. Aflatoxins are carcinogenic compounds that are causative agents in human hepatic and extra hepatic carcinogenesis. The chief attacking organ for aflatoxins B1 toxicity and carcinogenicity is liver. From the safety point of view aflatoxin management is important for the production of safe and excellent quality peanuts.
For this purpose present study was conducted to determine the level of aflatoxins in peanuts (roasted, un-roasted). Samples will be collected/purchased by simple random collection technique from local markets and vendors from different areas ( Sabzazar, Wahdat road , Shad bagh, Data darbar, Akbarimandi, Beaden road, Lohari gate, Ek-moria pull, Liberty, Firdous market, Siddiqiacoloney, Mughal pura, Faizbagh, Rehmanpura, Gulberg, Model town, Islam pura, Shahdara, Rang mahal, Muslim town, Township, Iqbal town, Awan town, Niazbegh, Mozang, Outfall road, Sanatnagar, Cantt, Secretriate and Shad man) of Lahore. The samples were analyzed by thin layer chromatography (TLC) to check the presence of aflatoxins (B1, B2,
G1 and G2). TLC analyses were further confirmed by high performance liquid chromatography (HPLC) to verify the accuracy of TLC. These analyses were performed in the Department of Food Science and Human Nutrition and WTO labs, University of Veterinary and Animal Sciences, Lahore. As out of 120 total samples of peanuts 60 samples were taken from vendors with 2 categories of roasted and unroasted while 60 samples were collected from shops with the same categories. Out of 120 samples, 55 (45.8%) were contaminated. In these 55 samples 48 (87.2%) samples were contaminated with aflatoxin B1.Aflatoxin G1 is also present in 3 samples (5.45%), aflatoxin B2 in 3 (5.45%) samples and Aflatoxin G2 is present only in one samples collected from vendors, and we can say that 1.8% samples were contaminated with aflatoxin G2.
Present study will be supportive for the investigation of aflatoxins in peanuts. Peanuts are widely consumed all over the world and occurrence of aflatoxins in this commodity is a major concern to human health. The present situation is too much worse about the levels of aflatoxins which are higher than the prescribed limit by the regulatory authorities. It was observed that TLC technique is good for the determination of aflatoxins in developing countries where the facilities of sensitive instruments are not accessible. Furthermore to quantify levels of aflatoxins by using sensitive instruments like HPLC, GC-MS and LC-MS is required for accurate detection of Aflatoxins in peanuts in markets to protect the consumers from exposure of aflatoxins high level which are carcinogenic and hepatotoxic.
Availability: Items available for loan: UVAS Library [Call number: 2614-T] (1).
123.
Tareekh-e-Musalmanan-e-Pakistan-o-Bharat / Vol.2
by Syed Hashmi Fareed Abadi.
Edition: Vol.2Material type: ; Literary form:
not fiction
Publisher: Karachi: Anjuman Tarraqqi-e-Urdu Pakistan; 2003Availability: Items available for loan: UVAS Library [Call number: 954 Hashmi 16860 Vol.2 2003 History] (1).
124.
Clinico-biochemical Studies on Detomidine Analgesia and Effects of its Combinatios on Animals
by Muhammad Arif Khan | Prof. Dr. Muhammad Ashraf | Dr. Khalid Pervez | Dr. Haji Ahmad Hashmi.
Material type: ; Literary form:
not fiction
Publisher: 2003Dissertation note: The overall objective of this study was to evaluate the newly introduced imidazole derivative, drug "detomidine" with alpha-2 adr2noceptor binding properties, and its various combinations in animals. A clinico-hiochemical study was carried out to explore the various aspects of a novel sedative and analgesic drug. Analgesia was evaluated by performing castration in small ruminants, rumenal fistulization in large ruminants, skin prick test, electric stimulation, and developing an experimental colic model in donkeys. The parameters used to evaluate analgesia revealed that detomidine has greater potential to lessen the pain during minor and major surgical interventions in different animals. However, its local usage to achieve paravertebral and epidural analgesia proved that detomidine produces general effect after getting into the circulation and very poor local effect. It has been concluded that the drug can be used as preanaesthetic with chloral hydrate and pentothal sodium anaesthesia to perform major surgical exercises in equine and canine respectively. In addition it has an edge over other sedative drugs on account of its undetrimental effect on various physiological parameters of the animals. Clinical trials have proved that detomidine "a novel sedative and analgesic" is a drug of choice for restraining, examination, and minor and major surgical manipulations on equine, bovine, caprine, ovine and canine species without any untoward effects. Availability: No items available Checked out (1).
125.
Azan
by Habib ur Rehman Hashmi.
Edition: 1stMaterial type: Publisher: Multan: Maktaba Qasmia: 2008Availability: No items available
126.
Islam ka Qanon e Shahadat
by Molana Syed Muhammad Mateen Hashmi.
Edition: 1stMaterial type: Publisher: Lahore; Dial Sing Trust Library; ndAvailability: Items available for loan: UVAS Library [Call number: 297.72 Mateen 11942 1st nd Islam] (1).
127.
100 Olia Karam
by Maaz Hashmi.
Edition: 1stMaterial type: ; Literary form:
not fiction
Publisher: Lahore: Nazria e Pakistan Academy ; 2012Availability: Items available for loan: UVAS Library [Call number: 297.1 Maaz 29518 1st 2012 Islam] (1).
128.
Effect Of Chicory (Cichorium Intybus L) Roots Powder On Blood Glucose Level Of Streptozotocin Induced Diabetic Rats
by Nazish Abid (2012-VA-782) | Dr. Sanaullah Iqbal | Ms. Tahreem Hussain | Dr. Abu Saeed Hashmi.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: The increasing numbers of elderly people, eating calorie rich diets, obesity and lack of physical activity have increased a great the number of patients with diabetes. Worldwide According to International Diabetes Federation (IDF) there were 6.9 million cases of diabetes in Pakistan in 2014 and occurrence of diabetes in adults of 20-79 years of age was 6.8 %.( International Diabetes Federation, cited on January 31, 2015). The feeding on chicory roots decreased the levels of plasma glucose, cholesterol, high density lipoprotein (HDL) cholesterol and also reduced liver cholesterol, triglyceride and total lipids of diabetic patient. In the present study, it was aimed to utilize a indigenous sources like prebiotics to overcome the representative burden on economy and estimating the outcome of administration of chicory powder for use as a substitute mediator of insulin in the control of diabetes mellitus.
In the present study, chicory roots were cleaned, oven dried and ground to fine powder. The proximate analysis of chicory powder was performed. For feeding trial, thirty two Albino rats 5 to 6 weeks age, almost of same weight and mixed sex were procured and were randomly divided into four groups i.e. (A) Control , (B) Diabetic control, (C) Chicory treated and (D) Metformin treated group containing six rats each (three male and three female). Each group of rats was fed with a high fat diet (20%) for the first 2 weeks of adaptation. Then Diabetes was induced in B, C and D groups of rats by injecting 65mg/kg STZ through intraperitoneally. The diabetic rats of group C and D were then used for chicory intervention (125mg/kg of body weight) and for Metformin medication (500mg/kg body weight) along with normal diet respectively. The blood glucose level and weighing of animals measured initially and then
Summary
39
weekly whereas glucose tolerance test of rats was performed initially and thereafter fortnightly. The feed and water consumption was measured on daily bases.
A significant (p<0.05) difference in blood glucose level was seen among the group B, C and D of rats. A non-significance difference (p>0.05) in blood glucose level was observed when compared between male and female rats of groups A, B, C and D. A significant (p<0.05) difference in live body weight was observed. The group B, C and D showed significant (p<0.05) decrease in live body weight from day 7 to day 28 as compared to group A whereas a non-significant (p>0.05) difference in live body weight of male and female rats of all groups was observed. A significant (p<0.05) difference in glucose tolerance between group C and D was observed whereas a non-significant (p>0.05) difference of glucose tolerance was seen between all male and female rats of groups A, B, C and D while a significant difference (p>0.05) in feed consumption within group A, B, C and D was seen whereas a significant difference (p<0.05) in feed consumption was seen between the subjects. A significant gender difference in feed consumption was observed (p<0.05) between the subjects but within the subjects a non-significant difference was seen. A non-significant (p>0.05) difference was observed within the subjects in water intake whereas significant (p<0.05) difference was seen between the subjects whereas a significant gender difference (p<0.05) was seen between the subject but a non-significant gender difference was observed within the subjects.
It is concluded that Cichoriun intybus root extract can be used as hypoglycemic agent to treat diabetic condition and has no adverse effects on body weight, feed intake and water intake. Availability: Items available for loan: UVAS Library [Call number: 2727-T] (1).
129.
Hashmi's Textbook of Medical Biochemistry/ 4th ed.
by Hashmi.
Edition: 4th ed.Material type: ; Literary form:
not fiction
Publisher: UK: CBS Publishing Co; 2010Availability: Items available for loan: IPS Library [Call number: 615.1 Hashmi 23943 4th ed 2010 IPS] (1).
130.
Impact of Arthrobotrys OIligospora, a Predacious Fungus, on the Epidemiology of Ruminant Trichostrongylid Parasites
by Haji Ahmad Hashmi.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 1989Dissertation note: Phd. Availability: Items available for loan: UVAS Library [Call number: 540-T] (1).
131.
Observations on Causative Agent(s) of Hydropericardium Syndrome (Angara disease) in Chickens
by Masood Rabbani | 1. Dr. Ata-ur-Rehman Rizvi | 2. Dr. Haji Ahmad Hashmi.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 1997Dissertation note: Phd. Availability: Items available for loan: UVAS Library [Call number: 594-T] (1).
132.
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).
133.
Use Of Activated Carbon Prepared From Agricultural Wastes For The Adsorption Of Azo Dye.
by Qaisera Parveen (2011-VA-378) | DR.Rahat Naseer | Dr.Saeed Hashmi | Dr.Muhammad Asad Ali.
Material type: Publisher: 2017Dissertation note: Dyes are the visible pollutants of water and have toxicological and carcinogenic effects. So, dyes should be removed from aqueous solutions/industrial effluents. Many techniques are used for dyes removal from industrial wastewater discharge. From economical point of view, by the use of low cost adsorbents, basic dyes were removed from aqueous solution. For this purpose, adsorbents easily available were used to prepare activated carbon by physical and chemical treatment. Low cost adsorbents were prepared from Agricultural waste materials like sugarcane bagasse and sawdust for the adsorption of Azo dyes.
Agricultural wastes were treated by physical and chemical means for preparation of activated carbon. Characterizations of adsorbents were done by FTIR and SEM. FTIR micrographs showed that the different functional groups present on fibrous materials are responsible for adsorbing the MB. SEM showed the surface morphology. The effect of contact time, adsorbent dose, pH and temperature was studied in batch experiment. Freundlich and Langmuir adsorption isotherm was verified. Adsorption kinetic studies were done at regular time intervals using fixed amount of adsorbents. Equilibrium isotherms were studied by adding different adsorbent doses at 150rpm to attain equilibrium. Effect of pH was studied after balancing pH of the dye containing solution and analyzing the remaining dye in solution for equilibrium contact time. Effects of different parameters were evaluated and statistical analysis was applied to explore the most efficient adsorbent for methylene blue removal. as an adsorbent for Azo dye removal from wastewater without causing detrimental effects on environment. Since sugarcane bagasse and sawdust, an agriculture solid waste, used in the study, easily available, the adsorption process is expected to be economically implemented for wastewater treatment Availability: Items available for loan: UVAS Library [Call number: 2854-T] (1).
134.
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).
135.
Potential Of Rice Husk And Rice Husk Ash For The Removal Of Malachite Green
by Nargis AfZAL (2011-VA-354) | Dr. Rahat Naseer | Dr.Abu Saeed Hashmi | Dr.Sameera Akhtar.
Material type: ; Literary form:
not fiction
Publisher: 2017Dissertation note: Potential Of Rice Husk And Rice Husk Ash For The Removal Of Malachite Green Availability: Items available for loan: UVAS Library [Call number: 2852-T] (1).
136.
Exploration Of Genetic Polymorphisms And Differential Expression Analysis Of Bovine Alpha-Lactalbumin And Osteopontin Genes Involved In Milk Composition
by Sidra Manzoor (2010-VA-92) | Dr. Asif Nadeem | Muhammad Imran | Dr. Abu Saeed Hashmi.
Material type: Publisher: 2017Dissertation note: Economically important traits of dairy animals are usually controlled by a large number of genes. The identification of the single nucleotide polymorphisms in potential genes has been associated with economically important traits. During lactation, mammary epithelial cells produced large amounts of specific milk proteins. Due to the expression sites, physiological properties and chromosomal localization, LALBA and SPP1 genes might be considered as candidate genes for milk composition in buffalo. Alpha-lactalbumin (LALBA) gene has been reported to be highly transcribed in transition and peak phase while late lactation exhibited its decline with progressive rise in SPP1 expression. This project was designed to investigate the effect of single nucleotide polymorphism that influencing the gene expression thus modulates the milk protein content in Nili Ravi. Samples of unrelated Nili-Ravi buffalo were collected from two Government, Buffalo Research Institute, Pattoki, and Livestock Production and Research Institute (LPRI) Bahadarnagar Okara, livestock farms. Milk samples were collected at 15, 90 and 250 days lactation for expression analysis. The genomic DNA was extracted by using the standard Phenol Chloroform Isoamyl alcohol (PCI) protocol. Specific set of primers was designed for the amplification of the LALBA and SPP1 genes. The amplified PCR products were sequenced for the identification of SNPs. To determine the differential expression of bovine LALBA and SPP1 genes, RNA was isolated from milk samples using the TRIzol reagent and converted it into cDNA. Taqman probes were used that are specifically designed to detect and target the DNA sequence. Five intronic polym orphic sites were identified in LALBA while exonic regions exhibited a complete homology with reference sequence. Additionally, eleven polymorphisms were identified in bovine SPP1 gene, six were in coding region and five were
Summary
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found in intronic portion of the gene. The analysis and correlation of all identified polymorphism was done by using SNPs data analysis software “SNPator”. Results obtained from expression study was stored in in-build software of Real Time PCR and Cycle threshold (Ct) values of LALBA and SPP1 mRNA were compared in individuals of Nili-Ravi buffalo to determine the variation in expression levels. The LALBA gene expression was observed highest in transition phase with a gradual decrease of expression in mid and late lactation. The sample, NR-5, was observed highly expressed (79.30) while NR-2 with low expression (19.28) for alpha lactalbumin in early lactation. The change in LALBA regulation at same stage was considered due to genetic variation of the respective animal. While the SPP1 gene expression was observed with the highest values in peak lactation and remains elevated in late lactation. NR-4 has the highest (72.27) expression among all mastitis free healthy animals while NR-2 was observed with low expression. Thus, the identified SNPs might be used as genetic marker for milk production traits. Gene expression patterns may also help us to understand the molecular mechanisms of bovine LALBA and SPP1 genes influencing milk composition. However, the expression of both genes was considered in a correlation with other genes involved in milk production pathway. Also, the mutational effects of other milk proteins might be involved in determining the expression pattern of both genes in selected animals. Therefore, further studies are likely to explore the regulation of milk protein genes and their translational efficiency during the course of lactation in dairy animals. Availability: Items available for loan: UVAS Library [Call number: 2830-T] (1).
137.
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).
138.
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).
139.
Bioconversion Of Agriculture Waste To Polyhydroxybutyrate Through Solid State Fermentation With Bacillus Thuringiensis
by Hafsa Yasmin(2015-VA-1065) | Dr.Abu Saeed Hashmi | Dr.Shagufta Saeed | Dr.Shahbaz yousaf.
Material type: ; Literary form:
not fiction
Publisher: 2017Dissertation note: Polyhydroxybutyrate (PHB) is a biopolymer,it can be used as a biodegradable thermoplastic material for waste management strategies.it can be produced by various microorganism bacterium, Bacillus thuringiensis accumulates PHB as intracellular granules Inside their cells in response to physiological stress such as excess of carbon sources and limitation of nutrients e.g. nitrogen and phosphorus etc. This research work PHB productionwas attempted from agriculture wastes like wheat bran, rice polishing and corn stover through solid state fermentation through optimization of different parameters like substrate water ratio, incubation period, volume of inoculum, inorganic salts concentrations, addition of molasses and corn steep liquor. A parent strain of Bacillus thuringiensis was maintained on nutrient agar plate. Fermentation media containing wheat bran, rice polishing and corn stover as substrates were used to check the production of PHB for the selected bacteria. Inoculum 0.5 mL was added into sterilized fermentation media and incubated for 0-96 hours. Thereafter growth media were harvested, culture was centrifuged. The extraction, determination and identification of PHB were carried out from the pellet.
It was found that Bacillus thuringiensisgave the maximum PHB yield (270mg/100g) with 10 mL of basal medium containing rice polishing, while the wheat bran produced (210mg/100g) at 72 hours and corn stover produced yield (130mg/100g) at 48 hours.Rice polishing gave the maximum PHB production yield (2000mg/100g) at 1mL of inoculum in the presence of optimized condition of 0.175% of KH2PO4.2H2O, 0.125% MgSO4.7H20, 0.75% urea, 1% corn steep liquor respectively.
In this study Bacillus thuringiensis produced higher yield of PHB using rice polishing as compared to wheat bran and corn stover.
So, it is concluded that PHB produced in this work can be used in various industries like pharmaceutics, food industry and also in medical field, it will also be helpful to reduce the pollution caused by other synthetic plastics.
Availability: Items available for loan: UVAS Library [Call number: 2964-T] (1).
140.
Love and revolution : Faiz Ahmed Faiz : the authorized biography
by Hashmi, Ali Madeeh.
Edition: 3rd Impression. Material type: ; Literary form:
not fiction
Publisher: New Delhi: Rupa; 2017Availability: Items available for loan: UVAS Library [Call number: 891.43917 Hashmi 32766 1st 2017 Eng.Literature] (1).
141.
Rasool e Khuda ki Beti: Seerat e Fatima tul Zahra R.A.
by Maaz Hashmi.
Edition: 1stMaterial type: ; Literary form:
not fiction
Publisher: Lahore: Dua Publication 2017Availability: Items available for loan: UVAS Library [Call number: 297.1 Maaz 32745 1st 2017 Islam] (1).
142.
Murshad Roomi Hazrat Shamas Tabraazi
by Maaz Hashmi.
Edition: 1stMaterial type: ; Literary form:
not fiction
Publisher: Lahore: Dua Publication; 2015Availability: Items available for loan: UVAS Library [Call number: 920 Maaz 32744 1st 2015 Islam] (1).
143.
Seerat Syedana Hazrat Usman Gani
by Dr. Muhammad Hussain Heacal | Maaz Hashmi.
Edition: 1st ed.Material type: ; Literary form:
not fiction
Publisher: Lahore: Dua Publications; 2017Availability: Items available for loan: UVAS Library [Call number: 297.77 Heacal 33151 1st 2017 Islam] (1).
144.
Dasht-e-Soos
by Jamila Hashmi.
Edition: 1st ed.Material type: ; Literary form:
not fiction
Publisher: Lahore: Sang-e-Meel Publications; 2018Availability: Items available for loan: UVAS Library [Call number: 891.4393 Jamila 33724 1st 2018 Urdu.Literature] (1).
145.
Bunyaadi khurd hayatyaat
by Bashir Ahmad Hashmi.
Material type: ; Format:
print
; Literary form:
not fiction
Availability: No items available
146.
The Mirror of Life: A Selection of Short Stories
by Hashmi B.A.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 1966Availability: Items available for reference: Old Books [Call number: 553 Eng.Literature 6221] (1).
