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1. 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 ( 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.]. 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).

2. Expression And Mutational Analysis Of Breast Cancer Susceptibility Gene 1 (Brca1) And Cyclooxygenase-2 (Cox-2) Gene In Feline And Canine Tumours

by Haleema Sadia (2007-VA-567) | Dr. Muhammad Wasim | Prof. Dr. TahirYaqub | Dr. Abu Saeed Hashmi.

Material type: book Book; Literary form: not fiction Publisher: 2014Dissertation note: Cancer is the first cause of death in cats and dogs while in human it is the second most cause of death (Jemal et al. 2008). According to an estimation, cancer related deaths in the world are 13% and 70% of these deaths are in poor countries (World Health Organization 2012). Such natural cases of cancers in cats and dogs especially, in dogs offer an opportunity to use the dogs for comparative cancer studies and as an animal model for anticancer drug development (Pawaiya 2008). Inu a series of more than 2000 autopsies, it was found that almost forty five percent dogs that lived for ten or more years expired because of cancer (Bronson 1982). Dogs are affected by skin cancer 35 times more often than humans. They are also affected 4 times more often by mammary gland cancer, 8 times more often by bone cancer, and twice more often by leukemia, than humans (Cullen et al. 2002). The regulation of cell proliferation, genome stability and programmed cell death are important for systemic homeostasis. 1.1Historical perspective on cancer causation Hippocratic and Galenic medicine attributed the spread of black bile (one of the four humours) in the tissue as the cause of the cancer (Diamandopolus 1996) is an idea survived intact through the Middle Ages and Renaissance. With the discovery of the lymphatic system by Gasparro Aselli in 1662, the black bile theory was superseded by the idea that cancer was an inflammatory reaction to extravasated lymph; a theory modified 150 years later by John Hunter who introduced the notion that contaminated coagulating lymph was the origin of the cancer (Kenneth 2003). A German pathologist Johannes Muller first time demonstrate that cancer is made up of cells (1838) but he also gave an idea that cancer cells were originated from a bud called Blastema instead of normal cells (Kardinal and Yarbro 1979). Following Schleiden and Schwann's cell theory of tissues,it was Rudolf Virchow (Muller’s student) who in 1855 demonstrated that every cell was derived from another cell (omnis cellula e cellula), including cancer cells (Mazzarello 1999; Porter 1999). In 1867 Wilhelm Waldeyer supported the theory of the normal cell for the origin of cancer and he believed that metastasis resulted from transportation of cancer cells by blood or lymph (Porter 1999). Around the turn of the twentieth century the beginning of tumour transplantation experiments led to the new view of the cancer cell as an autonomous cell. The first successful tumour transplants were described in 1876 by the Russian veterinarian Mstislav Aleksandrovich Novinski (Novinski 1876). He reported in his thesis entitled “On the Question of the Inoculation of Malignant Neoplasms” the first successful serial passage of tumours through transplantation in dogs. Novinski's transplantation experiments were based on the inoculation of canine transmissible venereal tumour (CTVT) in puppies. Novinski stated that successful tumour transplantation depends on the inoculation of a living element of the tumour and that the transplantation of the element of a cancerous tumour to healthy tissue acts as an infecting agent. In 1888 Wehr repeated Novinski's transplantation experiments in dogs with similar results (Shimkin 1955). It is interesting to note that the dogs used for transplantation of CTVT did not come from a single breed and were therefore not highly inbred. The allo-transplantation of tumours seemed less surprising in the late 19th Century than it does today with our modern knowledge of histo-incompatibility. The successful results obtained with CTVT served as model for tumour transmission in other animals. Hanau in 1898 inoculated two rats with vulvar epidermoid carcinoma and observed growth of the tumour in the recipients (Shimkin 1955). In 1901 Leo Loeb supported the transplant ability of tumours in rats (Witkowski 1983; Brent 1997). In 1903 a Danish veterinarian Carl O. Jensen determined the successful growth of transplanted tumours in mice by heredity (Brent 1997). The discovery that the tumour could be successfully transplanted into (Witkowski 1983; Brent 1997) other mice, led the scientists to use rodent system to supply tumours for experiments. The observation that a single tumour could be expanded through many generations exceeding the life span of the laboratory mouse led Leo Loeb to the "cancer immortality" concept (Witkowski 1983). The earliest observations reported by John Hill in 1759 and by Percival Pott in 1775 on the association of a specific tumour to a specific profession or work, led to the idea that some chemicals can cause cancer (Greaves 2000). In 1918 Yamagiwa and Ichikawa induced cancer by applying coal tar to rabbit skin(Greaves 2000; Luch 2005). After the discovery of the X rays by Wilhelm Conrad Roentgen in 1895, Frieben published data in 1902 indicating that cancer rates were increased among persons working with X-rays (Cassileth 1983; Greaves 2000) 1.2 Tumour Progression The first detailed characterization of the dynamic nature of cancer was described by Leslie Foulds (Foulds 1949). Foulds showed that tumours progress (evolve) through different stages, characterized by the acquisition of different phenotypic traits such as increased growth rate, hormone dependence, invasiveness, formation of metastasis (Foulds 1949; Fould 1954; Foulds 1957). With the progress of molecular biology the phenotypic view had been replaced with the somatic mutation theory, where cancer evolved through the accumulation of different mutations in several genes (Greaves 2000). The accumulation of mutations in somatic cells implicated the presence of different cells bearing different mutations and also the presence of natural selection, which selected the cells with advantageous mutations. One of the questions arising from the somatic mutation theory was whether a tumour had a single or a multiple origin. This observation was supported by a karyotype analysis in chronic myelogenous leukaemia (CML) by Peter Nowell and David Hungerford in 1960 (Nowell 2002). They described the presence of an unusually short chromosome 22 in all CML tumour cells analyzed, and the absence in the normal cells from the same patients. This observation suggested that this mutation was a somatic mutation that occurred in one cell in the bone marrow, which gave it a selective advantage to expand as a clone. Nowell postulated that a tumour develops by a Darwinian evolutionary process, where cells with mutations conferring a growth advantage are selected and expanded (Nowell 1976; Greaves 2002). In 1954 Peter Armitage and Richard Doll analyzed human cancer incidence over the age, and showed that chances of cancer increased in older people (Armitage and Doll 1954). The concept that cancer might be contagious also recurs throughout the past 300 years.In the 17th and 18th centuries, physicians Daniel Sennert and Zacutus Lusitanus supported the hypothesis that cancer was contagious. In fact in 1779 a hospital in Paris was directed to move the cancer patients from the city (Cassileth 1983; Kenneth 2003). 1.2.1 Exogenous and endogenous factors In 1844 the Italian physician Domenico Antonio Rigoni-Stern noted that cancer of the cervix was frequent among married ladies, rare among unmarried ladies and absent in Italians nuns. In contrast, breast cancer was more frequent among nuns (Greaves 2000). These observations led to the hypothesis that cervical cancer was sexually transmitted, and we now know that the cause is a papilloma virus (Hausen 2002).In 1908 Wilhelm E and Olaf B, transferred the leukemia in chicken by tissue filterates (Wyke 2003). In 1911, Peyton Rous demonstrated that viruses were the cause of solid tumours (Sarcoma) in chickens but it took many decades before his data were accepted (Dulbecco 1976). The notion that viruses can cause cancer was a discovery that brought back the fear that cancer was a contagious disease. There are many exogenous and endogenous risk factors that affect the tumor suppressor genes and oncogenes (Todorova 2006). Tumour viruses (Bishop 1980), chemical carcinogens (Loeb et al. 2000), natural chemicals, (Ames et al. 1990), herbicides (Glickman et al. 2004), physical carcinogens like radiation (Upton 1978) are exogenous factors while inherited genetic defects, immune system (Rosenthal 1998) and hormonal factors (Rodney 2001) are among endogenous risk factors. Although tumour cells are generally described as independent evolving units, recent results suggest that tumour cells are able to stimulate stromal cells to produce growth factors that increase tumour proliferation (heterotypic stimulation) (Kinzler and Vogestein 1998; Skibe and Fuseing 1998; Iyengar et al. 2003). It has been demonstrated that cells involved in the immune response to tumours may produce factors such as inflammatory chemokines that may also promote the tumour proliferation (Pollard 2004; Wyckoff et al. 2004) 1.2.2 Two hit hypothesis Retinoblastoma is a tumour that becomes manifested early in life. Retinoblastoma can be inherited or sporadic. According to the two hit hypothesis in the inherited form a single mutation in the Retinoblastoma (Rb) gene is present in the germ line which gives the genetic predisposition to develop cancer, but a second mutation in the normal Rb allele which occurs in the retinoblast must be acquired to develop cancer (Knudson 2001). In the sporadic form the two mutations in the Rb alleles occur in the somatic cells. Although the epidemiological and molecular observations have consolidated the multistage theory of cancer, the number of mutations and in which sequential order they have to be acquired to develop cancer is still an open question (Hanahan and Weinberg 2001; Hahn and Weinberg 2002b). 1.2.3 Oncogenes Early experiments involving transforming retroviruses and the transfer of genes from tumour cells into established rodent cells allowed the identification of several cancer causing genes called oncogenes. The result of these experiments suggested that cancer could be induced by the mutation of one proto-oncogene. However, the rodent cells used as recipient in the gene transfer experiments were not normal, but were immortalized, thus acquiring the ability to proliferate indefinitely. When the normal rodent cells were used, the transfer of a single oncogene failed to induce transformation, while the transfer of two oncogenes resulted in transformation. Human cells require more mutations than rodent cells and that there are differences also between cell types within the same species (Rangarajan et al. 2004). 1.3 Cancer Hallmarks Despite the enormous variety of tumours affecting different types of tissues in animals and humans, research over the past 50 years has revealed that all malignant cancers share the same essential alterations (Hanahan D and Weinberg RA 2000). These hallmarks include:  Immortalization  Evasion from programmed cell death (apoptosis)  Independence from growth stimulation  Resistance to growth inhibition  Angiogenesis  Invasion and metastasis  Genetic instability. These hallmarks are briefly described below. 1.3.1 Immortalization Telomeres contain DNA sequence repeats and protein. The repeat sequence consists of hexameric motifs such as GGGTTA in humans, extended for 10 –20 kilobases. The 3’ end has a 100-400 nucleotide over-hang (Mathon and LIoyd 2001). Telomeric DNA is generated by an enzyme called Telomerase Reverse Transcriptase (TERT) which has two subunits, RNA and catalytic protein subunit. This RNA binds the telomeres DNA ends thus acting as template for telomere elongation. The chromosome ends are protected by several proteins: TRF-1, TRF-2, and POT–1 (Mathon and LIoyd 2001; Hahn and Weinberg 2002a). Several experiments have shown that senescence is activated when the telomeres are shortened down to 5 kb and that senescence is triggered by the shortest telomere present in the cell (Hemann et al. 2001). Many reports have suggested that the replicative senescence is not activated by the erosion of the double strand repetitive sequence, but by the degradation of the 3’end single strand overhang, resulting in loss of protective capping (Stewart et al. 2003). Telomere length is maintained by the activation of telomerase or by an alternative mechanism called alternative lengthening of telomeres (ALT), where the telomeres are regenerated through recombination-based inter chromosomal exchange of sequence information (Bryan et al.1997; Dunham et al. 2000). In the normal cell telomerase is transiently expressed, since it can be detected only in S phase, but in neoplastic cells its expression is increased and is detectable throughout the cell cycle (Mathon and Lloyd 2001). In tumour cells the senescence and crisis barriers are avoided by the activation of telomerase regenerating the telomeres and by the inactivation of tumour suppressor and pro-apoptotic genes (Hanahan and Weinberg 2000; Hahn and Weinberg 2000b). 1.3.2 Apoptosis. The sensors detect the intra- and extra-cellular signals. The intracellular signals include DNA damage, hypoxia and oncogene overexpression (Evan and Littlewood 1998). The extracellular signals monitor the cell-cell and cell-matrix homeostasis (Aoshiba et al. 1997; Prince et al. 2002; Alberts et al. 2002a). The signals detected by the sensor are mainly conveyed to the mitochondria, where a series of cytoplasmatic proteins of the Bcl2 family control the release of cytochrome C from the mitochondria (Alberts et al. 2002a). The release of cytochrome C activates an array of intracellular proteases called caspases causing protein and DNA degradation (Hanahan and Weinberg 2000). The caspases can be directly activated by extracellular proteins such as FAS ligand, which binds to the death receptor FAS (Houston and O’ Connell 2004). Once the caspase cascade is triggered it cannot be inactivated (Alberts et al. 2002a). It has been reported that the tumour suppressor p53 can trigger the caspase cascade by the overexpression of the Bax protein, a member of the Bcl2 family, which in turn increases cytochrome C release thus inducing apoptosis (Hanahan and Weinberg 2000). In CTVT it is likely that expression of c-myc is up-regulated, due to insertion of a LINE-1 element as discussed later. Ectopic c-mycexpression can promote tumour growth and survival, as seen, for instance, in immunoglobulin gene c-myc chromosome rearrangements in Burkitt's lymphoma (Hemann et al. 2005). 1.3.3. Independence from growth stimulation Growth factors Thus the proliferation of a cell is dictated by the needs of the cells around it (Hanahan and Weinberg 2000). In contrast, a tumour cell escapes from the external dependence to become an autonomous evolving unit, by producing its own growth signals. Growth factor receptors Another mechanism selected by tumour cells is the overexpressions of growth factor receptors, which induce the tumour cells to become sensitive to concentrations of growth factor that normally, do not trigger proliferation (Hanahan and Weinberg 2000). Proliferation can also be induced by a mechanism independent of the growth factor, for example the alteration of the cytoplasmic tail of growth factor receptor causes self-activation of the receptor, which therefore becomes independent from the external microenvironment (Alberts et al 2002b). 1.3.4 Resistance of growth inhibition Like growth signals, the anti-proliferative signals derive from soluble factors or surface proteins that are produced by neighbouring cells, or are induced by components of the extracellular matrix (Hanahan and Weinberg 2000; Alberts et al. 2002d). These external inhibitory signals activate different intracellular pathways that regulate the cell cycle (Alberts et al. 2002c). The Rb protein and its related proteins, p107 and p130 play a key role in controlling this transition (Weinberg 1995). The association of Rb with the transcription factor E2F inhibits the transcription of genes involved in the G1-S progression (Alberts et al. 2002c). The hyper-phosphorylation of the Rb protein induces the dissociation with E2F, therefore allowing progression to S phase (Alberts et al. 2002c). Normally complexes of cyclin and cyclin dependent kinase (CDK) induce the phosphorylation of the Rb protein (Alberts et al. 2002c). Many tumours can avoid the antigrowth signals by altering Rb activity or the proteins involved in Rb phosphorylation (Mittnacht 2005). 1.3.5 Angiogenesis Although the majority of the new vessels in adult tissues are derived by sprouting from existing vessels, many evidences indicate that progenitor endothelial cells are derived from the bone morrow contributing to the vessel growth (Zhang et al. 2000; Contreras et al. 2003; Nishimura and Asahara 2005; Religa et al. 2005). Although endothelial cells are highly proliferative in response to several angiogenic factors, they have long half-lives up to several years (Carmeliet 2003). In order to adapt the vascular system to the tissue's requirements, several mechanisms regulate the process of angiogenesis (Carmeliet 2003). A key molecule involved in the angiogenesis process is the vascular endothelial growth factor (VEGF) (Carmeliet 2003). In addition it has been demonstrated that tumours can activate or inactivate pro- and anti-angiogenic factors respectively present in the extracellular matrix by producing several proteases (Gately et al. 1997; Harlozinska 2005). 1.3.6 Metastasis In cancer during tumour progression, some tumour cells acquire the ability to migrate and form new colonies at secondary sites and these cells then make new tumour cells (Hanahan and Weinberg 2000). It has been estimated that 90 % of mortality associated with cancer is due to metastasis (Sporn 1996). Results show that few cells in the primary tumour acquire the ability to grow in the secondary sites and that the tendency to metastasise is acquired in the early steps of tumour progression (Van’t Veer and Weigelt 2003). Progressive alteration of normal tissue homeostasis by tumour and stromal cells, allow tumour cells to move throughout degraded matrix, and to invade surrounding tissues (Hanahan and Weinberg 2000). Tumour cells are also aided to migrate by soluble factors (chemotaxis) and bound adhesion molecules (haptotaxis) (Nguyen 2004). In order to invade new organs, circulating tumour cells need to stop and exit the systemic circulation. In an unspecific manner, the extravasation may be due to the fact that large arteries progressively narrow in to arterioles and then capillaries and tumour cells can be trapped in this small vessel, thus allowing the migration in the new organ (Nguyen 2004). Although the exact mechanism behind the tumour homing is not completely understood, recent results suggest that the selective homing of cancer cells may be due to three mechanisms: 1) presence in the target tissue of specific growth factors or appropriate extra-cellular matrix that favour the selective tumour growth, 2) presence in the target organ vessel endothelium of specific adhesive proteins that interact with the tumour cells, favouring the tumour invasion, 3) production of a chemotaxis soluble factor by the target tissue that attract the tumour cells ( Fidler 2003). 1.3.7 Genetic instability Over the past 25 years numerous genetic alterations have been described in human and animal tumours. These genetic alterations can affect the DNA sequence and the chromosomes (Lengauer et al. 1998). The mutations of DNA include: substitution, deletion, translocation and insertion and they can affect one or more nucleotides. The necessity to transmit genetic information faithfully between generations demands genetic stability (Eisen and Hanawalt 1999) In normal conditions the genome is affected by spontaneous mutations caused by physiological DNA instability and by imprecision of the DNA polymerase proofreading activity during the DNA replication (Alberts et al. 2002e). In eukaryotic cells, several enzymes have been described with DNA polymerization activity, and five are the most important DNA polymerases involved in DNA replication and repair, alpha, beta, gamma delta and epsilon. To date the only polymerase involved in mitochondrial DNA replication is polymerase gamma. In vitro studies on the fidelity of DNA duplication has shown that the nucleotide mis incorporation rate varies among polymerases, with one in 5000 bases for beta and one in 10 000 000 for delta and epsilon polymerases (Umar and Kunkel 1996; Loeb and Loab 2000). To avoid non-complementary nucleotide incorporation, polymerase delta, gamma and epsilon contain a proofreading activity (Kunkel and Alexander 1986). Normally DNA replication is carried out by delta polymerase, but recent reports show that in some tumours this priority is shifted in favour of less accurate polymerases, thus increasing the mutation rate (Loeb and Loeb 2000). Environmental agents such as ultraviolet light, ionizing radiations and toxic substances in the dietary uptake can induce mutations (Loeb and Loeb 2000). 1.3.7a Single Base Excision Repair When a mutation effects on a single nucleotide then base excision repair take place. BER employs enzymes called DNA glycosylases, which are specific in removing a specific mutated base (Krokan et al 2000). 1.3.7b Nucleotide excision repair The nucleotide excision repair (NER) system is able to repair DNA damage induced by UV. In contrast to BER, the NER system recognizes altered nucleotides by scanning the DNA for a conformational alteration (bulky lesion) (Wood 1996). 1.3.7c Mismatch repair The mismatch repair (MMR) pathway includes a series of proteins that are involved in correcting errors that escape the DNA polymerase proofreading activity during DNA replication. They are also involved in suppressing recombination between non-identical sequences both in mitosis and meiosis (Kolodner and Marsischky 1999). Unlike BER and NER, MMR does not act on damaged or mutated sequences, but it targets only the newly synthesized DNA strand. Inactivation of the MMR system produces microsatellite instability (MSI) (Atkin 2001). 1.3.7d. Homologous recombination Homologous recombination repairs double strand breaks by using an intact and homologous DNA molecule as a template. In eukaryotes several proteins are involved in the homologous recombination process (Kanaar et al. 1998; Haber 2000). 1.3.7e. Non-Homologous End Joining Non-Homologous End Joining (NHEJ) is the more important repairing mechanism when there is break in DNA double strand and it is very important mechanism in mammals (Khanna and Jackson 2001). During the NHEJ process small deletions are generated. Given that majority of the mammalians genome is composed of non-coding regions, the probability that in normal situations the NHEJ process induces mutation in genes is low (Alberts et al 2002e). However, if there are multiple break points NHEJ increases the occurrence of illegitimate recombination (Rothkamm et al 2001). 1.3.7f Chromosome Instability (CIN) The cell reproduces by a series of events that allow DNA replication and cell division in a process known as the cell cycle. In order to check the correct order of events that take place in the cell cycle, a complex cell-cycle control system has evolved (Alberts et al 2002c). This system checks normal cell cycle progression by a series of stage-specific sensors known as checkpoints that are able to induce the arrest of the uncompleted stage until it is completed. The two fundamental processes in the cell cycle are the duplication and the division of the chromosomes, which take place during the Synthesis (S) and Mitosis (M) phase respectively. To prevent the possibility that two daughter cells have non-identical genomes, there are two checkpoints known as DNA replication and DNA damage checkpoints before mitosis, and one known as spindle-attachment checkpoint during mitosis (Alberts et al 2002c). Chromosome instability (CIN) is also associated with structural alteration of chromosomes, which include reciprocal and non-reciprocal translocations, amplifications, deletions and insertions (Cairns 2005). Structural chromosome instability, resulting from DNA breaks and rearrangements, is due to alteration of cell cycle checkpoints, DNA damage response and telomere integrity (Gollin 2005). Structural alterations may results in altered gene expression or produce fusion or chimeric proteins with dysregulated or new properties (Greaves and Wiemels 2003). Studies have shown that a large proportion of human tumours with chromosome instability have a high rate of loss of heterozygosity (Rajagopalan and Lengauer 2004). Therefore it has been argued that chromosome instability could accelerate the rate of inactivation or activation of tumour suppressor genes or oncogenes respectively (Rajagopalan and engauer 2004). CIN-associated genes can be classified on the basis of the mutations (Michor et al. 2005). Class I genes of CIN, e.g Mitotic Arrest Deficient gene (MAD-2 ) boost up CIN in case one allele is mutated or deleted. Class II genes of CIN e.g. Human Budding Uninhibited by Benzimidazoles (hBUB-1) gene boost up CIN if mutation is in one allele in a dominant negative fashion. Both Class I and Class II genes are required at the spindle assembly checkpoint (Amon 1999; Hoyt 2001). Class III genes of CIN e.g. Breast cancer gene BRCA1 and another Breast cancer gene BRCA2 boost up CIN if both alleles are mutated. BRCA genes have very important role at checkpoint and it is involved in DNA repairing and recombination (Yarden et al. 2002). 1.4 Evolutionary Dynamics of Tumour Development According to clonal evolution theory, cancer is the result of somatic mutations selected during tumour evolution (Nowell 1976). It has been argued that tumour cells cannot acquire the mutations needed for tumour progression at a physiological mutation rate, but that the tumour cell must acquire an increased mutation rate (Cairns 1998; Loeb and Loeb 2000). In order to induce cancer the mutations must affect a variety of genes that restrain somatic conflict (Frank and Nowak 2004). These genes are known as cancer related genes and can be subdivided in three categories: Gatekeeper, Caretaker, and Landscaper (Michor et al 2004). Gatekeeper mutations increase the cellular proliferation rate by the alteration of oncogenes, tumour suppressor genes and apoptotic genes (Michor et al 2004). Caretaker mutations increase genome instability by inactivating genes involved in maintaining genome integrity (Lengauer et al 1998). Landscaper mutations increase tumour proliferation by affecting genes involved in regulating the external cellular microenvironment (Bissel and Radisky 2001). While mutations affecting oncogenes behave in a dominant way, because only one mutated allele can induce a tumour phenotype, mutations affecting tumour suppressor genes can be neutral if the normal allele compensates the mutant allele, disadvantageous if the mutant allele triggers apoptosis, and advantageous if the mutated allele is inactivated and the second allele is insufficient to balance the wild type allele (Michor et al. 2004). In small compartments the inactivation of the two alleles of a tumour suppressor gene, is unlikely, unless the mutation rate is increased by genetic instability (Nowak et al. 2005). Loss of heterozygosity increases with chromosome instability (Michor et al. 2004). 1.5 Tumours of Feline and Canine included in this study 1.5.1Mammary Tumours Mammary gland tumours are most frequent in dogs (Moulton 1990) while in cats it is third in prevalence, after haemopoietic and skin tumours (Misdorp et al. 1999). The average age of peak prevalence of tumours in cats is approximately 9.3 years (Roccabianca et al. 2006). Mammary tumours can also affect male cats and dogs, with the average age for them being 12.8 years (Rutterman et al. 2000). Siamese has twice the risk in comparison to other breeds of cat (Weijer et al. 1972). Same predisposition was observed with our data, that all five cases collected in this study were belonging to Siamese breed. Mammary tumours are more prevalent in Pakistan and all the cats and dogs were between 5-11 years old. This suggests that there are more chances of mammary tumours in older cats and dogs. Mammary tumours included in this study were 23% all 22 tumours studied. 1.5.2 Canine Transmissible Venereal Tumours (CTVT) Canine transmissible venereal tumours first reported by Blaine in 1810 (Blaine, 1810) is a transmissible cancer in dogs. Studies found that CTVT was transmitted by transplantation of living cells (Novinski 1876), confirming it as a transmissible cancer. CTVT is of clonal origin, originating from a founder dog 11,000 years ago (Katzir et al. 1985; Murgia et al. 2006; Rebbeck et al. 2009; Murchison et al. 2014). It is one of only two transmissible cancers known (Murchison 2008) and is spread by allogeneic transfer of cells between dogs, usually during coitus. It manifests as a tumour, associated with the external genitalia of both male and female dogs, although tumours can also arise in the mouth, nose or skin. It is purported to be of histiocytic origin (Mozos et al. 1996; Mukaratirwa and Gruys 2003), and usually remains rather localised, except for rare cases of metastatic spread. Recorded cases of metastasis include involvement of the lymph nodes (Higgins 1966), skin (Dass 1986) and eye (Barron et al. 1963), among others. Experimental transplants of CTVT tumours into subcutaneous sites in experimental dogs are characterized by progressive and regressive phases. This is seen as a rapid volume increase, followed by tumour shrinkage, and eventually complete regression accompanied by serum-transferable immunity to reinfection (DeMonbreun 1934). In this project we collected 6 samples for BRCA1 and COX-2 studies in different tumours while 16 more samples for Department of Veterinary Medicine, University of Cambridge, UK. Although the prevalent rate of CTVT is in second number, many attentions were paid to collect CTVTs. For BRCA1 and COX-2 studies the 28% were CTVT out of 22 different tumours. 1.5.3 Perianal adenomas/Adenocarcinomas There are many glands present around the anus of dogs. These are sebaceous and non-secretary glands, while anal sac glands are positioned at 4 and 8 o clock to the anus and secrete their secretions into the lumen of theanal track (Yang Hai-Jie et al. 2008). Perianal adenomas are more frequent than adenocarcinomas (malignant form). In this study 3 tumours were collected which were 14% of total canine tumours collected. These tumours are mostly common in medium to older age dogs. 1.5.4 Granuloma Granuloma is also called as lick granuloma in dogs it is a type of skin cancer It typically results from the dog’s urge to lick the lower portion of one of her or his legs. This study reported 9% of total tumours included in this study. 1.5.5 Oral Tumours (Squamous cell Carcinoma) Oral tumours are 4th common cancers in canines. Male dogs have 2.4 times greater risk of developing oral tumours than female dogs (Dorn et al. 1968). This study reported 9% oral tumours in a period of 2 years. 1.5.6 Lymphoma Lymphoma is the second most prevalent intra –ocular tumours of dogs. Basic cause of lymphoma in dogs is unknown but genetic (chromosomal segregation), environmental and infectious factors such as retroviruses play vital role in developments of this cancer (Fighera et al. 2002). This study reported 9% Lymphomas of total collected tumours. 1.6 Rationale behind selection of genes 1.6.1 BRCA1 gene BRCA1 gene is tumour suppressor gene, it is involved in repairing the DNA double strands breaks and in case of failure it leads the cells towards apoptosis (Starita. 2003). BRCA1 forms BRCA1 Genome Surveillance Complex (BASC) when it combines with different types of tumour suppressor genes, DNA damage sensors and signal transducers (Wang et al. 2000). It is involved in Ubiqutination, transcription regulation (Friedenson 2007; Friedenson 2008). In humans BRCA1 was first identified at chromosome 17 (Hall et al. 1990) and it was isolated in 1994 (Miki et al. 1994). It is present at 17q21 with a length of 100 Kb. In canine it is located on chromosome 9. BRCA1 has 22 exons in canines and felines; it encodes a protein of 1882 amino acids in canine and 1871 amino acids in feline. Many scientists from different research showed that women who have famililal mutations in the BRCA1 or BRCA2 (BRCA1/2) genes have increased risk of breast cancer (Struewing et al. 1997). Fig 1: BRCA1 mechanism in DNA repairing. 1.6.2 Cyclooxygenase-2 Enzyme (Prostaglandins, COX-2). Cyclooxygenase-2 enzyme (Cox-2) is also called as Prostaglandins Endoperoxide synthase (PTGS). It is involved in the synthesis of prostaglandins which act as biological mediators in many body functions. It was first isolated from prostate gland that’s why it is called as Prostanglandin. Cyclooxygenase enzymes have two types, cyclooxygenas-1 and cyclooxygenase-2. Cyclooxygenase-1 is constitutively produced in the cell while cyclooxygenas-2 is inducible and it is constitutively produced only in kidneys, seminal vesicles and central nervous system. Its high expression has been recorded in many different types of tumours, it has been involved in anti-apoptosis, cell proliferation, tumour angiogenesis, cell invasion and immune suppression activities. In canine COX-2 is present on chromosome 7 having 604 amino acids and 10 axons. This correlation of cyclooxygenase-2 in cancer development suggests using new therapeutics against it. Studies have shown cycoloxygenase-2 high expression in number of different tumours (León-A 2008), such as intestinal, pancreatic, ovarian, prostatic, nasal cavity, oral cavity and mammary tumours of dogs (McEntee et al. 2002; Mohammed et al. 2004; Borzacchiello et al. 2007; Eplattenier et al. 2007; Mullins et al. 2004; Pireset al. 2010; Dore et al. 2003). Fig 2:COX-2 mechanism of action 1.6.3 DLADQA1 (MHCII gene) (Additional work performed at Department of Veterinary Medicine, University of Cambridge, UK). The Major Histocompatibility Complex (MHC) is a cell-surface protein mediating immune recognition through its interactions with T cells (Fig 3). There are three classes of MHC molecules in mammals - the classical MHC-I and II, and non-classical MHC-III Table 1). MHC-I interacts with CD8+ cytotoxic T cells, whilst MHC-II binds to CD4+ helper T cells. MHC molecules mediate antigen presentation to T cells. MHC-I typically presents self- peptides, whilst MHC-II presents foreign peptides. MHC molecules are extremely variable and polymorphic across the population, with a huge number of alleles at each MHC locus. This allows MHC molecules themselves to behave as antigens in transplant rejection, with the graft MHC peptide recognized as non-self by the recipient, and thus rejected. It would be expected that CTVT, an allergenic graft, should be rejected for two reasons: host MHC will present tumour antigens as foreign non-self to the host immune system, and tumour MHC will present a mismatch to the host immune system as a foreign antigen itself (Fig 3). This project focuses on the DLADQA1 locus (Wagner et al. 2002), a classical MHC-II gene on dog chromosome 12. There is high level of MHC allelic variability in any population (Niskanen et al. 2013). Fig 3:MHC is involved in graft rejection. This rejection (represented by the red arrow) occurs according to two principles. Firstly, host T cells may recognize the host MHC presenting a foreign peptide that should activate an immune response. Secondly, host T cells would also be able to recognize the tumour MHC presenting any peptide as foreign, since it is not self-MHC. It is thus surprising that CTVT is able to persist as an allogeneic graft. MHC expression was previously characterised molecularly by Murgia et al through RT-PCR of a MHC-I (DLA88) and MHC-II (DLADRB1) gene (Murgia et al. 2006). They found that there was downregulation of expression of both these MHC genes. DLA88 showed low levels of tumour-specific expression, whilst there was no detectable tumour expression of DLADRB1. Murgia et al. also performed MHC genotyping for a number of CTVT samples and confirmed that all CTVTs shared the same haplotype (Murgia et al. 2006). They identified two clusters at the DLADQA1 locus, with some CTVTs appearing to be haploid the locus, whilst others remained diploid. This is in contrast to evidence that suggests the DLADQA1 locus had undergone a copy-neutral loss of heterozygosity (LOH) (Murchison et al. 2014). 1.6.4 Technologies used in this research work. Different technologies are being used in cancer research such as PCR, flow cytometry, immunohistochemistry (IHC), in- situ hybridization (FISH, CSH) and microarray for diagnosis (Pawanet al. 2010). Here, I used Real time PCR for gene expressional analysis of BRCA1 and COX-2 and DLADQA1 (MHCII). Histopathology (Hematoxyline and Eosin staining) was performed for the diagnosis of tumours. CTVT diagnostics qPCR was also performed to measure the allele’s quantity of LINE-myc gene and CDKN2A gene. Conventional PCR measures at End-Point, while Real-Time PCR collects data during the PCR shows the data and quality of data during exponential growth phase also it has increase dynamic range of detection, it is very sensitive and no need for post PCR processing. Immunohistochemistry was performed to find out the expression of MHCII antigens in CTVTs. The serum protein electrophoresis and serum biochemistry was also measured. Western blotting was performed to detect antibodies in CTVTs (protein expression). It is a very good technique to measure the gene expression at protein level in fluidic material of cells. We performed capillary electrophoresis to find the mutations/SNPs in our genes of interest (BRCA1, COX-2 and DLADQA1). Genetic analyzer was used to find the sequence variations in our genes of interests. Other methods used for sequence variation studies, like SSCP, DGCG and HPLC miss the mutations (Rassi 2009). So the sequencing by capillary electrophoresis was the best option for this study. Availability: Items available for loan: UVAS Library [Call number: 2250-T] (1).

3. Development Of DNA Based Diagnosis Of Ancylostoma Caninumin Dogs And Its Specificity With Traditional Fecal Microscopy

by Abida Rehman (2012-VA-648) | Dr. WasimShehzad | Mr. Akhtar Ali | Dr. Imran Rashid.

Material type: book Book; Literary form: not fiction Publisher: 2014Dissertation note: The blood feeding, canine hookworms have gained importance due to their potential to cause a variety of diseases in animals and human communities (Traversa 2012). Almost all types of canine hookworms are involved in causing zoonotic diseases(Traub et al. 2008), infecting more than half a billion people worldwide (Fenwick 2012), which result in ~65000 deaths annually (Plotkin et al. 2008). Ancylostoma caninum is the most prevalent and pathogenic intracellular obligate hookworm parasite of dogs (Bojar and Klapec 2012). In Pakistan, parasitic infection byA. caninum is widely prevalent with variable distributions in different parts of the country. A microscopic based study in the Lahore areashowed 59.1% ofA.caninuminfestation in dogs (Ashraf et al. 2008). Clinically,A. caninum has been responsible for often neglected disease ancylostomiasis in its host.In this condition, it principally attacks on the mucosal layer of small intestine through its buccal capsule for sucking blood (Marquardt et al. 2000). Secretory anticoagulant proteins of A. caninum help in this process by blocking a wide variety of blood clotting factors including Xa. The inhibition of blood clotting factors causes greater blood loss which ranges from 1-2ml/worm/day (Cappello et al. 1995; Georgi et al. 1969; Stassens et al. 1996). The disease is indicated by the symptomsof weight loss, lethargy, roughness of the hair coat,infected pale mucous membranes, tarry feces and excretion of eggs (Marquardt et al. 2000).In chronic situation,A. caninum producesa devastating condition of iron deficiency anemia with intestinal bleeding (Loukas and Prociv 2001).A. caninum infection in the dog isaccompanied by other life threatening pathological conditions, these includegastrointestinal infections, hypoproteinemia, mental retardation,pneumonitis andacute fatalities (Schwenkenbecher and Kaplan 2007).Especially, puppies are more suscepted to aforementioned diseases because of transmammary transmission,low levels of body immunity and higher egg count(Anderson 2000; Olsen 1986). The life cycle of A. caninumis almost same both in human and dog. Itis most complex and critical for them as compared to other members of its genus. The cycle starts with the production of eggs by adult worms within intestine of an infectedhost which are passed out with feces. These eggs survive in soil without damages by variable environmental conditions,can become a source of reinfection for host species. Theinfective filariform larvae (hatch from eggs) get their route ina host bodyby penetrating through hair follicles on skin contact. In puppies these are mostly transmitted through transmammary or prenatal routes. After penetration, these larvae take way to lungs through the blood or lymphatic circulation. From here, these can be swallowed towards intestine where they attached to feed on blood and mucous (Marquardt et al. 2000). They also migrate to skeletal muscles via somatic circulation where they depositedas hypobiotic larvae. Warm and moist conditions cause their reactivation and migration towards gut (Prociv and Luke 1995; Traub et al. 2014). It is seenthat a healthy pet doggetsA. caninuminfection mainly due to lackof proper veterinary care, large population of infected stray dogs, poor sanitation, and by contamination of public parks and streets (Klimpel et al. 2010; Zewduet al. 2010). Infective dogs are responsible for transmission of this parasite to humans either playing role aspets, stray or rescue animals (Jafri and Rabbani 1999; Szabova et al. 2007) by shedding millions of eggs(Epe 2009). Children areparticularly and frequently attacked byA. caninumbecause they used to play in open contaminated areas (Farooqi et al. 2014). Transmission of this parasite to humans is mostly by penetration through the skin, when it comes in contact with its filariform larval stages present in contaminated soil.Humans also get infection through larval ingestion, and larvae canbe transmitted from the fur coat ofinfected companion animals (Caumes 2000; Hochedez and Caumes 2007; Provic 1998). In human, penetration of A. caninumfilariform larvae causessevere cutaneous larva migrans (CLM). It is the most devastating hypersensitivity reaction, characterized by long follicular, pustular, ephemeral lesions at infection sites with intense itching and pain (Kirby-Smith et al. 1926; Little et al. 1983). These sites can be attacked by bacterial species leading tothe damages ofsoft tissues (Chaudhry and Longworth 1989). CLM caused by A. caninum has achievedmore attentionmainly due to associated pathological conditions. These include eosinophillic enteritis, pneumonitis, and growth defects, etc. (Bowmanet al. 2003; Garcia et al. 2008; Tu et al. 2008). In childrenheart problems and mental retardation also havebeen reported (Albonico et al. 1999; Crompton 2000). As, A.caninumiscausing serious healthhazards, so improved and proper control of its pathogenesis is mandatory.The accurate diagnosisof infection helps to prevent its transmission by selection of appropriate precautions and vaccines. Specific identificationis alsohelpful to minimizedthe chances of anthelminticdrugresistance by A.caninumas reported in different studies (Bethony et al. 2006; Kopp et al. 2007; Peeling et al. 2008; Roeber et al. 2013). Different diagnostic methods are used to identify parasitic hookworms. Currently, fecal microscopy is the widely applied diagnosticmethod for the identification of hookworm infection,which depends on morphological based analysis of eggs (Katz et al. 1972; Ngui et al. 2012b). The main advantage of this traditional approach is its tendency to analyze sampleboth qualitatively (floatation, sedimentation) and quantitatively (McMaster, FLOTAC, Katokatz)(Cringoli et al. 2011; Eberl et al. 2002). However, the identifications based on morphological characteristics may prone to inaccurate diagnosis, because eggs of many hookworms e.g. Ancylostoma, Trichostrongylus, Unicinariastenocephala, Oesophagostomum, Necator americanus and Terniden species are morphologically indistinguishable,thiscan variate specificity and sensitivity of microscopy (Bajwa et al. 2014; Monis et al. 2002;Tan et al. 2014). Improvement in microscopic detection can be made by usingcopro-culture and immunodiagnostic techniques especially when results are confounding. In the copro-culture method, eggs are raisedto larval stages in appropriate growth conditions in the laboratory andthen classified up to genus level (Reiss et al. 2007). But in this technique as microscopic examination, morphological similarities between larval stages of related species hampers specific identification. Moreover, it is time consuming (takes about 7-14 days) and requires experienced technicians to handle larvae. Similarly, it is very difficult to evaluate exact burden of worms as there is significant variations in the number of excreted eggs in chronic cases, which is another reasonable disadvantage ofthis diagnostic technique (Booth et al. 2003; Schar et al. 2013). Immunological assays, including ELISA,based on the detection of coproantigens in the feces or serum of the infected dog using captured IgG have been used to increase the sensitivity of analysis (Kwon et al. 2003;Loukas et al. 1992). These methodsallowed effective and quick detection of parasistes as compared to fecal microscopy and coproculture technique.But issue of cross reactivity with other antigens like of Strongyloides stercoralis in mixed infections isan associated problem (Lindo et al. 1994). Moreover, immunological assays failed to provide information about past or current infection and also do not differentiate between species in mixed infection (Basuni et al. 2011). All the problems of traditional methods can effectively addressed by the adoption of DNA based molecular approaches. These methods have beenproved as worthwhile alternatives to identify parasitic species in any developmental stage (Muldrew 2009; Ndao 2009; Vasoo and Pritt 2013). Specifically, these techniques are very feasible for specific identification of genusAncylostomabecause ithasambiguous features.Advanced highly sensitive DNA based diagnostic procedures have potential to identify the causative agentfromminute quantity of DNA (from 0.2g of egg), as in low worm burden with no symptom of disease(de Carvalho et al. 2012; Wong et al. 2014).Rapid detection of parasites can be made in only one day even in case of mixed infections(Gasser et al. 2009). There are various available approaches in DNA based methods which can be applied for the detection of Ancylostomaspecies, theseinclude PCR-RFLP (e Silva et al. 2006; Traub et al. 2004), copro-PCR (Sato et al. 2010), single-strand conformation polymorphism (SSCP) (Gasser and Monti 1997; Monti et al. 1998),specific (conventional) PCR (Yong et al. 2007) and multiplex real time PCR (Jonker et al. 2012). The conventional PCR techniquehas worldwideapplications in specie specific identification of parasiticspecies(Gordon et al. 2011). For specific diagnosis,genetic markers have been identified in mitochondrial (cox1 gene) and nuclear genomes (internal transcribed spacers (ITS-1, ITS-2), 5.8S and 28S in ribosomal DNA (rDNA)) (Chilton 2004; Denver et al. 2000; Gobert et al. 2005; Ngui et al 2012a). Preferably, spacers are more suitable for diagnostic purposes because these regions havesequence variationsamong different species. Moreover, these are shorter in length (250-300) in comparison with mitochondrial DNA (Blouin 2002; van Samson-Himmelstjerna et al. 2002). Therefore, the availability of sequencing technologies,specific genetic markers and large amount of genetic data have increased the chances of implementation, development and effectiveness of DNA based diagnostic method for identification ofA. caninum(Taniuchi et al. 2011). Availability: Items available for loan: UVAS Library [Call number: 2262-T] (1).

4. Application Of Microsatellite Markers For Genetic Diversity Analysis Of Endangered Punjab Urial (Ovis Orientalis Punjabiensis) In Pakistan

by Anam Aftab (2012-VA-534) | Dr.Tanveer Hussian | Dr. Wasim Shehzad | Dr. Muhammad Tayyab .

Material type: book Book; Literary form: not fiction Publisher: 2014Dissertation note: Biological diversity is now recognized as common concern of mankind and genetic diversity is the major driver of variation within and across breeds, which helps populations to adapt to environmental changes. There is very little importance is given to conserve wild sheep in previous years and its genetic diversity is decreasing day by day. Every now and then breeds are being haunted for crossing to some other imported breed without attempting to see if such efforts will be sustainable. For any breed development efforts thus, available genetic resources need to be characterized both at phenotypic and genetic levels (Khan et al. 2007). Among the three levels of Biodiversity, one is Genetic variation which is suggested bythe International Union forNatureconservation (IUCN) for preservation (Mc Neely et al. 1990). The reason for it is that firstly; genetic diversity favors the changes as the environment changes and secondly; it prevents inbreeding depression (Reed and Frankham 2003). In this way genetic diversity increase the survival status and increase fitness of individuals. Among many other wild animals present in Pakistan, there are 6 to 9 species of wild sheep (Ovis orientalis) are present which have different color and size of their winter neck ruff of males, saddle patches and horns color. Urial is a picture of Marco Polo in texture and hue. In Pakistan, ladakh urial, Blanford urialand Punjab urial are found in Gilget, Baluchistan and Punjab respectively. The discrepancy lies in the color of ruff among these 3 sub species (Roberts, 1977). Urial is among those precious wild animals that were hunted severely for trophy and other purposes in the past, that’s why included in red list of IUCN in vulnerable category (IUCN 2000). Punjab Urial (A type of wild sheep – Ovis vignei punjabiensis) belongs to family bovidae which is the large family consisting of 140 species (Glazko et al. 2011) is facing serious threat of extinction in Pakistan is a medium-sized wild sheep which is included in IUCN red list of endangered animals (IUCN 2002). Urial is inhabitant in Western Central Asianregion stretching from northeast side of Iran and west side of Kazakhstan to Balochistan (Pakistan) and Ladakh regions of North India. The local name of Urial is Shapo, Arkar and Gad. Reddish-brown outstretched pelt that achromatizes during the winter is one of the distinct traits of urial (Aleem 1977; Schaller 1977). Urial is gregarious and sexually dimorphic as males are called rams and females as eves (Awan 2001). Male have weightof 40 kgand have large spiralled horns having height of 80 to 100 cm and females have comparatively less weight and height of 25 kg and 12 cm respectively and have uncurled horns.Females give birth to 1 or 2lambs in recent days of April (Awan, 2001). Males have a black ruff expanded from the neck to the trunk and notably longer horns. Table 1.1:Some physical features of the Punjab Urial (Awan et al. 2001) Features Urial Body weight 40 kg male; 25 kg female Shoulder size 31-35 inches or 80-90 cm Horn size 39 inches or 80-100 cm long male; 12 cm long female Urial are found in moderate to very arid habitats, especially grasslands including agricultural fields and woodland areas (Valdez, 1982). Urial is herbivorous and eats grasses, shrubs and grains. The patch of salt range of Pakistan which fall in the area of Pind Dadan Khan, Choa Saidan Shah and Kallar Kahar is considered like a paradise on earth for the wild fauna. The fascinating hills of these areas are covered with thick trees and different wild plants are also the sanctuary of urial. Table 1.2: The details of these sub species in IUCN list of endangered mammals (IUCN 2002). Subspecies Citation in IUCN list Ovis vegnei blanfordi VUC 1 Appendix ll Ovis vegnei punjabienses ENA1cde,c1+2a Appendix ll Ovis vegnei vegnei VUC 1 Appendix ll In Pakistan, Punjab Urial dispersed throughout the Kala Chitta and Salt Range in a very little number(Hess et al, 1997). The Afghan Urial inhabits Baluchistan, Khyber Pakhtunkhwa, and Sindh Provinces. In Chitral District, little segregated populations of Ladakh urial are stillextensively distributed near the west bank of the Kunar river from Chitral southwards to Drosh. Ladakh Urial existence at the east bank of Kunar river and north of Chitral are not proved (Malik 1987). Total population estimate of urial is recorded by conducting several surveys. Reasonably 2,500 to 3,000 urial existsin Baluchistan (Hess et al. 1997). In 1993 the overall population assessment of Northern Areas was four hundred to five hundred Urial (Rasool 1999). In Pakistan, there were seemingly less than 600 Ladakh urial (Hess et al. 1997;NWFP 1992; Schaller 1971, 1977) but the number of urial decreased to 200 to 300 urial in all over northern areas (Rasool 1999). Based on the facts mentioned above there is dire need to conserve the Urial population in the country. The urial is one the precious fauna of Pakistan and provides us with wool and meat (fat, flesh or any eatable part). It is also important in economic way and in maintaining of ecosystem balance. In the beginning, sheep were reared for meat, milk and skin (Ensminger and parker, 1986). After 3500 B.C. men learnt to spin wool and so used wool in textile industry (Smith et al. 1997). So, because of increasing world population, there is great demand of these products and increasing day by day. That’s why we have to conserve is natural resource of Pakistan.Habitat fragmentationleads to the risk of exaggerated genetic drift and inbreeding in isolated population. So, there is need to save urial from these threats by enforcement of law and conserving it for future. In all over the euchromatic genome Microsatellite markers are present. These markers are highly polymorphic (Ellegren 2000; Schlotterer 2000).A lots of polymorphic microsatellites have been analyzed in ruminants like domesticsheep, cattle etc. (de Gortari et al. 1997,1998 ;Hayes et al.1996; Jenkings et al. 1997) aiding the use of these in parentage testing.   Microsatellite markers are among the most reliable molecular markers for genetic characterization studies in animal species (Sunnucks, 2001) and are simple sequence repeats (SSRs) of 1-6 base pairs, repeated tandemly in coding as well as noncoding portion of DNA in prokaryotes and eukaryotes (Weber and May, 1989; Toth et al., 2000). Microsatellite markers have often used for genetic diversity studies because they areabundant, unbiased, widely distribution all over the DNA, highly polymorphic, easy in assessment like genotyping of these markers (Canon etal. 2001).Microsatellite markers aid in genetic differentiation and conservation studies (Peter et al. 2007;Rendo et al. 2004; Arranz et al. 2001).These are considered very useful markers for assessment of genetic diversity, parentage confirmation, genome mapping, disease research population genetic studies and conservation genetics. These are also reported to be efficient enough to identify within and among breed differentiation and population sub structuring in cattle (Glowatzki-Mullis et al. 1995; Ciampolini et al. 1995; Garcia-Moreno et al. 1996; Jarne and Lagoda, 1996; MacHugh et al. 1998).Therefore the conservation activities are very important to save Punjab Urial from extinction and the study is designed to explore its genetic diversity. Availability: Items available for loan: UVAS Library [Call number: 2261-T] (1).

5. 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).

6. Genetic Association Study Of Apolipoprotein A-V (Apoa5) And Sortilin (Sort1) Genes With Risk Of Coronary Artery Disease

by Irfan Basharat (2012-VA-802) | Dr. Akhtar Ali | Dr. Wasim Shehzad | Dr. Muhammad Tayyab.

Material type: book Book; Literary form: not fiction Publisher: 2015Dissertation note: In developed countries cardiovascular disorders are prominent cause of death. One third deaths in the world are due to cardiovascular disorders. Among cardiovascular disorders coronary artery disease responsible for one in five deaths in USA. Its main reason is the lipids values particularly cholesterol and triglycerides in the blood. An estimation made by WHO indicated that 9 million people die per year due to hypercholesterolemia. 100 blood samples were collected from patients of coronary artery disease and from normal patients with no myocardial history. Allele specific primers for SORT1 gene and APOA5 genes were designed using Primer 3 software web facility. Genomic DNA will be amplified by PCR then genotyping will be carried out and DNA will also be sequenced. Hardy-Weinberg principle and Fisher Exact test were used to assess the allele frequency and significant variations from results When patient of MI and normal group were genotyped and sequenced we find out that there are 34 AA homozygous, 1 GG homozygous and 12 heterozygous persons in case of APOA5. The SORT1 person shows 24 GG homozygous and 3 AA homozygous and 13 heterozygous persons. Our study shows a definite association between APOA5 and SORT1 with respect to MI disease persons. This study shows a significant association of single nucleotide polymorphism in APOA5 and SORT1 genes with coronary artery disease. Availability: Items available for loan: UVAS Library [Call number: 2326-T] (1).

7. Mutation Analysis Of Alpha-Synuclein Gene In Patients With Parkinson Disease

by Iffat Aleem (2009-VA-566) | Dr. Asif Nadeem | Prof. Dr. Tahir Yaqub | Ms. Huma Mujahid.

Material type: book Book; Literary form: not fiction Publisher: 2015Dissertation note: Parkinson disease is a complex, heterogeneous and chronic neurodegenerative disorder with a cumulative prevalence of greater than one per thousand, caused by neuronal loss, mainly affecting dopaminergic neurons of the substantia nigra. Parkinson disease is an idiopathic disorder of the extra pyramidal system characterized by tremors. Genetic factors contribute to its complex pathogenesis. A functional repeat polymorphism in the α-synuclein (SNCA) gene promoter conveys susceptibility for Parkinson disease. The α-synuclein (SNCA) has been implicated in rare autosomal dominant forms of Parkinson disease. The mutations in α-synuclein were associated with severe disease progression and a typical physical signs, indicative of neuro degeneration extending beyond the substantia nigra. Mutation in α-synuclein gene may have association with dopaminergic neuronal loss in Parkinson disease. Blood samples were collected from Parkinson disease patients. DNA was extracted by organic method. Primers were designed using Primer3 software. Amplification of gene was done by Polymerase Chain Reaction. PCR products were sequenced bi-directionally on ABI 3130XL Genetic analyzer. Sequence alignment was performed for polymorphism identification. The analysis of identified polymorphism has been done by CHROMAS software. Sequences were aligned by BLAST tool of NCBI. The results of analysis showed that no mutation found in exonic region of α-synuclein (SNCA) gene in Pakistani individuals selected for this study. Any change in exonic region of α-synuclein (SNCA) gene is a rare cause of sporadic and familial Parkinson disease in different populations. Availability: Items available for loan: UVAS Library [Call number: 2325-T] (1).

8. Polymorphism Of The Slc11a1 Gene Associated With Resistance To Bovine Tuberculosis.

by Qamar Raza Qadri (2009-VA-569) | Dr. Asif Nadeem | Dr. Tahir Yaqoob | Dr. Abu Saeed Hashmi.

Material type: book Book; Literary form: not fiction Publisher: 2015Dissertation note: Bovine Tuberculosis (bTB), caused by Mycobacterium Tuberculosis is a health threat to livestock. Information on genetic resistance or susceptibility because of polymorphisms of candidate genes could be used in making selection decisions. Solute carrier family 11 (protoncoupled divalent metal ion transporters), member 1 gene (SLC11A1), is a known candidate gene which is associated with natural resistance to infection by Mycobaterium spp in buffalo. Polymorphism in this gene can be studied for breeding disease resistance animals. Blood samples were collected from Nili Ravi buffalo breed of Pakistan. DNA was extracted by organic method. Primers were designed using Primer3 software. Amplification of gene was done by Polymerase Chain Reaction. PCR products were sequenced bi-directionally on ABI 3130XL Genetic analyzer. Sequence alignment was performed for polymorphism identification. The analysis of identified polymorphism has been done by CHROMAS software. Sequences were aligned by BLAST tool of NCBI. The results of analysis showed that no polymorphisms were identified in exonic region of gene. This might be due to less sample size. Genetics play important role in fighting against pathogens. Identifying the genes involved can lead to marker-assisted selection strategies. Availability: Items available for loan: UVAS Library [Call number: 2332-T] (1).

9. Identification Of Genetic Variations In Toll Like Receptor 1(Tlr-1) Gene To Evaluate Its Potential For Enhanced Resistance To Bovine Tuberculosis

by Shehar Bano (2013-VA-09) | Dr. Maryam Javed | Prof. Dr. Tahir Yaqub | Miss Huma Mujahid.

Material type: book Book; Format: print ; Literary form: not fiction Publisher: 2015Dissertation note: Bovine tuberculosis is a disease caused by the species included in the Mycobacterium tuberculosis complex. Toll-like receptors (TLRs) are a family of conserved innate immune recognition receptors that trigger adaptive immune responses. TLR1 play an important role in host defense against mycobacteria, especially by mediating the response to mycobacterial triacylated lipopeptides. The objective of this study is the identification of single nucleotide polymorphisms within the coding region of TLR1 gene to evaluate its potential for enhanced the resistance to bovine tuberculosis in Nili-Ravi buffalo breed. Fifty blood samples of Nili-Ravi breed were collected from UVAS Pattoki Campus, Research Farm B and Buffalo Research Institute (BRI) Pattoki. Inorganic method was used for DNA extraction, for amplification of the coding region of TLR1 gene PCR (Polymerase Chain Reaction) was used using specially designed primers and the PCR products were sequenced through Sanger’s Chain Termination method. For the analysis and alignment of sequencing the results obtained after sequencing were analyzed and aligned using the CLUSTAL W and BLAST software. After all these analysis Ten SNPs were identified in the coding region of TLR1 mentioned in table. The Ten SNPs identified in the coding region of TOLL LIKE RECEPTOR 1 were in this order P1 C>T, P2 T>C, P3 T>C, P4 T>C, P5 T>C, P6 C >T, P7 T>C, P8 C >T, P9 A>G and P10 A>G. The one SNP found in the current research is in compliance with the (Sun et al. 2012) research on TOLL LIKE RECEPTOR 1 hence Nine SNPs found in the current research are novel in Nili Ravi buffalo. The SNPs in the exonic region that is P1 C>T, P2 T>C, P3 T>C, P4 T>C, P5 T>C, P6 C >T, P7 T>C, P8 C >T, P9 A>G and P10 A>G were all transitions i.e. the conversion of purines to purines. Population genetic analysis and allelic distribution at all loci was analyzed using POPGENE 32 software indicated that at [P3=0.243009> 0.05] followed the assumptions of the Hardy-Weinberg equilibrium indicating that the alleles were randomly distributed throughout the population, no migration had occurred, no bottlenecks happened and population remained large in numbers.This Non-significant and obeying HWE, so can be potential marker for genetic selection.At [P1= 0.040418< 0.05], [P2=0.033603< 0.05], [P4=0.000649< 0.05], [P5=0.000262< 0.05], [P7=0.015112< 0.05] and [P9=0.000111< 0.05] the probability value below 0.05 indicated that population at these polymorphic sites was not obeying Hardy-Weinberg equilibrium. This indicated that at these positions alleles were not equally distributed in population. It can be concluded from my research that the SNPs identified in the current research may also hold potential for marker-assisted breeding programs, with the aim of breeding more BTB-resistant animals and herds within both the national farms and the private sector. Availability: Items available for loan: UVAS Library [Call number: 2335-T] (1).

10. Analysis Of Genetic Polymorphism In Exon 6 & 11 Of Glucosidase Beta Acid (Gba) Gene In Gaucher Diseased Patients From Punjab, Pakistan

by Aysha Arshad (2009-VA-571) | Dr. Muhammad Yasir Zahoor | Dr. Muhammad Imran | Dr. Imran Altaf.

Material type: book Book; Format: print ; Literary form: not fiction Publisher: 2015Dissertation note: Gaucher disease (GD) is amajor predominant heterogenic, inherited and metabolic lysosomal storage disorder. It is prompted by an alteration in glucosidase acid beta (GBA) gene. GBA gene encodes a 497 amino acid glucocerebrosidase enzyme. It is a lysosomal hydrolase, present in all mammalian cells membrane that carries the catalysis of complex ubiquitous sphingolipids called glucocerebrosides (GlcCer) into smaller and simpler molecules of sugar and ceramide. The human glucocereborside (GBA) gene is present in highly gene dense area on q arm of 21 chromosome and its fragment length is 7.8kb comprising of 11 exons. A pseudogene is also present in vicinity of GBA gene which shares 96% homology of sequence with functional gene. Genetic recombination and gene conversion among these two GBA genes are responsible for 10-20% GD mutations. >300 mutations of GBA have been described till 2014. GD has three different clinical forms depend on its heterogeneity. These are characterized by the age of onset and with or without the participation of CNS. In this study, 10 blood samples were collected of GD patients from repository at Molecular and Genomic Laboratory located at IBBT department, UVAS Lahore and from Children Hospital Lahore. DNA extraction was done by using organic method from blood samples. Amplification of GBA gene exons 1, 6 and 11 was performed using PCR. PCR products were sequenced using Sanger di-deoxy sequencing method. Different bioinformatics tools were applied for the sequence analysis of exon 1, 6 and 11. We found two variants of GBA gene. A deletion of CT nucleotide repeat in intron 1 was found. We also found a substitutional change of nucleotide T>A in intron 8. Availability: Items available for loan: UVAS Library [Call number: 2334-T] (1).

11. 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).

12. Observation Of Antibacterialactivity Of Human Salivary Histatin Against Staphylococcus Aureus

by Rizwan Irshad (2013-VA-10) | Professor Dr. Tahir Yaqub | Dr. Muhammad Wasim | Dr. Nisar Ahmad.

Material type: book Book; Literary form: not fiction Publisher: 2015Dissertation note: The saliva contains numbers of proteins which act as antimicrobial agents and these peptides called as antimicrobial peptides. The saliva contains numerous antimicrobial peptides the main salivary peptide present in saliva is Histatin protein rich in Histidine amino acid. Histatin is only antimicrobial peptides present in primates like monkey chimpanzees and human. It’s also contain anti-fungal as well as anti-bacterial effect against staphylococcus aureus. In this study showed the effect of human salivary Histatin against staphylococcus aureus. Salivary Histatin may show antibacterial activity against Staphylococcus aureus with its efficiency having link to age, gender and diabetic status. Total sixty-four saliva sample will be collected on the basis of gender, age and presence of diabetic status. The antibacterial activity of saliva was observed against Staphylococcus aureus by disc-diffusion method. DNA was extracted and HTN1 gene was amplified using specific primer. This study was helpful in demonstrating antimicrobial ability of Histatin proteins present in human saliva. It also provide insight with regards to age, sex and/or immunocompromising ailment (in this case, diabetes) having an effect on the ability of these proteins, thus, opening new doors when it comes to combating fungal infections in both human and animal subjects. The HTN1 gene sequenced and BLAST results proof that variation in Histatin anti-bacterial property in diabetic patients and was not due to mutation in the nucleotide sequences of the decreases in salivary Histatin was due to other reason not due to mutation in these individuals. The age bases study HTN1 gene BLAST results was found 99% similarity with the other age groups. Summary 40 The statistical analysis of healthy people with age and zone of inhibition was found ANOVA P<0.000. The increases of age will decreases the salivary Histatin anti-bacterial properties. The optimum antibacterial activity was measured 2cm in diameter. The present results indicated that healthy human saliva possess antibacterial ability against Staphylococcus aureus. The results indicated that salivary Histatin can be novel tool as antimicrobial peptides of future medical field. Availability: Items available for loan: UVAS Library [Call number: 2342-T] (1).

13. Mutational Analysis Of Hepatitis C Virus Ns4b Gene Encoding Protein

by Faiza Nisar Bukhari (2013-VA-12) | Dr. Muhammad Imran | Dr. M.Yasir Zahoor | Dr. Abu Saeed Hashmi.

Material type: book Book; Literary form: not fiction Publisher: 2015Dissertation note: Hepatitis C virus (HCV) infection has an estimated global prevalence of 2%–3% with approximately 270 million infected people worldwide of which 10 million belongs to Pakistan. HCV is a positive-sense single-stranded RNA virus from the family of Flaviviridea. Its genome encodes 10 proteins including a 261 amino acids protein NS4B. NS4B is a non-structural protein that performs hyperphosphorylation of NS5A, transactivates interleukin 8 promotor, suppresses HCV translation and modulates the ER stress response. Mutation in NS4B encoding gene may be targeted to develop an effective vaccine against HCV before virus invasion in host immune system. Serum was collected from 20 patients who are infected with HCV. RNA was isolated from these samples to reverse-transcribe it into cDNA. This cDNA were PCR-amplified and amplicons were detected by UV light after their separation through agrose gel electrophoresis followed by seguencing of NS4B encoding gene. Variations in HCV NS4B gene was analyzed by using Mega 6 software and sequence 2 blast analysis NCBI tool. Protein modeling was performed with phyre 2, DIANNA tool and I-Tasser online server and quality of model and antigenicity was checked by Vaxajen V 2.0 and Epijen softwares. Conservative analysis and epitope mapping were performed by using Immune Epitope Database (IEDB). A systematic approach was employed for the prediction of potential epitopes in NS4B protein. Vaxijen V 2.0 was used to determine overall antigenicity of NS4B using cut off value of 0.4. Values above this threshold level show probable antigens in the protein. The topology of NS4B was determined by TMHMM server with the help of membrane topology data. Regions outside membrane and transmembrane were eradicated for epitopes prediction. T cell epitopes propred was used with proteosome cleavage site filter of 4% threshold. From the following data analysis 12 to 15 possible T-cell epitopes can be predicted; only these T cell epitopes were included in conservation analysis. The present study provides information about mutational changes in HCV protein NS4B and thus preliminary data for the development of vaccine against HCV. This can also lead to future prospects for diagnosis, treatment and prognosis of HCV. Hepatitis C virus (HCV) infection has an estimated global prevalence of 2%–3% with approximately 270 million infected people worldwide of which 10 million belongs to Pakistan. HCV is a positive-sense single-stranded RNA virus from the family of Flaviviridea. Its genome encodes 10 proteins including a 261 amino acids protein NS4B. NS4B is a non-structural protein that performs hyperphosphorylation of NS5A, transactivates interleukin 8 promotor, suppresses HCV translation and modulates the ER stress response. Mutation in NS4B encoding gene may be targeted to develop an effective vaccine against HCV before virus invasion in host immune system. Serum was collected from 20 patients who are infected with HCV. RNA was isolated from these samples to reverse-transcribe it into cDNA. This cDNA were PCR-amplified and amplicons were detected by UV light after their separation through agrose gel electrophoresis followed by seguencing of NS4B encoding gene. Variations in HCV NS4B gene was analyzed by using Mega 6 software and sequence 2 blast analysis NCBI tool. Protein modeling was performed with phyre 2, DIANNA tool and I-Tasser online server and quality of model and antigenicity was checked by Vaxajen V 2.0 and Epijen softwares. Conservative analysis and epitope mapping were performed by using Immune Epitope Database (IEDB). A systematic approach was employed for the prediction of potential epitopes in NS4B protein. Vaxijen V 2.0 was used to determine overall antigenicity of NS4B using cut off value of 0.4. Values above this threshold level show probable antigens in the protein. The topology of NS4B was determined by TMHMM server with the help of membrane topology data. Regions outside membrane and transmembrane were eradicated for epitopes prediction. T cell epitopes propred was used with proteosome cleavage site filter of 4% threshold. From the following data analysis 12 to 15 possible T-cell epitopes can be predicted; only these T cell epitopes were included in conservation analysis. The present study provides information about mutational changes in HCV protein NS4B and thus preliminary data for the development of vaccine against HCV. This can also lead to future prospects for diagnosis, treatment and prognosis of HCV. Availability: Items available for loan: UVAS Library [Call number: 2339-T] (1).

14. 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: book Book; 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).

15. The Variability Analysis of The Gene Encoding HCV Non-Structural Protein NS2

by Abdul Rehman (2009-VA-546) | Dr. M. Imran | Dr. Muhammad Yasir Zahoor | Ms. Faiza Masood.

Material type: book Book; Literary form: not fiction Publisher: 2015Dissertation note: Theses submitted with blank cd. Availability: Items available for loan: UVAS Library [Call number: 2337-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: book Book; 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 Effect Of Cholesteryl Ester Transfer Protein (Cetp) Gene In Coronary Heart Disease Patients

by Zakiya Bano (2013-VA-554) | Dr. Akhtar Ali | Dr. Waseem Shehzad | Dr. Muhammad Tayyab.

Material type: book Book; Literary form: not fiction Publisher: 2015Dissertation note: Cholesteryl ester transfer protein (CETP) gene takes part with certain reverse cholesterol transport (RCT) pathway for the excess amount of accumulated lipid in peripheral tissues. The variations in this gene due to missense mutations on different exonic, intronic or on promoter regions alter CETP activity as well as impair the RCT pathway. By which, lipid metabolism also effects and causes atherosclerosis in vessels which trigger the blockage of blood flow and induces the imbalance for the supply of oxygen to the heart. So this atherosclerosis directly involves in addition of risk factor for coronary heart disease. Preferable study was made to highlight effect of CETP gene at molecular level by comparing control group with the selected patients having coronary heart disease. This study was appreciably made possible by targeting two reported polymorphisms, one in the intron 1 region Taq IB (rs708272) and on exon 14 region I405V (rs5882) of this CETP gene. The study was relatively speculated by the extraction of genomic DNA from all selected blood samples. By selecting two primers, certain segments were amplified for both rs708272 and rs5882 polymorphisms. Analysis of allelic frequencies distribution was calculated by Hardy Weinberg Equilibrium which showed no significance among control and CHD group and there was no association was analyzed in our population by using Fisher’s Exact Test. This is because of small number of samples studied in our population. But maximize concentrations of lipid parameters such as TC, LDL and TG with minimum variation in HDL-C concentration in CHD group as compared to control group that showed the effect of these polymorphisms on the activity of CETP gene with coronary heart disease. These determined missense mutations in CETP gene was helpful molecular tool for the screening purpose in coronary heart disease patients. Availability: Items available for loan: UVAS Library [Call number: 2345-T] (1).

18. Mutational Analysis of CaSR in Calcium Nephrolithiasis Affected Pakistani Families

by Asad Tufail (2013-VA-558) | Dr. Muhammad Yasir Zahoor.

Material type: book Book; Literary form: not fiction Publisher: 2015Dissertation note: Thesis submitted with blank cd. Availability: Items available for loan: UVAS Library [Call number: 2343-T] (1).

19. 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).

20. Study Of Wound Healing Effects Relating To Topical Application Of Human Saliva On Rabbits

by Sanila Amin (2013-VA-281) | Prof. Dr. Tahir Yaqub | Dr. Muhammad Imran | Dr. Habib ur Rehman.

Material type: book Book; Literary form: not fiction Publisher: 2015Dissertation note: Histatin proteins present in human saliva have been observed to show natural antibacterial and antifungal properties, as well as play a role in wound healing. These naturally occurring proteins can serve as effective agents when combating microbial infections of vulnerable wounds that have become drug resistant, without inducing negative side effects in the host. Focusing on these proteins can create a new outlook with regards to wound-healing medicine for both humans and animals. Subjects of this study were 30 fully grown adult male rabbits weighing 2.0 to 3.4 kg and ranging from 8 to 16 months in age. They were acclimatized for two weeks in stainless steel cages and fed commercial diets, vegetables, crushed wheat and corn all over the whole experiment. Out of all 30 rabbits 24 rabbits were experimental on which saliva was applied, three were negative control to check natural wound healing, and three were positive control on which wound healing medicine was applied. The 24 experimental rabbits were further divided into four groups with each group consisting of 6 rabbits to check the effect of age on wound healing. The age groups of human samples were divided as 15-25, 25-35, 35-45 and 45-55 (Verma et al. 2013). Saliva of human individuals belonging from these four age groups was applied on the wounds of experimental group. Furthermore, all age groups contained saliva from both gender i.e. each age group consisted of 3 male and 3 female saliva samples. Furthermore, DNA was extracted from blood samples of the same individuals from whom saliva samples were procured. HTN1 gene which is responsible for the production of salivary histatin protein was amplified using specific primers and PCR optimization. CHAPTER 6 SUMMARY 33 The results of this study demonstrated the wound healing properties of histatin proteins present in saliva and thus, providing a basis of using the natural ability of human saliva to act as a major component in the future of medicine for wound healing and preventing wound infections in both human and animals. Availability: Items available for loan: UVAS Library [Call number: 2344-T] (1).

21. Mutation Anlysis Of DTNBP1 Gene In Pakistani Patients With Schizophrenia Disorder

by Hafiza Sidrah Yasin (2013-VA-11) | Dr. Asif Nadeem | Dr. Maryam Javed | Dr. Abu Saeed Hashmi.

Material type: book Book; Literary form: not fiction Publisher: 2015Dissertation note: Schizophrenia (SCZ) disorder is a mental complex, heterogeneous and chronic neurodegenerative disorder with a cumulative prevalence of 1%. SCZ is an idiopathic disorder of the cortex and hippocampus. Environmental as well as genetic factors contribute to its complex pathogenesis. A functional repeat polymorphism in the Dystrobrevin Binding Protein 1 (DTNBP1) gene promoter conveys susceptibility for SCZ disorder. The DTNBP1 has been implicated in rare autosomal dominant forms of SCZ disorder because of mutations associated with severe disease progression and a typical physical signs and symptoms, indicative of neurodegeneration. Mutation in DTNBP1 gene has association with change in dysbindin protein which leads to change in abnormal neurotransmitter trafficking which leads to decrease in neuronal size, brain atrophy and reduced glutamate release in schizophrenia disorder. A systematic approach was applied to proceed the present study in order to identify the single nucleotides polymorphisms in schizophrenic patients. Blood samples (n=40) were collected from schizophrenia disorder patients. DNA was extracted by organic method. Primers were designed using Primer3 software. The amplification of gene was done by Polymerase Chain Reaction. PCR products were sequenced bi-directionally on ABI 3130XL Genetic analyzer. Sequence alignment was performed for polymorphism identification. The analysis of identified polymorphism was done by CHROMAS software. Sequence was aligned by Blast tool of NCBI. Difference between allele and genotype frequency of studied gene was evaluated and analyzed by using “SNPator”. The present study provides information about the susceptibility and genetic basis of the individual towards this disease and identified polymorphisms provides the opportunity to diagnose the disease earlier on the basis of particular SNPs in Pakistani patients. Availability: Items available for loan: UVAS Library [Call number: 2382-T] (1).

22. 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).

23. 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).

24. Expression And Purification Of A Potent Surface Antigen (Sag1) Of Toxoplasma Gondii In Prokaryotic Expression System

by Zunaira Zafar (2009-VA-542) | Dr. Wasim Shehzad | Dr. M. Yasir Zahoor | Dr. Imran Rashid.

Material type: book Book; Literary form: not fiction Publisher: 2015Dissertation note: Toxoplasma gondii, an intracellular obligate parasite infects almost all warm-blooded animals including human. Toxoplasmosis, caused by T. gondii, may show minute to severe clinical results in humans. Currently, there is no vaccine available for human use. SAG1 is a major candidate of interest for vaccine development that elicits humoral as well as cellular immune response against this devastating parasite. rSAG1 that had already been ligated in pET28/His expression vector, was transformed in E. coli (BL21) host and expression was confirmed by means of SDS-PAGE and western blotting. Nickel columns were utilized for affinity based chromatographic purification of rSAG1. This purified protein was then quantified via protein quantification kit. Immunogenic recombinant SAG1 can be used in diagnostic antigen-antibody tests e.g. in ELISA. Moreover, it might be used in vaccination against T. gondii. Vaccine against this parasite may alleviate socio-economic burden on human society that ultimately modulates the health parameters for better living. Availability: Items available for loan: UVAS Library [Call number: 2393-T] (1).

25. Mutational Analysis Of Hcv Gene Encoding E1 Glycoprotein

by Muhammad Saad Junjua (2013-VA-893) | Dr. Muhammad Imran | Dr. Wasim Shehzad | Dr. Abu Saeed Hashmi.

Material type: book Book; Literary form: not fiction Publisher: 2015Dissertation note: Hepatitis C virus (HCV) is a positive single stranded RNA virus that belongs to Flaviviridae family and causes liver related issues like hepatocellular carcinoma, cirrhosis and chronic liver disease. HCV is affecting people worldwide; more than 170 million peoples have been affected so far and the number is increasing day by day. Its prevalence in Pakistan is about 3% to 6%. There is lot of variation in its genome and it is classified into 6 major genotypes and these genotypes are further classified into many subtypes. Size of HCV is about 9500 bps which only encodes single polyprotein. This 3000 to 3300 amino acids polyprotein is processed by cellular and viral proteases to generate 10 polypeptides consisting of 4 structural (Core, E1, E2 and P7) and 6 non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A, NS5B). The most immunogenic gene from all the genes is E1. It involves in the interaction with the host cell and easily escapes from the immune system of host due to the presence of hypervariable regions in E1 gene. To isolate the E1 gene, RNA extraction was done using the kit method. RNA was converted to cDNA which was then amplified in two rounds of PCR using nested primers from HCV core region. After confirming the presence of HCV RNA in serum samples, PCR amplification of HCV E1 gene was carried out using gene specific nested primers. Amplified E1 gene products were Sanger sequenced and aligned with standard sequence to find out genetic variations. E1 gene sequences were converted to protein sequences for which secondary protein structures were made and analyzed. No noticeable change was seen in these secondary protein structures. The protein sequences were also analyzed for the presence of B-cell and T-cell epitopes; two T-cell epitopes (QAFTFRPRR, FLVGQAFTF) were found which may inform the development of a proper vaccine against HCV. Availability: Items available for loan: UVAS Library [Call number: 2399-T] (1).

26. 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: book Book; 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).

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