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1. Mutation Screen Of "Gamma-Aminobutyric Acid (Gaba)-A Receptor, Gamma 2" In Punjab Population

by Muhammad Javed Iqbal | Dr. Muhammad Wasim | Dr. Abu Saeed | Dr. Ali Raza Awan.

Material type: book Book; Format: print ; Literary form: drama Publisher: 2012Dissertation note: Epilepsy is a formidable form of neural disorder that can impose its long lasting effect on person's life and development. To date, it lacks any effective therapy and is multistep disease strengthened by an overwhelming number of genetic and epigenetic mechanisms that streamline epileptic attacks. This particular study encompasses two major types of epilepsy, CAE and GTCS by targeting a GABRG2 gene. Mutation analysis of the coding exons (exon 3, 5 and 9) was performed by direct sequencing of GABRG2 in order to sought out complex biological entities in both types of epilepsies. GABRG2 is a molecule that has recently been characterized as the culprit for epileptic seizures onset. GABRG2 encodes GABA receptor that is fundamental inhibitory neurotransmitter in mammalian brain and is a ligand-gated chloride channels. This ligand-receptor coupling results in the inward shuttling of chloride ions through the channels and this hyperpolarizes the neurons, which induce the inhibitory effect of neurotransmitters. Direct sequencing of candidate gene "GABRG2" traced out a single polymorphic site in the exon 3 of the CAE as well as GTCS cases. However, this single nucleotide alteration is more commonly identified in childhood absence epilepsy patients as compared to the generalized cases. Silent mutation was identified at locus 27909 C>T of 46.66% of the total screened or analyzed cases. Availability: Items available for loan: UVAS Library [Call number: 1500,T] (1).

2. Dna Barcoding Of Pakistan Avian Families (Sturnidae & Columbidae)

by Emma Umar | Dr. Ali Raza Awan | Dr. Muhammad | Ms. Saeeda Kalsoom.

Material type: book Book; Format: print Publisher: 2012Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1515,T] (1).

3. Genetic Characterization Of Pakistani Lathy Pigeons Using D-Loop, Cyt B And 16S Rrna Genes As Genetic Marker

by Muhammad Umair Latif | Ms. Sehrish Firyal | Dr. Ali Raza Awan | Dr. Muhammad.

Material type: book Book; Format: print ; Literary form: not fiction Publisher: 2013Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1399,T] (1).

4. Optimization For The Production Of Amylase By Geobacillus Sbs-4S

by Nasreen abdul jabbar | Dr. Muhammad Tayyab | Dr. Ali Raza awan | Ms. Asma waris.

Material type: book Book; Format: print ; Literary form: not fiction Publisher: 2014Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1905,T] (1).

5. Molecular Phylogeny And Diversity Analysis Of Gazella Bennettii (Chinkara) Deer Of Pakistan Using D- Loop Region Of Mitochondrial DNA

by Maleekah zaheer | Dr. Muhammad yasir zahoor | DR. Ali raza awan | Miss. Faiza.

Material type: book Book; Format: print ; Literary form: not fiction Publisher: 2014Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1985,T] (1).

6. Differential Expression And Mutation Analysis Of Heat Shock Proteins (Hsps) And Tumor Suppressor Gene (P53) In Differemt Cancer Types of Pakistani Dogs and Cats

by Rashid Saif | Dr. Muhammad Wasim | Dr. Ali Raza Awan | Dr. Muhammad.

Material type: book Book; Format: print ; Literary form: not fiction Publisher: 2014Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 2170,T] (1).

7. Phylogenetic Analysis Of Major Fresh Water Carps Of Pakistan Through DNA Barcoding

by Madeeha Awan (2012-VA-650) | Dr. Ali Raza Awan | Dr.Sehrish Firyal | Dr. Muhammad Tayyab.

Material type: book Book; Literary form: not fiction Publisher: 2014Dissertation note: Pakistan is bestowed with the land of geological and topographic diversity. The ecological variation is uniformly reflected in all water lands of the country. Pakistan has significantly huge natural inland water resources in the form of ocean, rivers, networks of canals and lakes (Mirza and Rafique 1994). The country is blessed with one of the largest freshwater resources in the world correspondingly large number of freshwater living vertebrates is available from which fishes are quite significant considering the ecological balance and its consumption as food. It is one of the food sources which solely provide all the essential nutrients, minerals and high quality protein which is not common from other food items (Muhammad Rafique 2007). Out of 33,100 fish species identified worldwide as per Fish Base organization report published in April 2015 (http://www.fishbase.org). Out of 233 (indigenous and exotic) freshwater fish species, 78 economically important indigenous fish species are available in the water bodies of the Pakistan. According to this report fishes are the largest vertebrate group, constituting about 50% of all vertebrate species. Systematically fishes are widely spread in nature, ranging from prehistoric jawless fishes to cartilaginous fishes and also from old to current day bony fishes. The taxonomic placement of these fishes shows their belonging to class Actinopterygii, sub-class Teleostei, 3 cohorts, 6 superorders, 13 orders, 30 families and 86 genera (Rafique 2007; Rafique and Khan 2012). Demand of fish is increasing day by day not only being the naturally available source of food rather the health benefits associated with its consumption. This necessitates to develop a more efficient and sustainable system to increase their growth. DNA based technologies are being competently employed in aquaculture production fields for pedigree information. Introduction 2 Moreover, tagging each fish individually is not an easy task so these DNA based methods help in avoiding intrusion of environmental factors which may result from raising fish families in separate reservoirs (Martinez 2007). Fish identification has been traditionally based on phenotypic features. However, due to high multiplicity and morphological similarity, in many cases, fish at its different developmental stages are hard to be identified by relying only on morphological characteristics (Victor et al. 2009). For phylogenetic studies of the animals the use of mt-DNA is very common and reliable compared to nuclear DNA due to its high evaluation capabilities, which results in gathering of differences even between closely related species (Moore 1995; Mindell et al. 1997).“Bar-coding gap" is the name given to the property that is inter-specific variation in this region is markedly higher than intra-specific variability (Hebert et al. 2003). Approximately each and every animal contain 13 protein-coding genes (PCGs) as an essential component of their mt-genome (mitochondrial genome), which helps in encoding of several proteins responsible for the oxidative phosphorylation machinery (Richly A et al. 2004, Song H et al. 2008). Being maternally inherited, mt-DNA is better as compared to genomic DNA such as quick evolution, less exposure to recombination, high copy number, high conservation, little duplications and negligible intergenic regions (Waugh J 2007, Xu J 2005). Clonal inheritance is the main property which makes it more worthy and suitable marker in comparison with other available molecular bio-diversity tools (Galtier et al. 2009). DNA barcoding is one of the taxonomic tools. It is being used to distinguish animal species based on the small segment of their genome such as mitochondrial DNA, designated as an identification tag or barcode of particular species (Herbert et al. 2003). Identification of species using DNA barcoding is quite debatable. Still many researchers consider it as a reliable Introduction 3 basic tool to ascertain the genetic characterization of diverse eukaryotic species, especially after establishment of the Consortium for the Barcode of Life (CBOL) in 2004 [http: //www. barcodeoflife.org/]. Ideally DNA barcoding should provide quick, reliable and cost effective species identification, even to those user who has little or negligible knowledge of taxonomy (Herbert et al. 2003, Hajibabaei M et al. 2005, Herbert et al. 2005). Identification of unknown source is possible by using distance based tree which can be created by comparing unidentified sequences against retrieved known sequences of different species (Hebert et al. 2003, 2004a, 2004b). DNA barcoding identification system has been recognized universally as standardized method to recognize species and unveil their genetic diversity (Herbert et al. 2003; Herbert et al. 2004). The ideal DNA barcoding is robust, with conserved, universal primer binding sites, reliable DNA amplification and sequencing. From whole mitochondrial genome, Cytb (Cytochrome b) is considered as one of the most promising gene due to its function and structure, even it is composed of both conserved and rapidly evolving regions which are more related to evolutionary studies (Farias et al. 2001). To identify unknown or ambiguous species it is considered more reliable as it contain sequences which provide the specific information about particular species (Parson W et al. 2000a, 2000b). It is also one of the most useful genetic marker to identify the linkage within families and genera (Meyer 1994; Teletchea 2009). Cytb gene is involved in comparative studies which results in development of new classification schemes and been used to assign a genus to a newly described species as well as improve the understanding of evolutionary relationships of genra (Castresana 2001). Introduction 4 One of the core objectives of this study is to identify and classify four freshwater indigenous fish species of Pakistan, which includes Labeo rohita (Rohu), Labeo calbasu (Calbans), Catla catla (Thalla) and Cirrhinus mrigala (Mori) using Cytb gene. Morphologically, Labeo rohita shows compressed body with convex dorsal profile while mouth bears a pair of barbells and fins are gray and orange in color. Catla catla shows compressed body with broad head. Mouth is wide with thick lower lip. Labeo Calbasu`s dorsal profile is more convex than that of abdomen and two pairs of barbells are present on fringed upper lip. Cirrhinus mrigala has elongated and streamlined body shape which is grayish and silver in color (Bhuiyan AL 1964; Rahman AK 1989). All of these species are found in freshwater bodies mostly lakes, rivers and ponds except Labeo calbasu which is a bottom dweller. These fishes are harvested by using rod and line or by using nets (Talwar PK and Jhingran AG 1991). These fishes are known as major carps and economically very important for the country due to their high consumption as food. These fishes are also used for fish farming due to their greater muscle mass thus also possess very high commercial value for fish farming business. Another objective of this study is to resolve the taxonomic anomalies related to above mentioned species. Selling of fish meat in mislabeled packaging is a serious issue now days. Most commonly Hypophthalmichthys molitrix (silver carp) and Ctenopharyngodon idella (grass carp) are sold under the label of Labeo rohita. DNA barcoding is also helpful in detecting such fraudulent mislabeling. It would be the first study in Pakistan to genetically characterize commercially important fish species. It would help scientists to know about their phylogenetic and taxonomic status and also assist fish fanciers to genuinely identify their species of interest. Identification of fish species is also important for conservation of biodiversity as it helps in preservation and Introduction 5 identification of endangered species by generating their barcodes from even minimal evidence available. This study has paved the way for molecular biologists to study taxonomic ambiguities at sub species level using SNP (Single nucleotide polymorphism) based identifying marker. Availability: Items available for loan: UVAS Library [Call number: 2207-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: book Book; 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. Genetic Characterization Of Pakistani Wild Quails Using Mitochondrial Coi Gene

by Wajiha Shakil (2012-VA-817) | Dr. Ali Raza Awan | Dr. Muhammad Yasir Zahoor | Prof. Dr. Tahir Yaqub.

Material type: book Book; Literary form: not fiction Publisher: 2015Dissertation note: A latest taxonomic tool termed as DNA barcoding is being used to genetically characterize animals. DNA barcoding is helpful in determining evolutionary relationship among species. Being a small sized genome and agile enough to show rapid mutation, mtDNA has been used as a pertinent marker of molecular biodiversity. DNA barcode works as an efficient tool in food manufacturing industry, diet investigation, forensics analysis, preventing unlawful trade and felonious poaching. The aim of this study was to develop DNA barcode for genetic characterization of Pakistani wild quail. Pakistani wild quail is important due to its demand for eggs, meat production, experimental purposes and gaming as well. Japanese quail was also included in this study because this quail is excessively produced in Pakistani farms. Japanese quail is present throughout the year and is comparatively bigger in size than wild quail. It has longer lifespan; farmers can easily breed this species in farms. It is suitable in poultry due to better meat yield. COI gene (500bp) was used as a molecular marker for identification at species level. DNA was extracted from blood samples of ten wild quails (Coturnix coturnix and fifteen japanese quails (Coturnix japonica). Reported bird universal primers were used to amplify COI region from the extracted mtDNA samples using PCR. Amplicon were then sequenced by Sanger sequencing method (Sanger et al. 1977). Forward and reverse DNA Sequences were aligned with the reference sequence using nucleotide BLAST on NCBI to observe the dissimilarity among the sequences. Consensus sequences generated were used to construct their phylogenetic tree to see their evolutionary relationship with other bird species. Japanese quail which is thought to be domesticated from Japan, its Pakistani population showed close relation with sequences Summary 90 generated in Japan for this particular species. Pakistani wild quail species showed its closest linkage with C. coturnix. In conclusion, COI barcode proved as an authentic tool for species identification and phylogenetic inference of Pakistani wild and farm grown quails. Wild quail species has been characterized using partial COI gene sequences. This study has provided a specific genetic marker which can differentiate Japanese quail from wild quail at molecular level as most of the time both species are confused with each other. It can be helpful to the farmers and bird fanciers because they can select the birds of their choice correctly. This is the first study reporting DNA barcode of this Pakistani quail species. It would help researchers to study about phylogenetic and taxonomic status and also assist quail fanciers and quail farmers to unaffectedly identify their species of interest in farming. Identification of quail species is also important for conservation of biodiversity as 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: 2311-T] (1).

10. Comparison Of Antifungal Activity Of Human Salivary Histatin Between Diabetic And Nondiabetic Individuals

by Farid-Ul-Haq (2013-VA-555) | Prof. Dr. Tahir Yaqub | Dr. Ali Raza Awan | Dr. Muhammad Tayyab.

Material type: book Book; Literary form: not fiction Publisher: 2015Dissertation note: Histatins are antimicrobial proteins found in human saliva. These proteins have also been observed to have the ability to aid in wound healing in various organisms. The genes HTN1 and HTN3 have been studied to govern these proteins. Histatin proteins have a vast array of antimicrobial properties. While a fungus, Candida albicans or C. albicans is a part of the human normal gut flora, it is a threat to people who have a compromised immune system. An overgrowth of the fungi belonging to the Candida family leads to candidiasis in humans, and oral candidiasis has been reported to a large extent namely in diabetic patients. The antifungal activity of histatin proteins laid the basis of the current research work. In this study, the antifungal activity of saliva from a total of 64 healthy and diabetic human samples against Candida albicans has been evaluated. The samples of both healthy and diabetic human samples belong from different age ranges: 15-25, 25-35, 35-45 and 45-55 years in order to change in antifungal activity with respect to age of an individual. Antifungal activity was observed through both agar well and agar disk diffusion methods, with agar disk diffusion methods showing positive results. According to the outcomes of this study at least 120μL of healthy saliva sample is required to create a zone of inhibition. Saliva from diabetic individuals showed no antifungal results. This occurrence led to the next part of this study involving amplification of HTN3 gene. The nucleotide sequences of both healthy and diabetic individuals were compared together and showed that the absence of antifungal activity in diabetic individuals might have reasons other than a genetic one, according to this study. The results observed from the present study indicate that healthy human saliva possesses antifungal activity against Candida albicans. In accordance Summary 39 to these results, the naturally occurring antimicrobial activity of histatin proteins present in human saliva can have immense use in the field of medicine. Availability: Items available for loan: UVAS Library [Call number: 2341-T] (1).

11. Mutational Screening Of The RB1 Gene In Pakistani Patients With Retinoblastoma

by Saeeda Kalsoom (2007-VA-555) | Dr. Muhammad Wasim) | Dr. Khushnooda Ramzan | Dr. Ali Raza Awan | Dr. Aftab Ahmad Anjum.

Material type: book Book; Literary form: not fiction Publisher: 2015Dissertation note: Retinoblastoma is a neonatal intraocular tumor caused by biallelic inactivation of RB1 gene. Rb patients and asymptomatic carriers undergo a series of clinical tests for diagnosis and tumor treatment. These clinical examinations prove to be expensive and time consuming. On the other hand if the proband’s RB1 gene mutation status is determined by genetic testing, it can prove as more significant and cost effective diagnostic methods. Secondly, only those asymptomatic or at risk carriers with the mutation, require clinical surveillance while those proven to be unaffected do not require additional clinical examinations. Furthermore early diagnosis of Rb by molecular testing can enable and enhance clinical management, earlier treatment, follow-up care, carrier screening, genetic counseling, prenatal diagnosis and reproductive planning in predisposed families. Irrespective of the importance of molecular testing of Rb patients, in Pakistan only a few clinical reports on Rb are available so, there was a dire need to find RB1 mutations in Pakistani Rb patients and to set a molecular based diagnosis for poor affected families. Keeping in view the importance of molecular diagnosis, in this study a reliable genetic test has been developed to detect the RB1 germline mutations in Pakistani Rb patients. During this study, 70 Rb patients including 38 unilateral and 32 bilateral cases were enrolled, from different regions of Pakistan. By using direct sequencing method, seven novel and twelve reported RBI mutations were found. The novel mutations included three frameshift mutations (c.1116_1119delCACT in exon 11, c.1436_1437delAC in exon 16 and c.2060_2061insTCATT in exon 20) and four substitutions (c.148G>T in exon 2, c.610G>T in exon 2, g.94G>C in exon 7, c.947A>T in exon 10 and g.1991G>C in promoter region) while twelve reported mutations in 146 22 patients included, 9 substitutions (c.160G>T in exon 2, c.289G>T in exon 3, c.751C>T in exon 8, c.920C>T in exon 9, c.967G>T in exon 10, c.1072C>T in exon 11, c.1654C>T in exon 17, c.2063T>C in exon 20 and c.2359C>T in exon 23), one frameshift mutation (c.772_776del in exon 8) and two splice site mutations (c.380+1G>T and c.1215+1G>A in intron 3 and 12 respectively). Mutation detection rate was found to be 77.8% in (7/9) bilateral familial, 50% in (2/4) unilateral familial, 56.5% in (13/23) bilateral sporadic and 14.7% in (5/34) unilateral sporadic patients while overall rate of mutations in bilateral and unilateral patients was detected as 62.5% (20/32) and 18.4% (7/38) respectively. Beside mutations one novel c.940-64C>T (intron 9) and nine reported intronic variants c.380+45 C>T (intron 3), c.501-77G>A (intron 4), c.1128-72T>G (intron 11), c.1695+99A>T (intron 17), c.1695-1696delAA (intron 17), c.1815- 104A>G (intron 18), c.1961-10T>C (intron 19), c.2663+33T>C (intron 25) and c.2664-10T>A (intron 25) were also found. Carrier screening facility was also provided to six asymptomatic siblings (as possible carriers) of familial proband but none of them was found to be diseased. Hopefully, in future the findings and developed protocol of this study will help to reveal the molecular basis of Rb in Pakistani Rb patients which additionally help to secure vision and life of Rb patients. Further, in Pakistan there is dire need to develop “National Rb Registry Centre”, to register all new Rb cases for finding incidence rate and prevalence of Rb in Pakistan. Beside this other related issues like financial constraints, health education, planning and awareness about Rb, occupational training for health providers, capacity building for neonatal ophthalmologic screening and cosmetic rehabilitation for surviving Rb patients are important and should consider. Availability: Items available for loan: UVAS Library [Call number: 2370-T] (1).

12. Assessment Of Evolutionary Rate In Different Serotypes Of Foot & Mouth Disease Virus

by Muhammad Farooq (2011-va-823) | Dr. Ali Raza Awan | Prof. Dr. Thair Yaqub | Dr. Muhammad Tayyab.

Material type: book Book; Literary form: not fiction Publisher: 2015Dissertation note: FMDV belongs to the family Picornaviridae with seven serotypes around the world. Nevertheless, serotypes prevalent in Asia includes A, O and Asia-1. Because of evolution in genomic sequence of FMDV, it is becoming difficult to control the problem through conventional methods. Changes in the genome can be detected using software through sequence analysis. In the software, evolutionary models are used to measure the evolutionary change for the identification of new sub lineages. In current study genomes sequence data (1998 - 2011) of bovine FMD serotypes (A, O and Asia 1) was collected through NCBI in FASTA format. This data was converted into PHYLIP format. On Dell workstation, with Microsoft Windows 8.1 operating system, BioEdit, TipDate V.1.2 was deployed. Sequence data was aligned through CLUSTAL W algorithm of Multiple Sequence Alignment using BioEdit. Using TipDate, genome sequence data was analyzed using three evolutionary models (F84, HKY and REV) and phylogenetic trees were produced showing evolutionary rate and likelihood ratio of FMDV serotypes O, A and Asia-1.. Results of the current study showed higher values of evolutionary rate in bovine FMD virus which was estimated 7.49 x 10-4 with likelihood value -1429.507680 in serotype A, 2.418 x 10-3 with likelihood -3707.168484 in serotype O and2.16 x 10-3 with likelihood value -3723.344884 in serotype Asia-1, respectively. Markove Reversible Model showed higher rates of evolution in all three serotype with best likelihood values. Phylogenetic results showed higher rate of evolution or substitution in viruses. Furthermore serotypes A, O and Asia-1are mutating with passage of time and new variants are being observed. It was also observed that this evolutionary process is continued in these three serotypes during 1998-2011. This study confirmed the evolutionary changes in FMDV serotypes prevalent in Pakistan during the period 1998 – 2011. This study showed that isolate are evolving with increasing rate. High rate of mutation in Asia-1 was observed than serotype A and serotype O. F84 and HKY85 models produced close results but these models are not identical works on equal and unequal base frequencies. Markove model estimated average base substitution with mutation and depicts good phylogenetic trees of sequence data. Availability: Items available for loan: UVAS Library [Call number: 2372-T] (1).

13. 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: book Book; 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).

14. 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: book Book; Literary form: not fiction Publisher: 2015Dissertation note: CD not available. Availability: Items available for loan: UVAS Library [Call number: 2379-T] (1).

15. 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: book Book; 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).

16. Genetic Identification And Characterization Of Pakistani Birds Of Perdicinae Subfamily (Partridge) Through Dna Barcoding Method

by Asim Iqbal Jutt (2013-VA-557) | Dr. Ali Raza Awan | Dr. Muhammad Wasim | Prof. Dr. Kamran Ashraf.

Material type: book Book; Literary form: not fiction Publisher: 2016Dissertation note: Pakistani birds of Perdicinae sub family are cage and game birds. Birds includes Altectoris chukar, Ammoperdix heyi, Ammoperdix griseogularis, Francolinus francolinus and Francolins pondicerianus. Traditional methods of identification were based on the phenotypical characterization of birds, which may lead to incorrect identification, so there was need to explore their characters at DNA level for accurate identification and to establish a DNA reference. Birds of sub-family Perdicinae have not been genetically characterized in Pakistan. A new precise method “DNA barcoding” was applied using COI gene of mDNA for authentic identification and classification of these birds. Blood and tissue samples of five species (fifteen samples) were obtained. DNA of each sample was extracted by organic method. Amplification of CO1 gene was done by using a universal set of primers BIRDF1, BIRDR1. Sequence of 450bp were analyzed using bioinformatics softwares. Each sample was aligned with its reference sequence of COI gene available on NCBI. Every nucleotide position which did not align with the reference sequence was studied to identify SNPs. A common phylogenetic tree of all partridges showed that they have common ancestor about 0.7 million year ago, F.francolinus, F.pondicerianus and A.heyi shared a common clade whereas A.chukar made a separate clade from the ancestor. A.heyi and F.pondicerianus showed closed resemblance. It has been proved that DNA barcoding is an efficient and accurate molecular tool for species identification and phylogenetic implication. This study established a DNA Data Bank that helped scientists to investigate the biodiversity, taxonomic classification, species identification and also established foundations for molecular biologists to study taxonomic uncertainties at sub species level using SNP based identifying marker. Availability: Items available for loan: UVAS Library [Call number: 2714-T] (1).

17. Snp Genotyping Of Cacna1a Gene Implicated In Childhood Absence Epilepsy (Cae)

by Wajeeha Tariq (2010-VA-487) | Dr. Muhammad Wasim | Dr. Ali Raza Awan | Dr. Muhammad tayyab.

Material type: book Book; Literary form: not fiction Publisher: 2016Dissertation note: Childhood absence epilepsy (CAE) is more pediatric epileptic syndrome. It is about 5 to 15% of all childhood epilepsies. CAE is polygenic and multifactorial syndrome. Many different genes other than CACNA1A gene are involved to cause the CAE collectively. Mutation in P/Q type alpha 1 A subunit channel (Cav2.1) gene CACNA1A, leading to the reduction of Cav2.1 activity in both neurons and in expression system. Reduction in Cav2.1 channel activity altered the neurotransmitter release at neocortical synapses. Molecular genetics techniques have identified various mutation in the genes of ion channels such (CACNA1A, CACNA1G, CACNA1H, CACNB4), sodium channel genes (SCN1A, SCN1B and SCN2A) and GABA receptor genes (GABRD and GABRG2). CACNA1A ion channels are the standard mediator of neurotransmission in Central nervous system (CNS) and mutations in this gene play significant role in the generation of absence seizures. Pore forming alpha 1 a (Cav2.1) channels encoded by CACNA1A gene and are usually located in presynaptic neuron. Present study was aimed to examine coding regions of CACNA1A gene for analyzing the mutations involve in epilepsy. Blood samples (n = 40) of true CAE representatives were collected from Children hospital Lahore. DNA was isolated from all blood samples through standard organic method. Amplification of CACNA1A gene exon 36 regions was done with specially designed primers. Later on, results were analyzed through sequencing of target region. Sequenced samples were analyzed through BioEdit software and alignment was done through Clustal Omega software. It has been identified that absence epileptic patients of Pakistan showed Mutation in exon 36 of CACNA1A gene at position 281258bp and 281285bp which alter the protein sequence. Due to frame shift mutation a stop codon was detected at position 1813 in protein sequence. So a truncated and loss of function Cav2.1 channel might be formed. In epileptic patients, mutation is responsible for the absence seizures. In the conclusion, we can say that additional study with large number sample is required to amend the effects of these mutations and their associated factors are precisely and perfectly identified. Further, there is need to investigate the other gene variation causing epilepsy in the local population of Punjab Pakistan. This study will ultimately help to develop genetic counseling strategies, gene therapies and parental diagnostic procedures for the Pakistani population. Availability: Items available for loan: UVAS Library [Call number: 2746-T] (1).

18. 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: book Book; 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).

19. Mutational Analysis Of Cacna1ggene Implicated In Childhood Absence Epilepsy And Its Comparative Genomics In Mice

by Fiza Idrees (2015-VA-803) | Dr. Muhammad Wasim | Dr. Ali Raza Awan | Prof. Dr. Aftab Ahmed Anjum.

Material type: book Book; Literary form: not fiction Publisher: 2017Dissertation note: Childhood absence epilepsy (CAE) is the subtype of Idiopathic generalized epilepsy (IGE). It accounts for 2-8% of patients with epilepsy. The frequency of CAE is more in girls than boys. The percentage of CAE in youngsters is 10-12%. In addition to CACNA1G, many other genes can be the possible cause of CAE. The pattern of inheritance of CAE is polygenic and complex. SNP might be a gain of function mutation in T- channel genes that results in increase T-type calcium channel activity. Ion channel genes and genes for GABA receptors are affected in epilepsy. By using various techniques of molecular genetics mutations have detected in genes of calcium channels (CACNA1H,CACNA1I, CACNA1A, CACNA1G and CACNB4), in genes of sodium channels like (SCN1B, SCN2A, SCN1A ) and genes for GABA receptor (GABRG2 and GABRD ). Gain of function mutation in CACNA1G gene and increased activity of α1G channels are the possible reason for abnormal SWD in absence epilepsy. Aim of this study was to assess acknowledged and/or the novel mutations in CACNA1G gene obtained from local childhood epileptic patients. Blood samples (n=20) were obtained from CAE patients. These samples were collected from children hospital Lahore. Organic method was used to extract DNA from these collected samples. Specific primers were designed for exon 13 and 17 and these exonic regions were amplified using PCR. After PCR, sequencing of PCR products was performed and then sequencing results were analyzed using chromas lite software. It has been observed that CACNA1G gene has two mutations in exon 17. It was noticed that protein sequence was altered and the positions of mutations were 38594bp and at 38635bp 38594bp and at 38635 bp. So SNP was detected and there was a gain of function mutation α1G channel activity. In conclusion, these mutations are responsible for absence seizures in CAE patients. So, it can be concluded that to find out how individuals get affected by these mutations and what factors are involved in causing such mutation, a large scale study should be conducted.In addition, other genes involved in causing epilepsy should also be investigated in local Pakistani Punjab population. As a result of such studies, various diagnostic procedures, strategies for counseling and gene therapies can develop. Availability: Items available for loan: UVAS Library [Call number: 2925-T] (1).



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