1.
Identidiation Of Genetic Susceptiblity Of Myopic Loci In Families From Punjab
by Maria Fareed Siddique | Prof.Dr.Masroor Elahi Babar | Dr. Sehrish Firyal | Prof. Dr. Abu.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 2010Dissertation note: Myopia, or nearsightedness, is a condition in which the eye cannot focus on distant objects and sometimes closer ones too. In past different authors reported different loci responsible for myopia. They used specifically synthesized markers for different loci and after conducting linkage analysis through genotyping the myopic families were found to be linked for those loci, whereas, in some studies the cause of myopia was environmental. Till now, linkage studies have identified at least 18 possible loci in 15 different chromosomes associated with myopia, although some of these remain to be confirmed. In past, no study was done in Pakistan on myopic families for finding responsible myopic locus in this regard. So, more conclusive and well-designed studies on family pedigrees of individuals with high myopia were needed to be conducted in Pakistan by using genetic markers associated with myopia.
In this study, a panel of microsatellite markers was developed. Blood samples were taken from six myopic families. DNA was extracted. PCR was performed for amplification of these I microsatellite markers on 34 samples belonging to 6 families. Genotyping analysis was performed for the PCR products of microsatellite markers. These results were studied by constructing and analyzing haplotypes on the basis of PAGE gel bands. Heterozygosity, homozygosity, polymorphism with all microsatellites markers, specific for two loci were checked.
One family MYO-4 was found to be potentially linked with markers for the locus MYP-18. Another family MYO-5 showed potential linkage for the locus 2q37.2. Remaining four families (MYO-l, MYO-2, MYO-3 and MYO-6) were totally unlinked with all the markers (D14S984, D14S63, D14S999, D2S2202, D2S2968 and D2S338 for both loci demonstrating genetic insusceptibility of myopic loci in developing myopia and thus suggesting the complex genetic variability of myopia.
This study will serve as the pioneering database for further research on identifying the genetic heterogenic complexity of myopia. Results of this study lead to development of a panel of microsatellite markers which can be used for linkage studies of more myopic families in Pakistan. This study opens the door for new geneticists as the results can also be helpful in carrying out genetic counseling for the myopic persons who are going to be married and specifically for those who have dominant inheritance. This was a preliminary study on myopic patients in Pakistan and data produced during this study will be helpful for drawing and determining genetic inheritance of expected babies with affected parents and siblings. Moreover this study can become the basis for further research investigations on myopics in Pakistan.
CONCLUSION
This was a pioneering study to develop panel of microsatellite markers for conducting linkage analysis and genetic characterization of myopic patients in Pakistan. As a result of this successful study a reliable, efficient and very informative panel of microsatellite markers was successfully developed which was capable to interpret individual diseased allelic identity, to be used for conducting linkage analysis through genotyping of myopics in Pakistan. This study will serve as the database for further research on identifying the genetic heterogenic complexity of myopia and also these successful results can be further analyzed in future on more myopics from different areas of Pakistan. This work provokes the need for further research purposes in identifying the genes influencing myopia that could help develop targeted treatments for children who are genetically predisposed to developing myopia.
Availability: Items available for loan: UVAS Library [Call number: 1177,T] (1).
2.
Leptin Mutations In Morbidly Obese And Severely Lean Individuals From Pakistan
by Muhammad Wasim | Dr. Sehrish Firyal | Dr. Muhammad | Dr. Muhammad Imran.
Material type: ; Format:
print
; Literary form:
drama
Publisher: 2013Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1623,T] (1).
3.
Isolation Identification & Molecular Based Investigation Of Bovine Rotavirus
by Ambreen Masood | Prof. Dr. Tahir Yaqoob | Dr. Jawad Nazir | Dr. Sehrish Firyal.
Material type: ; Format:
print
; Literary form:
drama
Publisher: 2013Dissertation note: Livestock is an important part of the economy of Pakistan. Calf's diarrhea due to group A bovine rotavirus causes high morbidity and mortality, which results in significant economic losses to livestock. In Pakistan overall prevalence of bovine rotavirus infection is 2.6%. As Pakistan is a developing country, survival of calves is really important to produce milk, meat and hides for propagation of livestock. The aim of current study was to isolate bovine rotavirus from faecal samples of diarrheic calves by antigen capture ELISA and molecular investigations. So, it was helpful to check the prevalence of bovine rotavirus in Lahore district.
This study will be a milestone for better treatment strategies of calf diarrhea problem. It will also pave the way for better vaccine development strategies to cure the disease. A total of 100 diarrheic faecal samples of cattle and buffalo's calves less than three months of age were collected from Lahore district. Rotavirus screening was done by direct sandwich ELISA by using commercial Rotavirus detection kit (Cypress Diagnostics, Belgium). ELISA confirmed 12 samples to be positive for bovine rotavirus. Among 12 positive samples, 7 were found positive in buffalo calf and 5 in cattle calf. After RNA extraction and cDNA synthesis, the PCR was done for amplification of VP4 gene of all ELISA positive bovine rotavirus samples. But only 5 samples (3 buffalo calf samples and 2 cattle calf samples) give desired product of 880 bp of VP4 gene. After sequencing and bioinformatics analysis, phylogenetic tree was constructed. It is evident that Pakistani bovine rotavirus VP4 gene (BRV/QOL/13) has maximum identity of 98% with Indian bovine rotaviruses VP4 gene.
Availability: Items available for loan: UVAS Library [Call number: 1707,T] (1).
4.
Mitochondrial Nd2 Gene Based Molecular Classification Of Various Pakistani Domestic Pigeons
by Muddasar Saeed khan | Dr. Sehrish firyal | Dr. Ali raza awan | DR.Muhammad.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 2014Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1983,T] (1).
5.
Molecular Variability Analysis Of Mitochondrial Dna Hypervariable Region L And Ll In Four Consecutive Human Generations of Punjab
by M. Faaras iqbal | Prof. Dr. Tahir yaqub | DR. Sehrish firyal | Miss Faiza.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 2014Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 2008,T] (1).
6.
Meca Gene Based Methicillin-Resistant Staphylococcus Aureus Typing Isolated From Poultry And Their Potential
by Muhammad Sohaib iftikhar baig | Dr. Sehrish Firyal | Dr. Abu Saeed | Dr. Muhammad Zubair Yousaf.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 2013Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 2082,T] (1).
7.
Effect of Ginger and Turmeric Against Cadmium Induced Hepato-Renal Toxicity in Albino Rats
by Hafiza Sajda Ashraf (2012-VA-578) | Ms.Asma Waris | Dr. Muhammad Tayyab | Dr. Sehrish Firyal.
Material type: ; Literary form:
not fiction
Publisher: 2014Dissertation note: Metal compounds and metal is natural elements of all ecosystems, moving between biosphere, hydrosphere, atmosphere and lithosphere. Metal complexes are increasingly introduced in the environment and could finally accumulate in a/biotic systems (Florea et al. 2005).
Contact to heavy metals is potentially damaging particularly for those metal compounds, which do not contain any physiological function in the metabolism of cells. A heavy metal is a part of an ill-defined subset of constituents that show metallic properties, which would mostly include the some metalliods, actinides, lanthanides and transition metals. Heavy metals have a high density and atomic weight much greater at least 5 times than water. Anthropogenic basis of heavy metals, i.e. contamination, have been introduced to the ecosystem waste-derived fuels are particularly prone to have heavy metals. More than 20 heavy metals, but inorganic arsenic, lead and cadmium are of particular concern (Gornal 1949).
Although, carcinogenic and toxic effects of metals have been observed in animals and humans, and that these metals form a key part in the normal functioning of biological cells. Some necessary transition metals like manganese, iron, zinc and copper contribute in controlling a variety of signaling and metabolic pathways. On the other hand their redox properties and coordination chemistry gave them an additional advantage that these metals might escape from the control mechanism such that homeostasis, partitioning, transport and binding to the designated cell elements and they interrelate with protein sites other than those which are tailor- made for them by displacing other metals from their natural binding sites. While, this process does not take place regularly, but the toxicity of metals can lead to impairment and dysfunctioning of cells (Leonard et al. 2004).
Oxidative stress is one of the main mechanisms of heavy metal toxicity. These metals are able to interact with DNA causing oxidative worsening of biological macromolecules and nuclear protein (Chen et al. 2001).
Metals like mercury, iron, cadmium, lead, copper and nickel, have the capability to produce reactive radicals, leading to cell damage like damage to lipid bilayer, depletion of enzyme activities and DNA (Stohs 1995). Moreover, these reactive radical species comprise a broad diversity of sulfur-, oxygen, nitrogen- and carbon radicals, initiating not only from lipid peroxides, hydrogen peroxide and superoxide radical but also in chelates of proteins complex peptide and amino acid, with the toxic metals. Metals produce reactive species, which in turn can cause nephrotoxicity, hepatotoxicity and neurotoxicity in humans and animals (Chen and Sthos 1995).
Cadmium is a natural metal located in the Periodic Table of the elements between mercury and zinc and the chemical behavior of cadmium is like a Zn. There is usually a divalent cation, complexd through other constituents (e.g CdCl2). Cadmium in the soil crust around 0.1ppm (Hans 1995) frequently being found as a contaminant in Pb or Zn deposits. In Zn or Pb smelting cadmium produced as a by product. Commercially, Cd is used in batteries, galvanizing steel, lasers, ink color, television screens, cosmetics and was used as an obstacle in nuclear fission and zinc to weld seals in water pipes made of lead before 1960. In the United States, approximately 600 metric tons are produced annually and about 150 tons are imported (US 2012).
Contact of Cd in human occurs mainly through ingestion or inhalation. Absorption through the skin contact is negligible. Intestinal absorption of cadmium is greater in individuals with zinc, calcium or iron deficiency (Nordberg et al. 2007).
The main source of cadmium exposure in human is considered to be the cigarette smoking (Friberg et al. 1983). Cd levels in blood and kidney are consistently elevated in smokers than nonsmokers. Inhalation exposure due to industry can be major occupational settings for example, soldering or welding and can cause a severe chemical pneumonitis (Nordberg et al 2007).
Exposure to cadmium from getting unhygienic food (eg, shellfish, leafy vegetables, rice regions of Japan and China and organ meats,) or water (either the old tap closed Zn / CD or a long-term industrial pollution) and can produce long-term effects on health (Abernethy et al. 2010).
After absorption, Cd is transported all over the body, often linked to a sulfhydryl group of protein such as metallothionein and about 30% deposits in the kidneys and 30% in the livers, and the rest scattered throughout the body (Argonne et al 2001). Half life of cadmium in the blood was estimated 75 to 128 days. (Jarup et al 1983). As a result urine, blood and hair Cd levels are poor substitutes for body burden and primarily reflect current contact; it is also true with the other heavy metals. Urine provocation test will require the estimation of cadmium in the body (Bernhoft et al. 2012).
The toxicity of cadmium has been shown in parts of body, cadmium induces tissue damage by creation of oxidative stress (Matovic et al. 2011; Patra et al. 2011; Cuypers et al. 2010) epigenetic changes in DNA expression (Wang et al. 2012; Martinez et al. 2011; Luparello 2012) mainly in the proximal segment of the renal tubule S1 (Vesay et al. 2010) inhibition or up regulation of transport routes (Therenod et al. 2012; Wan et al. 2012; Vankerkhove 2012).
Other pathologic mechanisms comprise competition disruption of the physiologic effects of Mg or Zn (Abdulla et al. 1989; Moulis et al. 2010; Shukla et al. 1984), destruction of mitochondrial function and inhibition of heme synthesis (Schauder et al. 2010), and potentially inducing apoptosis (Cannino et al. 2009). Glutathione reduction is observed, as structural deformation of proteins due sulfhydryl groups bind to the cadmium (Valko et al. 2005). Moreover, these effects are amplified by contact with other toxic metals such as As and Pb (Whittaker et al. 2011) and may be ameliorated by Se or Zn and by factors increasing levels of Nrf2 (Wang et al. 2012; Kcwill 2012).
Medicinal plants are plants having inherent active components used to treat disease or relieve pain (Okigboet et al. 2008). In most developing countries traditional medicines and medicinal plants are used as healing agents for the maintenance of good physical condition (UNESCO 1996) and in developing countries 80% of the peoples relies on traditional medicines, usually herbal remedies, for their prime health care needs (Schmincke et al. 2003). Plants extracts and their products are used in medicines as herbal remedies and they are being used to cure diverse infections (Arekemase et al. 2011). Moreover, there has been an increased concern in the beneficial potential of medicinal plants or plant products containing antioxidant properties in plummeting free radical induced tissue injury (Gupta & Flora 2005). Plants make a vital contribution to health care. The medicinal properties of plants could be based on the antimicrobial, antipyretic, antioxidant, effects of the phytochemicals in them (Cowman 1999; Adesokan et al. 2008).
Natural antioxidants also in the form of crude extracts or their chemical ingredient are very efficient in retarding the devastating processes create by oxidative stress (Zengin et al. 2011) and the toxicity analysis of the majority of the medicinal plants are not yet fully appreciated it is usually accepted that drugs which are derivative of plant products are safer than their imitative counterparts (Oluyemi 2007).
Ginger (Zingiber officinale), is a part of the Zingiberaceae family, is a eminent spice used in your daily diet (Demin et al. 2010) and also utilized for the traditional treatment of several infirmities (Afzal et al 2001). Major components of ginger like shogaol, gingerol, diarylheptanoids and volatile oil, work as antioxidant, anti-diabetic, analgesic, antipyretic, anti-inflammatory, anti-lipid and anti-tumor (Penna et al. 2003; Kadnur et al. 2005; Islamr et al. 2008; Shim et al. 2011; Kim 2008; Wangw et al. 2009). Latest scientific research has exposed that ginger has many therapeutic such as anti-oxidant effects, a capability to restrain the formation of inflammatory complexs and direct anti-inflammatory effects (Thomson et al. 2002). Ginger extract have antioxidative features, since it can scavenge hydroxyl radicals and superoxide anion. Z. officinale was found to slow down the activity of peroxidation and lipoxygenase (Topic et al. 2002).
Another, frequently used spice of Zingiberaceae: ‘curcuma longa’ (turmeric) has shown its strong intrinsic activity as a healing agent for several ailments. The active ingerdient of turmeric is the Curcumin that (Curcuma langalinn) shows antioxidant property. It is a yellow coloured phenolic pigment yield from the turmeric rhizomes (family Zingiberaceae).The most significant characteristic of curcumin is that it has no side consequences, regardless of the therapeutic agent in a number of useful purposes. It acts as a scavenger of free radicals (Khanna et al. 1999). Curcumin is considered to be an efficient antioxidant against oxidative tissue damage. It can considerably restrain the generation of reactive oxygen species (Joe et al. 1994) Moreover, curcumin is considered to be a powerful inhibitor tumour cells proliferation (Joe et al. 2004) a powerful cancer chemopreventive agent (Duvoix et al. 2005; Aggarwal et al. 2005) an dexhibits anti carcinogenic, anti-infective and anti viral properties (Araujo et al. 2001).
Availability: Items available for loan: UVAS Library [Call number: 2199,T] (1).
8.
Polymorphisms Of Bovine Tumor Necrosis Factor Alpha Gene And Its Association With Mastitis In Sahiwal Cows
by Huma Sattar (2013-VA-03) | Dr. Sehrish Firyal | Dr. Ali Raza Awan | Dr. Muhammad Tayyab.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Mastitis is one of the shocking maladies of milch animals causing high production losses to livestock industry of Pakistan (Kenyanjui et al. 2011). It is an inflammatory condition of udder; represent a major problem in dairy cow management. It is one of the most common and frequent disease of dairy industry. Producers suffer a huge loss due to veterinary treatment costs and necessary culling of the infected animals. It negatively affects the milk production, quality of milk, and farm economics (Fourichon et al. 2005). Increasing the disease resistance among dairy cattle is therefore desirable because without controlling mastitis, the national goals of developing dairy farming on commercial and scientific lines and production of wholesome milk which conforms to the standards of WTO Accord would remain elusive.
Mastitis is inflammation of udder that caused by physiological and metabolical changes (Schalm and Noorlander 1957). There are two main types of mastitis; clinical mastitis (characterized by classical symptoms i.e., swelling of udder, redness, clumps and clots in milk etc) and sub-clinical mastitis (not show any symptoms, Milk appear normal, udder appear normal) (Schrick et al. 2001). Mastitis is ranked as a top disease of dairy herds (Rinaldi et al. 2010). This mammary gland infection caused by pathogenic micro organisms such as Staphylococcus aureus, Streptococcus uberis, and Esherichia coli in the mammary gland (Heringstad et al. 2000).
India, China and United States are the larger producer of milk and Pakistan is on forth number in milk yield. Pakistan almost produces 36.5 million tons of milk yeild per year (Cady et al. 1983).The Sahiwal breed is well known among for its superior dairy qualities (Barker et al. 1998). Both cross and pure breed Sahiwal cows have high milk production rate (Khan et al. 2013).
It is very difficult to comprehend this disease because numerous environmental and genetic factors are involved in the origin and development of mastitis (Bradley 2002; Carvajal et al. 2013). Susceptibility and resistance to mastitis is a complex trait influenced by genetic variation of animals. Among these variations, the polymorphisms in immunity genes are principal key factors in defensive mechanism of mammary gland (Ibeagha-Awemu et al. 2008).
The mammary gland tissue is protected by immune system by two defense system; innate and acquired immunity. Innate immunity response by the host is a quick response of bacterial defense system (Mesquita et al. 2012). Innate system is a rapid and effective mechanism that activated on recognition of antigen (Akira et al. 2006). Innate immune system is activated when specific pattern recognition receptors (PRR) that are present on the surfaces which are attach to the specific pathogen (Shuster et al. 1996). PRR are presnt on leucocytes in milk and on the epithelial cells lining of udder. It is reported that T- lymphocyte subset i.e., CD4+, CD8+ and ɤδT are present in infected bovine mammary glands. (Goldammer et al. 2004; Strandberg et al. 2005).
Innate defense (nonspecific) of the mammary gland is stimulated by the physical barrier such as teat end, natural killer (NK) cells, neutrophils, macrophages and certain other soluble factors. The teat cannals are considering the main line of defense. Microorganisms enter from teat canal in milk. The main roles of teat sphincter muscles are to remain orifice close so that bacteria cannot enter. This teat canal also lined with keratin, whose estrified and non estified fatty acid function as bacteriostatics that provide protection and play role to eliminate bacteria causing mastitis (Oviedo-Boyso et al. 2007).
If a pathogen is not eliminated by the physical barrier, the acquired immune system is triggered. In comparison, this system is much faster than other immune response. The memory response is significantly stronger, long durable and more efficient to kill the pathogen. The acquired immune system (memory response) have ability to differentiate self or nonself cells and produce antibodies only against antigens through membrane bound protein called major histocompatibility complex (MHC) molecules. Specific immune system activate only when antigens bind with an MHC that is present on the surface of certain cells, this process is referred as antigen presentation. Recognition of pathogenic factors for elimination is mediated by macrophages, several lymphoid, and immunoglobulins (Ig) or antibodies (Sordillo and Streicher 2002).
The most acute responding macrophages and T-cell cytokines are TNF-α, LTF, IL1, IL6, IL8, and IFN-ɤ present in intramammary infection in cows. These genes play important role in improvement of immunity to mastitis (Burton and Erskine 2003).
Tumor necrosis factor alpha is main pro-inflammatory adipokine that is part of systematic immune defense. The main function of TNF-α gene is responsible for proliferation, differentiation and activity of many immune system cells; B lymphocytes, NK (natural killer). It also induces the production and release of many other cytokines (Wojdak Maksymiec et al. 2013) and also enhances the chemotactic and phagocytic effects of immune response. TNF-α gene contains four exons and three introns that are present on chromosome BTA23q22 (Bannerman 2009; Moyes et al. 2009).
TNF-α is a member of a group of cytokines that stimulate the specific immune system. TNF consist of 212 amino acid arranged in stable homotrimers (Kriegler et al. 1988; Tang et al. 1996). The 17-kilodalton (kDa) TNF protomers are composed of two β-pleated sheets and β-strands, joined together antiparallel (Tang et al. 1996).
TNF-α is a component of natural protection systems of humans and animals. Milk gives nourishment and disease resistance to the new born. Various cellular and soluble immune components are important for protecting the mammary gland from infectious diseases like mastitis. Mastitis affects one third of all dairy cows and cost the dairy industry about 2 million dollars annually (National Mastitis Council (1996). Dairy cattle are especially susceptible to mastitis due to diminished mammary gland defense mechanisms (Sordillo and Streicher 2002).
TNF-α is not only produced by activation of macrophages, but also other cell types such as CD4+ lymphocytes, NK cells, neutrophils, mast cells, eosinophils, and neurons. Large amounts of TNF are released in response to lipopolysaccharide, other bacterial products, and Interleukin-1 (IL-1).TNF-α stimulates the proliferation, differentiation and activity of many immune system cells; B lymphocytes, NK (natural killer). TNF-α induces the release of many other cytokines (Wojdak-Maksymiec and Mikolajczyk 2012). TNF-α also enhance the chemotactic and phagocytic effects of immune response.
. The present study is designed to determine the genetic polymorphism in exon 4 of TNF-α gene of mastitic cows and its association resistance and susceptibility towards mastitis.
Availability: Items available for loan: UVAS Library [Call number: 2224-T] (1).
9.
Dna Based Characterization Of Triacyl Glycerol Lipase Gene From Geobacillus Sp. Sbs-4s
by Maheen Aslam (2012-VA-803) | Dr. Muhammed Tayyab | Ms. Asma Waris | Dr. Sehrish Firyal.
Material type: ; Literary form:
not fiction
Publisher: 2014Dissertation note: Lipases are hydrolases responsible for the liberation of fatty acids from triglycerides (Akoh et al. 2004). With the exception of hydrolysis, lipolytic enzymes can also catalyze transesterification, esterification and interesterification in low aqueous conditions (Goldberg et al. 2005). Under micro-aqueous conditions, lipases have exceptional ability to catalyze the reverse reactions that leads to acidolysis, alcoholysis and esterification (Jaegar and Reetz 1998). Previously production of lipases has been reported from various sources like microorganisms, animals and plants (Lee et al. 2006). Lipases extracted from different sources have broad spectrum properties depending on their sources regarding pH optima, positional specificity, thermostability, fatty acid specificity, etc (Gupta et al. 2004). Thermostable lipases are important for many industries due to their distinct feature (Demirjian et al. 2001). Psychrophilic lipases have high activity at low optimum temperature so they are fascinated for the production of relatively frail compounds and their use has been increased in the organic synthesis of chiral intermediates (Joseph et al. 2008). Alkali stable lipases have ability to work optimally at alkaline pH and are highly suitable to be used in detergents (Sarethy et al. 2011). Lipases are the component of additives in biotransformations, environmental bioremediations, molecular biology applications, food and detergent industry and heterologous gene expression in psychrophilic hosts to prevent formation of inclusion bodies (Houde et al. 2004). Lipases occur in almost all organisms from bacteria to complex organisms. In complex eukaryotes, pig and human pancreas are the main source for lipase production. In eukaryotes, lipases carry out lipoproteins metabolism, fat digestion, reconstitution and adsorption. Lipases have also been extracted from plants. They are found in higher plants and energy reserve tissues.
(Treichel et al. 2010). However, microorganisms are preferred for the production of enzymes over plants and animals because of their shortest generation time, the high yields, great flexibility in environmental conditions, ease of cultivation conditions, variety in catalytic activities, regular supply due to absence of seasonal fluctuations, simplicity in genetic manipulation and quick growth of microorganisms on economical media (Gurung et al. 2013). The production of microbial enzymes is safer and more expedient and they have more stability than their corresponding animal and plant enzymes (Messaoudi et al. 2010). Lipases share a common architecture of α/β-hydrolase fold and a highly conserved pentapeptide catalytic triad G-X1-S-X2-G, where G for glycine, S for serine, X1 for histidine and X2 for glutamic or aspartic acid (Widmann et al. 2010). In the highly conserved catalytic triad there is a nucleophilic residue comprising serine and a catalytic residue containing aspartic or glutamic acid and histidine (Anobom et al. 2014). Lipases have alkyl groups on the surface of their structure due to which they are strongly hydrophobic. Broad substrate specificity is another remarkable characteristic of lipases. Also they catalyze the hydrolysis of alcohols with various chain lengths and esters of fatty acids. The long chain fatty acids of varying chain lengths hydrolysis form triglycerides correspondingly (Patil et al. 2011). Lipases are biotechnologically important enzymes and they have vast applications in leather, food, textile, pharmaceutical, detergent, paper, cosmetic industries and in biodiesel formation (Gupta et al. 2004). Lipases are used in processing of food by the esterification and transesterication of oils and fats. These enzymes are involved in the enhancement of flavor, prolong shelf life and improves aroma of bakery goods, beverages, dairy products, fruits and vegetables. In food
Introduction
3
industry egg yolk is treated with phospholipase to hydrolyze egg lecithin and isolecithin which improves its heating stability and emulsification capacity. This treated egg yolk is then used for the processing of mayonnaise, baby foods, custards, salad or food dressings and sauces. Lipases are also used to remove fats from meat and fish (Aravindan et al. 2006). In textile industry lipases are used in processing of fabrics, thus improving its quality and absorbing ability by removing size lubricants. Polyethylene terephthalate is an important synthetic fiber in the textile industry (Araujo et al. 2008). Lipases action on that fiber improves its hydrophilicity and anti-static ability (Contesini et al. 2010). Lipases in therapeutics are involved in the synthesis of macrolide products. Macrolide products have potential antitumor activity against a broad spectrum of human tumor lines including multidrug resistant cell lines. In pharmaceutical industries, lipases are used for esterification, transesterication and asymmetric hydrolysis of racemic alcohols and carboxylic acids to produce their enantiomeric forms. Many β-blockers, nonsteroidal anti-inflammatory and anti-asthamic drugs are pharmacologically active in their one enantiomeric form while toxic in other form like “profens and ibuprofen” are pharmacologically active in their (S)-enantiomeric form whereas (S)-thalidomide has severe side-effects (Jegannathan and Nielsen 2014). Leather manufacturing industries use lipases for degreasing which is the process of removing fats and grease from skins and hides of cattle. Organic solvents and surfactants are also used to process leather but these methods are not eco-friendly and results in the emission of volatile organic compounds. Besides fat dispersion lipases also improve the quality of leather by making it water proof and low fogging (Horchani et al. 2012). Lipase is used as a catalyst in the tranesterification of vegetable oil or alcohols to form emollient esters like myristyl myristate. Emollient esters due to their moisturizing properties are
Introduction
4
used in beauty creams. Lipases have also been used in anti-obese creams and they are added as texturing agents to improve the consistency of creams and lotions (Sharma and kanwar 2014). Laundry detergents have surfactants as their primary constituent which remove stains. But they require a considerable amount of energy and also they are toxic to our environment, released in water even they are harmful to aquatic life. The detergent industries are developing trends to use such agents that are eco-friendly and require less energy. Nowadays enzymes are being used in the detergents to remove tough stains and give softness, resiliency to fabrics, antistaticness, dispersible in water and mild to eyes and skin. Lipases are used specially to remove oil and grease stains (Ghuncheva and Zhiryacova 2011). The demand of industries for lipases has grown in the past decade for their environment friendly nature, biodegradability, high specificity and high catalytic efficiency. The commercial applications of lipases are a billion-dollar business that comprises their use in a broad spectrum of industries. Many techniques are being used nowadays to improve the features of lipases e.g., stability, activity, specificity and selectivity, reduction of inhibition (Rebeiro et al. 2011). The main advantage of using immobilized lipases is that it is possible to reuse them, since they can be easily recovered, thus making the process economically feasible, not interacting chemically with the polymer, thus avoiding its denaturation in detergent industry and ester formation (Sharma and Kanwar 2014). Genetic engineering has been used to modify the industrial enzymes to enhance its properties (Adrio and Demain 2014). For lipases as potential candidates of detergent industry, these have to be thermostable, alkali stable, stable against proteolysis, action of oxidative compounds and other chemicals used in detergents. In food and pharmaceutical industry usage
Introduction
5
lipases should be more stable in organic solvents and they must show high stereospecificity (Verma et al. 2012). Geobacillus sp. SBS-4S is a thermophillic microorganism that was isolated from Gilgit bultistan, Northern areas of Pakistan. It was found to be gram positive, rod shaped aerobic endospore-forming bacterium. It grows optimally on pH 7 and temperature 55 °C. It produces several industrially important extracellular enzymes including amylases, proteases and lipases (Tayyab et al. 2011). The present study deals with the characterization of triacylglycerol lipase gene responsible for the hydrolysis of triglycerides. Availability: Items available for loan: UVAS Library [Call number: 2234-T] (1).
10.
Detection And Analysis Of Improvised Explosive Devices Used In Terrorism Activities In Pakistan
by Arslan Nazar (2012-VA-630) | Dr. Sehrish Firyal | Dr. Muhammad Sarwar | Dr. Muhammad Wasim | FaizaMasood.
Material type: ; Literary form:
not fiction
Publisher: 2014Dissertation note: Defence and security organizations are in steadyrequirement of finding new options for the detection of explosives. Fundamental applied research in this area focuses on uncovering of highly energetic substances as well as home-basedexplosives that could be a weapon of mass devastation (Marshal and oxley, 2009 yinon and Zitrin 1996, Scubert and Rimiski-Korsakove, 2006). Current methods of detection for explosives or highly energetic materials are based on a wide variety of technologies that focus on either massexplosives or little portions of explosives. Mass explosives can be distinguishedin some way by imaging features,character of the explosives charge, wires and detonators or unswervingly by spotting the chemistry and dielectric characteristics of the explosive substance. Trace recognition method relay on revealing of vapours given off by the explosives or on explosive’sconstituent part that are set down on nearbyexteriors (national Academy of sciences Committee, 2004). Even though, numerous published material is available about methods of sensing of explosives present in air,water, clothing,soiletc and these put forward the benefit of providing traceconfines of sensation at ppb intensities (Caron et al,. 2010; Hilimi and Luonge). Inthe bulk of the criminal acts, sampling is done at the scene trailed by a sample preparationmove, to be shortly processed by a particular technique for analysis. Sampling and samples preparation are amidmajor, shortcomings in explosive uncovering in many cases frightening the physical condition and life of examiner and the responding officer.
Improvised explosive devices are widely used by military in wars and police to keep up regulation and command. Gush of terrorist activities and increase in criminal conduct have been a matter of great concern worldwide and particularly for Pakistan. There have been motiveless
annihilation of private and community properties as well as industrial centres, causing irretrievabledamages to state and local markets and imperillinghumanexistence(Shen et al. 2005). Potentially perilous explosives like dynamite, varied military explosives havingnitroglycerine (NG), Cyclotrimethylenetrinitramine commonly known as RDX, Cyclotetramethylenetetranitramine and alternativehome-produced low explosives and provocative devices are now currently promptly obtainable to scandalous and terrorists. The haphazard and deliberate uses of these explosives consist ofextortion of cash and taking vengeance, unlawful transportation of prohibited substances, assassinations, terrorist and delinquent activities in numerous regions of the country (Sharma and Lahiri 2005).
Recognition of detonating method, estimating the path ways taken by explosive transportation andarresting the anti-social charactersconnected with unstable materials and explosions is primary aim of explosive analysis. For this purpose various explosive substances and explosive remains are to be examined qualitatively and the ingredients are to be approximated quantitatively using primarily by thin layer chromatography (TLC). TLC is a technique employed for the screening of organic constituents at hand in the post blast samples. The identification of explosives containing alkylammonium nitrate is done by TLC. Secondly Gas chromatography with mass spectrometry (GC-MS) technique with the benefit of anelevated resolving supremacy is avitalapparatus for the analysis of chemical composition of explosives. The extremelyproficient GC analysis withcapillary columns authorizes the examination of explosive hydrocarbons, identical substances of nitroaromatics, hexogen (RDX) and the high explosive pentaerythritoltetranitrate (PETN) in a single run. The spectroscopic identification of explosive materials by FTIR is striking due to the intrinsicpotential of real-time detection, non-vicious analysis, and nominal sample preparation, thirdly the scanning electron microscope (SEM) produces an increased image of the sample based on the contact of an electron beam with the sample’s exterior. Finding of minutemasses of explosive remains play an important part in forces, inhabitant, and counter terrorism requests(Pacheco-Londono et al. 2005). To press on explosives sensor methods, present methods need to become affordable and transportable without disturbing the integrity of the devices. The uncovering of ordinary explosives as well as trinitrotoluene (TNT), RDX, HMX, 2,4,6 Trinitrophenyl-N-methylnitramine (TETRYL)Pentaerythritoltetranitrate (PENT), and NG were carried out using diverseprocedures(Sanchez et al. 2007).
Detection of explosives is anparticularly relevant analytical concern for law enforcement personals and for the environmental protection agencies. As the use of explosive substances have been increased by the terrorists, problems have increased for law enforcement and environment and security agencies regarding the detection ofexplosives residues in baggage, parcels vehicles, aeroplane, on travellers, etc. In bomb scene investigations, it is important to find debris that includes detection of explosive residues. Mobile and hand held explosives detectors, similar to those used for detecting hidden explosives, can be of great help in detecting such residues. Several methods i.e. GC/MS, SEM, FTIR were used in Punjab Forensic Science Agency (PFSA) to analyze residues of explosives. The detection of landmines is an acute, urgent worldwide problem that needs specific and effective detection methods (Yinon 2002). Keeping in mind the above said situations, the project was designed with following objectives
Availability: Items available for loan: UVAS Library [Call number: 2235-T] (1).
11.
Epidemiological Investigation And Risk Factor Analysis Of Brucellosis In Large Ruminants And Their Attendants At Govt. Livestock Farms In Punjab
by Muhammad Raashid (2007-VA-496) | Prof. Dr. Mansur-ud-Din Ahmad | Dr. Muhammad Hassan Mushtaq | Dr. Sehrish Firyal.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Pakistan has been renowned as an agricultural country. It is rich in livestock sector having fairly large populations of domestic animals. Among these, populations of cattle and buffalo are 38.3 and 33.7 million respectively. The importance of cattle and buffalo cannot be denied at any level as these are the principal farming animals and milk and beef are widely consumed locally in the country. The estimated annual milk production of cattle and buffalo include 17.372 and 30.462 million tonnes respectively and a combined 1.829 million tonnes beef for human consumption during 2012-2013 (Pakistan Economic Survey 2012-2013).
Brucellosis, a worldwide bacterial zoonosis, is one of the most serious diseases causing huge loss to national economy and human beings among developing countries (Wu et al. 2013). The disease is endemic in Middle East, Central Asia, Africa, Mediterranean region and parts of Latin America (Gwida et al. 2010). Brucellae are Gram-negative bacteria, facultative anaerobic and intracellular pathogens. These show a wide range of host specificity. These coccobacilli measure from 0.6 to 1.5 µm long and 0.5 to 0.7 µm wide. Eight species have been identified in the genus Brucella such asBrucella abortus(B. abortus affecting cattle and buffalo), B. melitensis (sheep and goats), B. ovis (sheep), B. suis (swine), B. canis (dog), B. neotomae (desert rats), B. ceti (cetaceans) and B. pinnipedialis (pinnipeds) (Blasco 2010). This wide range of species covers almost all domestic animals however cats have found resistant. Generally it is considered as a reproductive problem in both male and female animals (Ficht 2003).
Brucellosis has been listed as the second most serious zoonotic disease in the world after rabies by OIE (OIE 2009). B. abortus, the primary cause of Brucellosis in large ruminants, (cattle and buffalo), remains not only a significant threat as a source of human illness but also risks economy of the country (Makita et al. 2011). Present estimates of economic loss in meat and milk production resulting from Brucellosis are $800 million annually in the United States(OIE 2009). The incidence of the disease can be correlated to several factors including demographic and geographic factors(Soomro et al. 2014). Seroprevalence of the disease has been reported in different regions of Pakistan and ranges 3.25 to 4.4%(Naeem et al. 1990).
Brucellosis in cattle and buffaloes can be recognized clinically by an abortion usually occurring form 6 months and onwards i.e. last trimester of pregnancy (Soomro et al. 2014). Brucellosis is principally a disease of sexually mature animals as it affects mainly the reproductive system and fertility of the animals. It significantly reduces the survival rate of newborns and also the milk yield (Sikder et al. 2012). Greyish white mucoid or mucopurulent discharges from the vagina, prior to parturition of cow, may show the clinical patterns of disease along withnormal patterns of parturition like swelling of the vulva, relaxation of pelvic ligament, enlargement of udder and discharge from the vulva (Shafee et al. 2012).
Human infections as a result of Brucellosis range more than 500,000 annually round the world (Abo-Shehada and Abu-Halaweh 2011). Brucellosis can cause a wide range of symptoms similar to the flu and may also include fever (39-40°C), night sweats, headache, back pain and physical weakness. Severe form of infection may result in involvement of the central nervous system or the lining of the heart (Soomro et al. 2014). It is one of the principal public health problems for an agricultural country like Pakistan, where majority of the population is engaged in livestock farming (Shafee et al. 2012).
Brucellosis in humans is a severely debilitating condition that usually requires prolonged treatment involving a combination of strong antibiotics. The treatment results in permanent and disabling sequel, and also in significant medical expenses along with loss of income due to loss of working hours. Brucellosis can be transmitted to humans by ways of inhalation, direct contact with infected animals or contaminated products of conception and ingestion of unpasteurized dairy products and undercooked meat or meat products (Gwida et al. 2010; John et al. 2010). Brucellosis can also be transmitted from infected animals to human beings who are in close contact with animal secretions like infected vaginal secretions, blood, urine, feces, aborted fetus, or those who consume unpasteurized milk or other raw milk products. Shepherds, milkmen, butchers, knackers, veterinary assistants, and abattoir workers are at high risk (Agasthya et al. 2007)
Availability: Items available for loan: UVAS Library [Call number: 2245-T] (1).
12.
Detoxification Of Aflatoxins Using Different Organic Acids
by Sana Ejaz (2013-VA-14) | Dr. Mateen Abbas | Dr. Muhammad Tayyab | Dr. Sehrish Firyal.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: From global prospective of food safety and food security, mycotoxin contamination of foods has gained much attention as potential health hazards for humans and animals. Cereals and other crops are exposed to fungal attack in the field or during storage and this attack may result in mycotoxin contamination of crops. Animal feed is basic necessity for all the live stock, poultry and other animals. AF is the most important for human and animal health perspective and in developing countries such as Pakistan where climate conditions favor the formation of these toxic metabolites. Governments and private organizations of international level have established maximum residue levels (MRIs) which usually guide to control AF in feed. Therefore, the current study was planned to detoxify AF by using different organic acid treatments in animal feed collected from different dairy farms of Punjab.
The samples of cotton seed cake, maize oil cake and animal feed were collected and checked the presence of AFB1 qualitatively by TLC and quantitatively by HPLC. The samples which gave positive results were treated with different acidic treatments applied on it. Firstly checked the results of citric acid, acetic acid and lactic acid on feed sample qualitatively by TLC. TLC plates were checked under UV box and the samples which showed the detoxification of AF were quantitatively analyzed by HPLC in Toxicology Laboratory, QOL, UVAS, Lahore, Pakistan.
The average concentration of AFB1 found in the cotton seed cake, maize oil cake and mixed feed were 279.8 ppb, 34.2 ppb and 25.5 ppb, respectively much greater than permissible levels proposed by European Union. Treatments of varying concentration of citric acid, acetic acid and lactic acid were applied on positive samples (≥20 ppb) and checked their effect on rate of detoxification.
All the above mention treatments applied on the feed samples in order to obtained in vitro detoxification of AFB1. Sprayed different concentration of acetic acid, citric acid and lactic on positive samples by varying volumes and placed them over night then extracted and analyzed.
It has been observed that 1N concentration of citric acid, acetic acid and lactic acid showed complete detoxification. However, when these samples were treated with 0.5N solution of organic acids then variation was seen in rate of detoxification.
Statistically these results were analyzed by ANOVA which showed that effect of these treatments on rate of detoxification was highly significant (P<0.05). In vitro detoxification of AF by these organic acids was proved beneficial in order to reduce the animal and human health risks. However, in vivo detoxification of aflatoxin by using these organic acids should be studied in future.
Availability: Items available for loan: UVAS Library [Call number: 2283-T] (1).
13.
Genetic Diversity Among Different Isolates Of Pasteurella Multocida From Poultry
by Arslan Sardar (2013-VA-282) | Dr. Imran Altaf | Prof. Dr. Aftab Ahmad Anjum | Dr. Sehrish Firyal.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Fowl cholera is an acute bacterial disease of broiler breeders and layer breeders caused by Pasteurella multocida. In the present study, 10 isolates from different areas of Punjab were purified. These samples were confirmed by API Kit. Different molecular techniques like PCR and RFLP were used to investigate variation at the molecular level among 10 isolates collected from different areas of Punjab. Different mutations were observed among 10 field isolates at different mutation sites by sequencing. Phylogentic tree was also made using MEGA6 software that supported the sequencing results. ‘Msp1’ endonuclease cleaved bacterial whole genome at different cutting sites, all 10 isolates collected from different districts of Punjab cleaved into 3 to 5 fragments ranging from 600 to 10000 base pairs which showed the genetic variation among 10 isolates of P.mulocida. Availability: Items available for loan: UVAS Library [Call number: 2315-T] (1).
14.
Variation Analysis Of Hepatitis C Virus Gene Encoding E2 Glycoprotein
by Saimoon Theeen (2009-VA-565) | Dr. Muhammad Imran | Dr. Sehrish Firyal | Dr. Muhammad Tayyab.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Hepatitis C virus (HCV) major cause of liver infections was discovered in 1989. It is positive stranded RNA virus and belongs to Flaviviridae family. Its genome shows high rate of variations due to which, its rate of infection is high. As in Pakistan 3% to 6% population and 170 million people worldwide are affected by it. Due to variations in its genome 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). Whereas E2 is considered the most immunogenic gene from all the genes. It involves in the interaction with the host cell and easily escape from the immune system of host due to the presence of hypervariable regions in E2 gene.
To isolate the E2 gene RNA extraction was done using the kit method and then it was converted to the cDNA which is then followed by the PCR amplification. The amplification products were then purify and sent for the sequencing to CAMB. Then the bioinformatics tools were applied on the results. In which the protein structural analysis and epitope mapping was done. Then the conserved epitopes were predicted using the IEDB conservancy analysis tool. The conserved B-cell epitopes (TElAILPCSFTPMPAL and RGERCDIEDRSEQH) and T-cell epitope (TPMPALSTG) are now considered valuable to produce the antibodies against E2 protein.
For diagnosing HCV genotype 3a, these conserved epitopes may be highly useful and may also help in developing a successful vaccine that can target 3a genotypes. Availability: Items available for loan: UVAS Library [Call number: 2333-T] (1).
15.
Study Of Genetic Polymorphism In Exon 7 And 9 Of Glucosidase Beta Acid (Gba) Gene In Gaucher Diseased Patients From Punjab, Pakistan
by Ayesha Khalid (2013-VA-07) | Dr. M. Yasir Zahoor | Dr. Sehrish Firyal | Mr. Tariq Mahmood.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Gaucher disease (GD) is an inborn metabolic disease transmitted through recessive
pattern of inheritance and it is a pan-ethnic disease. It is the most common lysosomal storage
disease caused by the deficiency of glucocerebrosidase (GCase), a lysosomal enzyme use in the
degradation of macromolecules into simpler molecules.
Glucosidase beta acid (GBA) gene encode glucocerebrosidase enzyme and mutations in
this gene is responsible for glucocerebrosidase deficiency which results in an accumulation of
unbroken glycolipids in those organs rich in monocyte-phagocyte immune system elements i.e.
spleen, liver, bone marrow and leads to histological changes. GBA is located on chromosome
1q21 consisting of 11 exons and 10 introns having 7.8kb length. It is divided into three types (I,
II and III) on the basis of neurological involvement. More than 300 mutations have been reported
in GBA and cause the GD.
The present study was performed in order to characterize GBA gene in GD patients from Punjab.
Blood samples of 10 patients,enrolled in Children Hospital, Lahore, were taken from DNA
repository of Molecular and Genomic Lab at IBBT, UVAS Lahore. The DNA was extracted
using organic method. Next step was the amplification of extracted DNA using PCR. After it, the
PCR product is purified and this purified PCR product was sent for sequencing. Sequencing of
exon 4, 7 and 9 was done using dideoxy sequencing method. After applying different
bioinformatics tool, it was found that there was no muttaion in these exons but a heterozygotic
variation G>A was found in intron 8. This finging will help in demonstration of molecular
pathogenesis of Gaucher disease. Availability: Items available for loan: UVAS Library [Call number: 2338-T] (1).
16.
Antiviral Effect Of Human Saliva Against Avian Influenza Virus Strain H9n2
by Maryam Riaz (2008-VA-340) | Prof. Dr. Tahir Yaqub | Dr. Sehrish Firyal | Prof. Dr. Kamran Ashraf.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Saliva is an important body fluid that contains a complex array of proteins, peptides and various substances that help in maintaining the health of the oral cavity. Saliva exhibits a broad-spectrum of antiviral activity against enveloped viruses as it disrupts the viral membrane. Influenza is a common virus that has been diagnosed in humans and avian species due to AIV. This study has demonstrated the naturally occurring antiviral activity of human saliva against the H9N2 influenza virus that serves as a serious threat to poultry and has been shown to possess high zoonotic potential which can cause a new pandemic.
In this study saliva samples from healthy individuals were taken and the natural antiviral ability of saliva was observed against AIV (Pk-UDL/01/08 H9N2) of calculated EID50 106.66. Inoculum prepared from saliva and H9N2 virus was injected in 9 days old embryonated eggs using CAS route and incubated at 37°C for 48 hours. A negative control (only saliva) and positive control (only virus inoculum) was also determined in the current study. The antiviral activity of saliva was observed through haemagglutination test. The HA test of harvested fluid showed that human saliva indeed possesses antiviral activity against H9N2 virus and can be used as a natural antiviral agent in medicine.
Furthermore, the genomic DNA was extracted from the blood samples. HTN3 gene responsible for histatin production, was amplified using gene specific oligonucleotides. The obtained HTN3 gene sequences were analyzed using Chromas software. The sequence alignment showed 99% similarity to the available sequences in NCBI database and 100% similarity to each individual sample. To conclude, this study has demonstrated that human saliva possesses antiviral activity against H9N2 virus. The nucleotide sequence analysis from each sample
CHAPTER 6
SUMMARY
Summary
47
showed no particular change which shows that antiviral activity of glycoproteins present in saliva does not vary at a genetic level. This innate antiviral activity can open a new frontier when it comes to combating viral infections that have grown resistant to conventional drugs in both human and animal subjects. Availability: Items available for loan: UVAS Library [Call number: 2336-T] (1).
17.
Genetic Identification And Molecular Classification Of Sub-Family Phasianinae Of Pakistani Bird Species Through Dna Barcoding
by Maryem Hussain (2008-VA-349) | Dr. Ali Raza Awan | Dr. Sehrish Firyal | Dr. Muhammad Tayyab.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: DNA barcoding is a precise technique that uses molecular genetics tools for accurate identification, categorizing, relating and separating the phylogenies of species. Being a small sized genome and agile enough to show rapid mutation, mtDNA has been used as a pertinent marker of molecular biodiversity.The aim of this study was to develop DNA barcode for genetic characterization and classification of Sub-family Phasianinaeof Pakistani bird species. Theyhave not been genetically identified yet in Pakistan. It includes birds like domestic chicken(Gallus gallusdomesticus), aseel chicken(Gallus gallusdomesticus strain),blue peafowl(Pavo cristatus), green peafowl (Pavo muticus), white peafowl (Pavo cristatus leuticus), Kalij pheasant (Lophura leucomelanos),monal pheasant (Lophophorus impejanus),koklass pheasant(Purcrasia macrolopha), ring necked pheasant (Phasianus colchicus), Tragopan (Tragopan melanocepals) andred junglefowl (Gallus gallus). These birds are considered an important part of an ecosystembecause they play a significant role in seed dissemination, pollination of plants and disease spread which are the basic constituents of an ecosystem. They are used for food, hunting and entertainment purposes.
Mitochondrial geneCytochrome c oxidase subunit 1 (CO1)of 500bps was used as a marker for identification at specie level.Genomic DNA was extracted by each blood and tissue sample of eleven bird species (33 samples). Amplification of CO1 gene was a done by using a universal set of primers (BIRDF1 and BIRDR1)containing region of almost 750 bps (Hebert et al. 2003).Amplicons were purified and sequenced Sanger sequencing method (Sanger et al. 1977). Forward and reverse sequences were analyzed using softwaresEMBOSS merger,ClustalW, BioEdit and nBLAST. Phylogenetic analysis of selected bird species was done. Each sequence was aligned
with its reference sequences of CO1 gene present on NCBI. Every nucleotide position which did not align with the reference sequence was studied to identify SNPs. Fixation index (FST) were used to measure species diversity within a same sub population relative to that found in the entire population. Consensus sequences (500bps) generated was used to construct their phylogenetic tree to see their evolutionary relationship with other bird species. All species showed their closest linkage with their respective species. Pakistani population of peafowl and chicken species showed the close relation with same sequences generated in China. Tranopans showed its closest linkage with T. temminckii.
In conclusion, seven species ofPhasianinaesub-family of Pakistani bird species was genetically characterized first time in Pakistan by using CO1 as a barcode. It proves that DNA barcoding is an efficient and accurate molecular tool for species identifica¬tion and phylogenetic implication. This study leads to establish a DNA Data Bank that helped scientists to investigate the biodiversity, taxonomic classification, specie identification, in forensic purposes and to study the genetic and phenotypic evolution of these species. DNA barcoding through CO1 gene works as a functional tool for detectingmeat mislabeling and preventing illegitimate trade. This study has established foundations for molecular biologists to study taxonomic uncertainties at sub species level using SNP based identifying marker. It helps in preservation and identification of endangered species by generating their barcodes from even minimal evidence available.
Availability: Items available for loan: UVAS Library [Call number: 2376-T] (1).
18.
Molecular Characterization of Pakistani Common Leopard
by Muhammad Usman Ijaz (2012-VA-908) | Dr. Sehrish Firyal | Dr. Ali Raza Awan | Dr. Muhammad Tayyab.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: CD not available. Availability: Items available for loan: UVAS Library [Call number: 2379-T] (1).
19.
Identification Of Single Nucleotide Polymorphism In Toll Like Receptor 4 Gene And Its Association With Mastitis In Sahiwal Cows
by Hafiz Kamran Rizwan Ullah (2013-VA-557) | Dr. Sehrish Firyal | Dr. Muhammad Wasim | Prof. Dr. Habib Ur Rehman.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Several factors militate against realizing the milk production potential of cows. Mastitis is one of the shocking maladies of milch animals causing high production losses to livestock industry in Pakistan and elsewhere in the world. Mastitis has been familiar as one of the most inexpensively important diseases affecting dairy animal’s worldwide.
Susceptibility and resistance to mastitis is a complex trait and influenced by genetic variation of the immunity genes of animals. Among these variations, the polymorphisms in Toll-like receptor 4 gene (TLR4) play important role in the immune response to mastitis. Polymorphism in exon 3 of TLR4 gene is associated with mastitis susceptibility and resistance. It is a potential candidate gene for screening of the mastitis susceptible and resistant dairy cows.
The present study was designed for the identification of polymorphism in TLR4gene associated with mastitis. Blood samples from 20 Sahiwal cows having clinical and subclinical mastitis were sampled. Blood sample of 10 normal Sahiwal cows was also collected. DNA was extracted. Specific primers for amplification of TLR4 gene were designed from NCBI.
TLR4gene was amplified and sequenced to get the desire sequence of this gene. Comparative analysis of the resulted sequences using NCBI BLAST was done.
Availability: Items available for loan: UVAS Library [Call number: 2392-T] (1).
20.
Lactoferrin Gene Polymorphism in Dairy Cattle
by Syeda Iqra Aiman Bukhari (2009-VA-556) | Dr. Sehrish Firyal | Dr. Ali Raza Awan | Dr. Muhammad Tayyab.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Several factors militate against realizing the milk production potential of cows. Mastitis is the most costly and the prevalent production-limiting disease of dairy animals in Pakistan and elsewhere in the world. It is accompanied by elevated Somatic cell count (SCC) in the milk and estimated genetic correlation between SCC and mastitis ranges between 0.53-0.77. Susceptibility and resistance to mastitis is a complex trait and influenced by genetic variation of animals. Among these variations, the polymorphism in Lactoferrin gene (LTF) plays an important role in the immune response to mastitis.
Polymorphism in intron 6 of LTF gene is associated with mastitis susceptibility and resistance. It is a potential candidate gene for imparting resistance mastitis in dairy cows.
The present study was designed for the identification of polymorphism in LTF gene associated with mastitis. Milk and blood samples were collected from 20 Sahiwal cows having clinical and subclinical mastitis. SCC of milk samples was performed using serial dilutions. 10 normal Sahiwal cows as control were included in present study. DNA was extracted from blood using organic extraction and kit method followed by DNA quantification. Amplification of LTF gene was designed by using already reported primers obtained from NCBI.
LTF gene was amplified and sequenced to get the full length sequence of this gene. Comparative analysis of the resulted sequences using NCBI BLAST was done.
Outcomes:
The results obtained from polymorphisms in LTF gene can play an important role for selection of mastitis resistant and susceptible dairy cows. This can be useful in selective breeding of cattle for enhanced immune response, as a tool to improve inherent animal health, which ultimately can lay the foundations to contain the magnitude of economic loss due to mastitis.
Develop a biological response modifier that will promote a sustained immunity of the mammary teat and protect the gland from invading pathogens. Availability: Items available for loan: UVAS Library [Call number: 2416-T] (1).
21.
Polymorphism Analysis Within Tata-Box Of Bovine Lactoferrin Gene And Its Association With Mastitis In Sahiwal Cows
by Kashmala Haroon (2014-VA-04) | Dr. Sehrish Firyal | Dr. Immad Rashid | Dr. Muhammad Tayyab.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: Mastitis is one of the most important diseases in dairy cows throughout the world, and is responsible for significant economic losses to the dairy industry especially in Pakistan. Several factors are responsible for this disease and about 20% bovines are suffering with this disease. Mastitis susceptibility and resistance is influenced by genetic variation of animals. Variations to polymorphisms in LF gene assume critical part of the immune response to mastitis. Polymorphism within LF gene may influence immune response to the mastitis in bovines. Recent study shows that promoter region of LF gene is highly polymorphic among bovines.
Present study was planned to identify polymorphism analysis within TATA-box of bovine LF gene and its association with mastitis. Multiple blood samples were collected from Sahiwal cows having clinical and sub-clinical mastitis. 10 samples were collected as a control. DNA extraction was done by organic extraction method and then quantification was done by Nanodrop. Amplification and sequencing was performed to get desire sequence of the gene. Comparative study of obtained sequence results were analyzed by using NCBI blast. Bioinformatics analysis was done with the help CLUSTAL W and BioEdit softwares.
Two novels and one reported SNPs were discovered within TATA-box of LF gene that might be having strong genetic association with mastitis in Sahiwal cows. This gene is strong candidate gene to differentiate between mastitis susceptible and resistant Sahiwal cows.
Availability: Items available for loan: UVAS Library [Call number: 2584-T] (1).
22.
Polymorphism Analysis Of Exon 2,5 And 10 Of Bovine Lactoferrin Gene And Its Association Within Mastitis In Sahiwal Cows
by Sidra Mukhtar (2014-VA-223) | Dr. Sehrish Firyal | Dr. Sultan Ali | Dr. Muhammad Wasim | Dr. Muhammad Avais.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: A few factors militate against understanding the milk production capability of bovines. Mastitis is the most costly and the prevalent production-limiting disease of dairy animals in Pakistan and somewhere else on the world. Susceptibility and resistance to mastitis is a complex characteristic and impacted by hereditary variation of animals. Among these variations, the polymorphisms in LF assumes critical part in the immune responses to mastitis.
Susceptibility and resistant to mastitis is a complex trait and influenced by genetic variation of the immunity genes of the animals. Among these variations, polymorphism in Lactoferrin gene (LF) play important role in immune responses to mastitis. Polymorphism in exons 2, 5 and 10 of Lactoferrin gene are associated with mastitis susceptibility and resistance. It is a potential candidate gene for screening of the mastitis susceptible and resistant dairy cows.
The present study was designed for the identification of polymorphism in LF gene associated with mastitis. Blood samples from 20 Sahiwal cows having clinical and subclinical mastitis were sampled. Blood samples of 10 normal Sahiwal cows were also collected. DNA was extracted. Specific primers for amplification of LF gene were designed by using Primer 3 software.
LF gene was amplified and sequenced to get the full length sequence of the gene. Comparative analysis of resulted sequences was done with the help of NCBI BLAST. Multiple sequence alignment was done by using CLUSTAL W and BioEdit softwares. Protein analysis was done with ExPasy translate tool and the development of 3D structure were using PYMOL software.
Availability: Items available for loan: UVAS Library [Call number: 2582-T] (1).
23.
Evaluation Of Inhibition Activity Of Indigenous Lactobacilli Spp. On Ammonia Emitting Bacteria
by Fatima Sajjad (2010-VA-312) | Dr. Muhammad Nawaz | Prof. Dr. Masood Rabbani | Dr. Sehrish Firyal.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: Poultry is the 2nd largest industry in Pakistan which is growing at an amazing rate of more
than 10% from last few years. It provides jobs for more than 1.5 Million people. Although, it is
growing at excellent pace, it still faces many problems. One of the important problems in poultry
farming is the production of ammonia by urease producing microbes. Ammonia is health hazard
for both poultry and human. Urease producing bacteria which are the major problems in poultry
are Pseudomonas aeruginosa, Proteus mirabilis, Klebsiella pneumoniae and Staphylococcus
aureus in poultry droppings. Probiotics such as Lactobacilli can inhibit the urease producing
bacteria in poultry intestine as well as environment thus mitigate the ammonia emission. Present
study was designed to isolate Lactobacilli from indigenous poultry, screen them for
antimicrobial activity against urease producing microbes and determine their effect on growth of
Proteus mirabilis during co-culture experiments. A total of 71 Lactobacilli isolated were
recovered from 20 samples of droppings (10) and caeca (10) of back-yard poultry on MRS agar
plates. Twenty seven (27) isolates demonstrated antimicrobial activity against ammonia emitting
bacteria by agar spot and well diffusion assays. Seven isolates (FSL19, FSL25, FSL39, FSL45,
FSL51, FSL63 and FSL71), having better antimicrobial activity, were selected for co-culturing
with Proteus mirabilis in nutrient broth. FSL25, FSL45, and FSL51 showed more than 2 log10
reduction of Proteus mirabilis in co-culture experiments. FSL25, FSL45, and FSL51 were
identified as Lactobacillus plantarum, Lactobacillus fermentum and Lactobacillus salivarius,
respectively by amplifying and sequencing their partial 16S rDNA. It is concluded that
Lactobacillus plantarum FSL25, Lactobacillus fermentum FSL45 and Lactobacillus salivarius FSL51 may be used to mitigate ammonia emitting bacteria in poultry environment after further
investigations. Availability: Items available for loan: UVAS Library [Call number: 2594-T] (1).
24.
Determination Of Comparative Effect Of Two Eeg Yolk Based Extenders On Post Thaw Semen Quality Of Sahiwal Bull
by Shahid Ali (2010-VA-05) | Prof. Dr. Main Abdul Sattar | Prof. Dr. manzoor Ahamd | Dr. Sehrish Firyal.
Material type: ; Literary form:
not fiction
Publisher: 2017Dissertation note: Cryopreservation of semen is the principal step for its usage in artificial insemination. Freezing of semen leads to reduction in post thaw semen quality. Glycerol has the cryoprotective properties that led to preserve spermatozoa. Egg yolk is also a basic constituent of semen extenders. Beneficial effect of egg yolk on sperm cryopreservation is plasma membrane protector. Tris based extenders are commonly used for semen cryopreservation of bulls, rams and bucks. Based on the economics and beneficial effects of extenders on bovine post thaw semen quality, tris based commercial as well as custom made semen extenders requiring egg yolk addition, needs to be considered for further studies. This study had been designed to determine the comparative effect of two egg yolk based extenders on post-thaw semen quality (Triladyl™and TCEY) on Sahiwal bulls. Semen was collected using an artificial vagina having temperature of 42 ºC from five adult Sahiwal regular donor bulls, raised at Center of Excellenc for Bovine Genetics (CEBG) Renalakhurd, Okara.Seven replicates of the experiment were performed. Volume, concentration and motility of ejaculates was evaluated. Semen samples having motility >60% and sperm/ml >500x106 were included in study. After evaluation, semen samples were pooled, divided into two aliquots of equal volume and kept in water bath at 37ºC. One aliquot was extended with tris citric acid egg yolk extender (TCEY) and other was extended with commercially available extender (Triladyl™). Pre-freeze CASA sperm motility parameters and kinematics of these extended aliquots were assessedat CEBG. After that, extended semen was cooled to 4 ºC for 4hr, equilibrated for 2hrs at 4ºC and packaged into 0.5 ml French straws (20 x 106 spermatozoa/straw). All semen straws were placed into automatic programmable freezer having liquid nitrogen vapors for 10 min. Afterward, shifted to liquid nitrogen for freezing and were stored until post-thaw semen evaluation carried out. The experiment was
repeated for seven times (replicates, n=seven). For post-thaw semen evaluation, four semen straws per treatment group were thawed (30 seconds) in water bath at 37ºC and post-thaw CASA sperm motility parameters and kinematics were checked. Post-thaw motility (PTM%) , Plasma Membrane Integrity (PMI %) , acrosome integrity (AI %) , live Sperm Percentage (LSP % )and sperm abnormalities (SA %) were checked by phase contrast microscope. Similarly AI (%), PMI (%), mitochondrial integrity (MI%) and DNA integrity (%) were checked by fluorescence microscope at Department of Theriogenology, University of Veterinary and Animal Sciences, Lahore.
Pre-freeze CASA sperm motility parameters;progressive %, rapid %and kinematics;average path velocity (VAP um/s), straight line velocity (VSLum/s), linearity (LIN %) were significantly better in Triladyl thanTCEY.Post thaw CASA sperm motility parameters; motile %, progressive %, rapid % and kinematics; VAP (um/s),VSL (um/s), straightness (STR %) and LIN (%) were also significantly better in Triladyl than TCEY. Post thaw semen quality parameters containing PTM (%), PMI (%), LSP (%), DI (%) andMI (%) were significantly better in Triladyl as compared to TCEY.
Availability: Items available for loan: UVAS Library [Call number: 2785-T] (1).
25.
Mutation Detection Of Cacnb4 Gene Involved In Childhood Absence Epilepsy And Its Comparative Genomics In Mice
by Ayesha Amin (2015-VA-1048) | Dr. Muhammad Wasim | Dr. Sehrish Firyal | Prof. Dr. Aftab Ahmad Anjum.
Material type: ; Literary form:
not fiction
Publisher: 2017Dissertation note: Childhood absence epilepsy (CAE) is due to multiple factors affecting almost 9-12 years children. Depolarization of ion channel activates the channel. CACNB4 gene is affected by epileptic seizures. Disturbance in ion channel can affects different genes as CACNA1G, CACNA1H, CACNA1A, SCN1B, SCN1A,SCN2A and GABA receptor genes. CACNB4 gene has a major role in influencing epilepsy in human.
In present study,it is directed to analyze the mutations in epilepsy present in coding region of CACNB4 gene.
Collection of blood samples were from Children Hospital, Lahore, Punjab Pakistan from CAE patients of epilepsy. By using standard DNA extraction method, DNA was extracted from samples. Primers were designed for the amplification of exon 3 and 13 of CACNB4 gene.
Results were examined after sequencing the samples. BioEdit software was used to study the samples thoroughly. NCBI BLAST was used to align the sequences.
It is investigated that the sequences of CAE patients of epilepsy of CACNB4 gene has mutation at position position 258023bp which changes A>G. In protein sequence, the mutation is at position 413 which changes L (Leucine) to L (Leucine). This mutation has no effect because this is a synonymous mutationwhere the codon CUG is changes to CUA, both codes for same amino acid that is leucine, so no effect at all by this change in exon 13.
Three mutations are present in the intronic region of exon 13 first, second and third at positions 258184bp A deleted, 258289bp and 258191bp of CACNB4 gene respectively. These all mutations are present in intronic region so has no effect in phenotypes of individual.
In conclusion, maximum numbers of samples were needed to observe the effect of mutations and factors that causes epilepsy. This study will now help the researchers to investigate genetic therapies, strategies of genetic counseling and parental diagnosis for the population of Pakistan.
Availability: Items available for loan: UVAS Library [Call number: 2924-T] (1).
26.
Study On Polymorphism Of Promoter Region Of Bovine Lactoferrin Gene And Its Relation With Mastitis In Nili Ravi Buffalo
by Muhammad Asim (2012-VA-636) | Dr. Sehrish Firyal | Dr. Muhammad Yasir Zahoor | Dr. Muhammad Tayyab.
Material type: ; Literary form:
not fiction
Publisher: 2017Dissertation note: Dairy animals in Pakistan and worldwide are facing most persistent and economically affecting
disease mastitis. Only a healthy buffalo can produce good quality milk of physiologically normal
composition. Mammary gland inflammation due to mastitis badly affects the quantity and quality
of milk and this causes big loss to dairy industry. Even province Punjab bears economic losses of
240 million per annum due to mastitis.
Susceptibility and resistance to mastitis is affected by the variation in immunity genes. Among
immunity genes Lactoferrin (LF) have important role in immune defense system and perform
antibacterial, antiviral and anti-inflammatory function. LF is found in most of body fluids like,
milk, blood, tear, saliva, bile and mucous. Polymorphism in promoter region of Lactoferrin gene
is associated with mastitis susceptibility and resistance. For screening of the mastitis
susceptibility and resistance of dairy buffaloes, LF is a potential candidate gene.
The present study was designed for the identification of polymorphism in LF gene associated
with mastitis. Blood samples from 20 Nili Ravi buffalos having clinical and subclinical mastitis
were selected. Blood samples of normal Nili Ravi buffalos were also collected. DNA was
extracted; specific primers for amplification of LF gene were designed with Primer-3 software
by using already reported sequence on NCBI.
Amplification of LF gene performed by PCR and sequenced the amplicon. A comparative
analysis of sequence result was performed by using NCBI BLAST. BioEdit software was used to
perform multiple sequence alignment. Comparison analysis of LF gene promoter region shows
multiple mutations in in clinical and subclinical as compare to reported sequence (accession no.
EF650854) at NCBI. While normal samples sequence results are similar to reference data. These
results show LF is a candidate gene for mastitis resistance. Availability: Items available for loan: UVAS Library [Call number: 2923-T] (1).
27.
Polymorphism Analysis Of Bovine Tumor Necrosis Factor Alpha Gene And Its Association With Mastitis In Nili Ravi Buffaloes
by Samia Tanveer (2011-VA-362) | Dr. Sehrish Firyal | Dr. Muhammad Wasim | Dr. Muhammad Nawaz.
Material type: ; Literary form:
not fiction
Publisher: 2017Dissertation note: Various number of factors cause hindrance in the milk production potential of buffalos. Mastitis is the costly and most prevalent disease causing production losses of dairy herds in Pakistan and elsewhere in the world. Susceptibility and resistance to mastitis is complex trait influenced by genetic variation of animals. Among these immunity gene variations, the polymorphism in tumor necrosis factor alpha gene (TNF-α) play important role in immune response to virus. Polymorphism in TNF-α gene is associated with mastitis susceptibility and resistance. It would be a potential candidate gene for imparting resistance mastitis in dairy buffalos. Blood sample were taken from the 20 Nili Ravi buffalos having clinical and subclinical mastitis. Extraction of DNA was done from frozen blood after thawing, using organic extraction method & also kit method followed by DNA quantification (i.e. gel electrophoresis and nanodrop). Total 5 primers were designed using Primer3 bioinformatics tool. All these primers were optimized using different protocols and a set recipe was obtained for each primer. The amplification of DNA samples was done one by one using all these five primers on optimized protocol. The amplicons obtained were subjected to agarose gel electrophoresis to check whether we have the required product or not using 100 kb ladder and then amplicones were send for the sequencing.
Summary
110
The sequencing analysis of resulted amplicon sequence was done using Bioinformatics software Finch TV. Total of 6 mutations were found while 5 were same in all the samples whereas 6th mutation was found only in clinical samples. It is valuable in accomplishing genetic progress for resistance and to improve the immune response. This study will paved the way for animal breeder for selection of Nili Ravi mastitis resistant buffalos for breeding. TNF-α gene polymorphism based marker is now available for screening of resistant bulls as well. Availability: Items available for loan: UVAS Library [Call number: 2936-T] (1).
