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: ; Literary form:
not fiction
Publisher: 2014Dissertation note: Pakistan is bestowed with the land of geological and topographic diversity. The ecological variation is uniformly reflected in all water lands of the country. Pakistan has significantly huge natural inland water resources in the form of ocean, rivers, networks of canals and lakes (Mirza and Rafique 1994). The country is blessed with one of the largest freshwater resources in the world correspondingly large number of freshwater living vertebrates is available from which fishes are quite significant considering the ecological balance and its consumption as food. It is one of the food sources which solely provide all the essential nutrients, minerals and high quality protein which is not common from other food items (Muhammad Rafique 2007). Out of 33,100 fish species identified worldwide as per Fish Base organization report published in April 2015 (http://www.fishbase.org). Out of 233 (indigenous and exotic) freshwater fish species, 78 economically important indigenous fish species are available in the water bodies of the Pakistan. According to this report fishes are the largest vertebrate group, constituting about 50% of all vertebrate species. Systematically fishes are widely spread in nature, ranging from prehistoric jawless fishes to cartilaginous fishes and also from old to current day bony fishes. The taxonomic placement of these fishes shows their belonging to class Actinopterygii, sub-class Teleostei, 3 cohorts, 6 superorders, 13 orders, 30 families and 86 genera (Rafique 2007; Rafique and Khan 2012).
Demand of fish is increasing day by day not only being the naturally available source of food rather the health benefits associated with its consumption. This necessitates to develop a more efficient and sustainable system to increase their growth. DNA based technologies are being competently employed in aquaculture production fields for pedigree information.
Introduction
2
Moreover, tagging each fish individually is not an easy task so these DNA based methods help in avoiding intrusion of environmental factors which may result from raising fish families in separate reservoirs (Martinez 2007). Fish identification has been traditionally based on phenotypic features. However, due to high multiplicity and morphological similarity, in many cases, fish at its different developmental stages are hard to be identified by relying only on morphological characteristics (Victor et al. 2009).
For phylogenetic studies of the animals the use of mt-DNA is very common and reliable compared to nuclear DNA due to its high evaluation capabilities, which results in gathering of differences even between closely related species (Moore 1995; Mindell et al. 1997).“Bar-coding gap" is the name given to the property that is inter-specific variation in this region is markedly higher than intra-specific variability (Hebert et al. 2003).
Approximately each and every animal contain 13 protein-coding genes (PCGs) as an essential component of their mt-genome (mitochondrial genome), which helps in encoding of several proteins responsible for the oxidative phosphorylation machinery (Richly A et al. 2004, Song H et al. 2008). Being maternally inherited, mt-DNA is better as compared to genomic DNA such as quick evolution, less exposure to recombination, high copy number, high conservation, little duplications and negligible intergenic regions (Waugh J 2007, Xu J 2005). Clonal inheritance is the main property which makes it more worthy and suitable marker in comparison with other available molecular bio-diversity tools (Galtier et al. 2009).
DNA barcoding is one of the taxonomic tools. It is being used to distinguish animal species based on the small segment of their genome such as mitochondrial DNA, designated as an identification tag or barcode of particular species (Herbert et al. 2003). Identification of species using DNA barcoding is quite debatable. Still many researchers consider it as a reliable
Introduction
3
basic tool to ascertain the genetic characterization of diverse eukaryotic species, especially after establishment of the Consortium for the Barcode of Life (CBOL) in 2004 [http: //www. barcodeoflife.org/].
Ideally DNA barcoding should provide quick, reliable and cost effective species identification, even to those user who has little or negligible knowledge of taxonomy (Herbert et al. 2003, Hajibabaei M et al. 2005, Herbert et al. 2005). Identification of unknown source is possible by using distance based tree which can be created by comparing unidentified sequences against retrieved known sequences of different species (Hebert et al. 2003, 2004a, 2004b). DNA barcoding identification system has been recognized universally as standardized method to recognize species and unveil their genetic diversity (Herbert et al. 2003; Herbert et al. 2004). The ideal DNA barcoding is robust, with conserved, universal primer binding sites, reliable DNA amplification and sequencing.
From whole mitochondrial genome, Cytb (Cytochrome b) is considered as one of the most promising gene due to its function and structure, even it is composed of both conserved and rapidly evolving regions which are more related to evolutionary studies (Farias et al. 2001). To identify unknown or ambiguous species it is considered more reliable as it contain sequences which provide the specific information about particular species (Parson W et al. 2000a, 2000b). It is also one of the most useful genetic marker to identify the linkage within families and genera (Meyer 1994; Teletchea 2009). Cytb gene is involved in comparative studies which results in development of new classification schemes and been used to assign a genus to a newly described species as well as improve the understanding of evolutionary relationships of genra (Castresana 2001).
Introduction
4
One of the core objectives of this study is to identify and classify four freshwater indigenous fish species of Pakistan, which includes Labeo rohita (Rohu), Labeo calbasu (Calbans), Catla catla (Thalla) and Cirrhinus mrigala (Mori) using Cytb gene. Morphologically, Labeo rohita shows compressed body with convex dorsal profile while mouth bears a pair of barbells and fins are gray and orange in color. Catla catla shows compressed body with broad head. Mouth is wide with thick lower lip. Labeo Calbasu`s dorsal profile is more convex than that of abdomen and two pairs of barbells are present on fringed upper lip. Cirrhinus mrigala has elongated and streamlined body shape which is grayish and silver in color (Bhuiyan AL 1964; Rahman AK 1989). All of these species are found in freshwater bodies mostly lakes, rivers and ponds except Labeo calbasu which is a bottom dweller. These fishes are harvested by using rod and line or by using nets (Talwar PK and Jhingran AG 1991). These fishes are known as major carps and economically very important for the country due to their high consumption as food. These fishes are also used for fish farming due to their greater muscle mass thus also possess very high commercial value for fish farming business.
Another objective of this study is to resolve the taxonomic anomalies related to above mentioned species. Selling of fish meat in mislabeled packaging is a serious issue now days. Most commonly Hypophthalmichthys molitrix (silver carp) and Ctenopharyngodon idella (grass carp) are sold under the label of Labeo rohita. DNA barcoding is also helpful in detecting such fraudulent mislabeling.
It would be the first study in Pakistan to genetically characterize commercially important fish species. It would help scientists to know about their phylogenetic and taxonomic status and also assist fish fanciers to genuinely identify their species of interest. Identification of fish species is also important for conservation of biodiversity as it helps in preservation and
Introduction
5
identification of endangered species by generating their barcodes from even minimal evidence available. This study has paved the way for molecular biologists to study taxonomic ambiguities at sub species level using SNP (Single nucleotide polymorphism) based identifying marker. Availability: Items available for loan: UVAS Library [Call number: 2207-T] (1).
2.
Polymorphisms Of Bovine Tumor Necrosis Factor Alpha Gene And Its Association With Mastitis In Sahiwal Cows
by Huma Sattar (2013-VA-03) | Dr. Sehrish Firyal | Dr. Ali Raza Awan | Dr. Muhammad Tayyab.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Mastitis is one of the shocking maladies of milch animals causing high production losses to livestock industry of Pakistan (Kenyanjui et al. 2011). It is an inflammatory condition of udder; represent a major problem in dairy cow management. It is one of the most common and frequent disease of dairy industry. Producers suffer a huge loss due to veterinary treatment costs and necessary culling of the infected animals. It negatively affects the milk production, quality of milk, and farm economics (Fourichon et al. 2005). Increasing the disease resistance among dairy cattle is therefore desirable because without controlling mastitis, the national goals of developing dairy farming on commercial and scientific lines and production of wholesome milk which conforms to the standards of WTO Accord would remain elusive.
Mastitis is inflammation of udder that caused by physiological and metabolical changes (Schalm and Noorlander 1957). There are two main types of mastitis; clinical mastitis (characterized by classical symptoms i.e., swelling of udder, redness, clumps and clots in milk etc) and sub-clinical mastitis (not show any symptoms, Milk appear normal, udder appear normal) (Schrick et al. 2001). Mastitis is ranked as a top disease of dairy herds (Rinaldi et al. 2010). This mammary gland infection caused by pathogenic micro organisms such as Staphylococcus aureus, Streptococcus uberis, and Esherichia coli in the mammary gland (Heringstad et al. 2000).
India, China and United States are the larger producer of milk and Pakistan is on forth number in milk yield. Pakistan almost produces 36.5 million tons of milk yeild per year (Cady et al. 1983).The Sahiwal breed is well known among for its superior dairy qualities (Barker et al. 1998). Both cross and pure breed Sahiwal cows have high milk production rate (Khan et al. 2013).
It is very difficult to comprehend this disease because numerous environmental and genetic factors are involved in the origin and development of mastitis (Bradley 2002; Carvajal et al. 2013). Susceptibility and resistance to mastitis is a complex trait influenced by genetic variation of animals. Among these variations, the polymorphisms in immunity genes are principal key factors in defensive mechanism of mammary gland (Ibeagha-Awemu et al. 2008).
The mammary gland tissue is protected by immune system by two defense system; innate and acquired immunity. Innate immunity response by the host is a quick response of bacterial defense system (Mesquita et al. 2012). Innate system is a rapid and effective mechanism that activated on recognition of antigen (Akira et al. 2006). Innate immune system is activated when specific pattern recognition receptors (PRR) that are present on the surfaces which are attach to the specific pathogen (Shuster et al. 1996). PRR are presnt on leucocytes in milk and on the epithelial cells lining of udder. It is reported that T- lymphocyte subset i.e., CD4+, CD8+ and ɤδT are present in infected bovine mammary glands. (Goldammer et al. 2004; Strandberg et al. 2005).
Innate defense (nonspecific) of the mammary gland is stimulated by the physical barrier such as teat end, natural killer (NK) cells, neutrophils, macrophages and certain other soluble factors. The teat cannals are considering the main line of defense. Microorganisms enter from teat canal in milk. The main roles of teat sphincter muscles are to remain orifice close so that bacteria cannot enter. This teat canal also lined with keratin, whose estrified and non estified fatty acid function as bacteriostatics that provide protection and play role to eliminate bacteria causing mastitis (Oviedo-Boyso et al. 2007).
If a pathogen is not eliminated by the physical barrier, the acquired immune system is triggered. In comparison, this system is much faster than other immune response. The memory response is significantly stronger, long durable and more efficient to kill the pathogen. The acquired immune system (memory response) have ability to differentiate self or nonself cells and produce antibodies only against antigens through membrane bound protein called major histocompatibility complex (MHC) molecules. Specific immune system activate only when antigens bind with an MHC that is present on the surface of certain cells, this process is referred as antigen presentation. Recognition of pathogenic factors for elimination is mediated by macrophages, several lymphoid, and immunoglobulins (Ig) or antibodies (Sordillo and Streicher 2002).
The most acute responding macrophages and T-cell cytokines are TNF-α, LTF, IL1, IL6, IL8, and IFN-ɤ present in intramammary infection in cows. These genes play important role in improvement of immunity to mastitis (Burton and Erskine 2003).
Tumor necrosis factor alpha is main pro-inflammatory adipokine that is part of systematic immune defense. The main function of TNF-α gene is responsible for proliferation, differentiation and activity of many immune system cells; B lymphocytes, NK (natural killer). It also induces the production and release of many other cytokines (Wojdak Maksymiec et al. 2013) and also enhances the chemotactic and phagocytic effects of immune response. TNF-α gene contains four exons and three introns that are present on chromosome BTA23q22 (Bannerman 2009; Moyes et al. 2009).
TNF-α is a member of a group of cytokines that stimulate the specific immune system. TNF consist of 212 amino acid arranged in stable homotrimers (Kriegler et al. 1988; Tang et al. 1996). The 17-kilodalton (kDa) TNF protomers are composed of two β-pleated sheets and β-strands, joined together antiparallel (Tang et al. 1996).
TNF-α is a component of natural protection systems of humans and animals. Milk gives nourishment and disease resistance to the new born. Various cellular and soluble immune components are important for protecting the mammary gland from infectious diseases like mastitis. Mastitis affects one third of all dairy cows and cost the dairy industry about 2 million dollars annually (National Mastitis Council (1996). Dairy cattle are especially susceptible to mastitis due to diminished mammary gland defense mechanisms (Sordillo and Streicher 2002).
TNF-α is not only produced by activation of macrophages, but also other cell types such as CD4+ lymphocytes, NK cells, neutrophils, mast cells, eosinophils, and neurons. Large amounts of TNF are released in response to lipopolysaccharide, other bacterial products, and Interleukin-1 (IL-1).TNF-α stimulates the proliferation, differentiation and activity of many immune system cells; B lymphocytes, NK (natural killer). TNF-α induces the release of many other cytokines (Wojdak-Maksymiec and Mikolajczyk 2012). TNF-α also enhance the chemotactic and phagocytic effects of immune response.
. The present study is designed to determine the genetic polymorphism in exon 4 of TNF-α gene of mastitic cows and its association resistance and susceptibility towards mastitis.
Availability: Items available for loan: UVAS Library [Call number: 2224-T] (1).
3.
Sequence Analysis Of Mitochondrial Atpase 8/6 Gene Variants In Equine
by Kashif Hameed Anjum (2012-VA-905) | Dr. Asif Nadeem | Mr.Maryam Javed | Dr. Muhammad Tayyab.
Material type: ; Literary form:
not fiction
Publisher: 2014Dissertation note: Human has been using horses for doing different jobs like transportation, hunts, carrying loads, warfare and sports (Zhang et al. 2012). In Pakistan, horses and donkeys are mostly used for transportation whilehorses are also used for racing and playing games like polo.There are two main types of horses:Equuscaballusare domesticated horses and Equusferus are the wild horses. There are more than 300 breeds of horses in the world today (Barbara and Dafydd, 2007). The horse population is estimated as 0.32 million and has been decreasing over the years in Pakistan. Main breeds of horses that are found all over the Pakistan are Kajlan, Kakka, Balochi, Morna, Shien, Anmol, Makra, Pak-thoroughbred,Heerzaiand Waziri (Khan, 2004).
Seventy percent of the population earns living from the land. Agriculture contributes nearly 21% to gross domestic product and generates 43% of all jobs. Over 30 million people in rural areas derive their livelihood from livestock production. The number of impoverished communities moving from the country to find work in Pakistan’s towns and cities is rising. Many of these people rely on working equine animals to earn a living.
Nuclear and mitochondrial genomes are frequently used in animal genetic research. Nuclear genomeis generally a huge and complicated molecule and is not well studied in many species. However mitochondrial DNA being small sized and having high mutation rate is used frequently for the purpose of genetic research (Stanley et al. 1994). Characteristic of having fast evolution rate as compared to nuclear DNA makes mitochondrial genes a good tool for genetic studies (Avise, 1994).
Several studies have investigated the genetic relationship among horse and donkey breeds using mitochondrial sequences as a marker for breed characterization and phylogenetic. Each mitochondrion contains its own circular DNA, replication, transcription and translation machinery and serves as semi-autonomous organelle. Mitochondria perform so many important functions in our body like metabolism(oxidative phosphorylation), apoptosis and aging(Weinberg, 2007).
The advent ofpolymerase chain reaction and direct sequencing techniques with the use of mtDNA as a phylogenetic marker has been extended to much greater levels of phylogenetic inclusiveness (Zardoya and Meyer,1996). The special features of mtDNAi-e,lack of introns, maternal inheritance, absence of recombination events and haploidy have made it the most common type of sequence information used to estimate phylogenies among both closely and distantly related texa(Meyer, 1993).
Four of the five mitochondrial respiratory chain complexes, namely C1, C3, C4 and C5 (ATP synthase) contain subunits encoded by mitochondrial DNA (Kadenbach, 2012). ATP synthase (Complex5) functions to make ATP that is used by the cell (Von et al. 2009). ATP synthasecomprisesan integral membrane cylindrical, the F0 particle and a peripheral matrix-facing F1 particle, the catalytic ATP synthase domain (Boyer, 1997). All aerobically respiring organisms possess ATP synthase enzymes and are located inthe cell membrane in prokaryotes, the mitochondrial inner membrane in eukaryotes and the chloroplast thylakoid membrane (Ackerman and Tzagoloff, 2005). This enzyme is responsible for the final step of oxidative phosphorylation. The protons move down their concentration gradient from inter membrane space to matrix through F0 particle while F1particleuses the energy provided by influx of these protons and converts ADP molecule into ATP. ATPase 6 and ATPase 8 proteins are components of F0 particle where they play direct role in maintaining the structure and function of ATP synthase (complex 5). All five subunits of F1 and most of the F0 subunits are nuclear encoded(Collinson et al. 1996). Only two proteins i-e, ATPase 6 and ATPase 8 are encoded by mtDNA (Boyer, 1993).
The present study is designed to investigate the diversity and phylogenetic analysis of Thoroughbred Pakistani horse and donkey breeds on the basis of ATPase 6 and ATPase 8 genes.
Availability: Items available for loan: UVAS Library [Call number: 2236-T] (1).
4.
Expression And Mutational Analysis Of Breast Cancer Susceptibility Gene 1 (Brca1) And Cyclooxygenase-2 (Cox-2) Gene In Feline And Canine Tumours
by Haleema Sadia (2007-VA-567) | Dr. Muhammad Wasim | Prof. Dr. TahirYaqub | Dr. Abu Saeed Hashmi.
Material type: ; Literary form:
not fiction
Publisher: 2014Dissertation note: Cancer is the first cause of death in cats and dogs while in human it is the second most cause of death (Jemal et al. 2008). According to an estimation, cancer related deaths in the world are 13% and 70% of these deaths are in poor countries (World Health Organization 2012). Such natural cases of cancers in cats and dogs especially, in dogs offer an opportunity to use the dogs for comparative cancer studies and as an animal model for anticancer drug development (Pawaiya 2008). Inu a series of more than 2000 autopsies, it was found that almost forty five percent dogs that lived for ten or more years expired because of cancer (Bronson 1982). Dogs are affected by skin cancer 35 times more often than humans. They are also affected 4 times more often by mammary gland cancer, 8 times more often by bone cancer, and twice more often by leukemia, than humans (Cullen et al. 2002). The regulation of cell proliferation, genome stability and programmed cell death are important for systemic homeostasis.
1.1Historical perspective on cancer causation
Hippocratic and Galenic medicine attributed the spread of black bile (one of the four humours) in the tissue as the cause of the cancer (Diamandopolus 1996) is an idea survived intact through the Middle Ages and Renaissance. With the discovery of the lymphatic system by Gasparro Aselli in 1662, the black bile theory was superseded by the idea that cancer was an inflammatory reaction to extravasated lymph; a theory modified 150 years later by John Hunter who introduced the notion that contaminated coagulating lymph was the origin of the cancer (Kenneth 2003). A German pathologist Johannes Muller first time demonstrate that cancer is made up of cells (1838) but he also gave an idea that cancer cells were originated from a bud called Blastema instead of normal cells (Kardinal and Yarbro 1979). Following Schleiden and Schwann's cell theory of tissues,it was Rudolf Virchow (Muller’s student) who in 1855 demonstrated that every cell was derived from another cell (omnis cellula e cellula), including cancer cells (Mazzarello 1999; Porter 1999). In 1867 Wilhelm Waldeyer supported the theory of the normal cell for the origin of cancer and he believed that metastasis resulted from transportation of cancer cells by blood or lymph (Porter 1999).
Around the turn of the twentieth century the beginning of tumour transplantation experiments led to the new view of the cancer cell as an autonomous cell. The first successful tumour transplants were described in 1876 by the Russian veterinarian Mstislav Aleksandrovich Novinski (Novinski 1876). He reported in his thesis entitled “On the Question of the Inoculation of Malignant Neoplasms” the first successful serial passage of tumours through transplantation in dogs. Novinski's transplantation experiments were based on the inoculation of canine transmissible venereal tumour (CTVT) in puppies. Novinski stated that successful tumour transplantation depends on the inoculation of a living element of the tumour and that the transplantation of the element of a cancerous tumour to healthy tissue acts as an infecting agent. In 1888 Wehr repeated Novinski's transplantation experiments in dogs with similar results (Shimkin 1955). It is interesting to note that the dogs used for transplantation of CTVT did not come from a single breed and were therefore not highly inbred. The allo-transplantation of tumours seemed less surprising in the late 19th Century than it does today with our modern knowledge of histo-incompatibility.
The successful results obtained with CTVT served as model for tumour transmission in other animals. Hanau in 1898 inoculated two rats with vulvar epidermoid carcinoma and observed growth of the tumour in the recipients (Shimkin 1955). In 1901 Leo Loeb supported the transplant ability of tumours in rats (Witkowski 1983; Brent 1997). In 1903 a Danish veterinarian Carl O. Jensen determined the successful growth of transplanted tumours in mice by heredity (Brent 1997). The discovery that the tumour could be successfully transplanted into (Witkowski 1983; Brent 1997) other mice, led the scientists to use rodent system to supply tumours for experiments. The observation that a single tumour could be expanded through many generations exceeding the life span of the laboratory mouse led Leo Loeb to the "cancer immortality" concept (Witkowski 1983).
The earliest observations reported by John Hill in 1759 and by Percival Pott in 1775 on the association of a specific tumour to a specific profession or work, led to the idea that some chemicals can cause cancer (Greaves 2000). In 1918 Yamagiwa and Ichikawa induced cancer by applying coal tar to rabbit skin(Greaves 2000; Luch 2005). After the discovery of the X rays by Wilhelm Conrad Roentgen in 1895, Frieben published data in 1902 indicating that cancer rates were increased among persons working with X-rays (Cassileth 1983; Greaves 2000)
1.2 Tumour Progression
The first detailed characterization of the dynamic nature of cancer was described by Leslie Foulds (Foulds 1949). Foulds showed that tumours progress (evolve) through different stages, characterized by the acquisition of different phenotypic traits such as increased growth rate, hormone dependence, invasiveness, formation of metastasis (Foulds 1949; Fould 1954; Foulds 1957). With the progress of molecular biology the phenotypic view had been replaced with the somatic mutation theory, where cancer evolved through the accumulation of different mutations in several genes (Greaves 2000). The accumulation of mutations in somatic cells implicated the presence of different cells bearing different mutations and also the presence of natural selection, which selected the cells with advantageous mutations. One of the questions arising from the somatic mutation theory was whether a tumour had a single or a multiple origin.
This observation was supported by a karyotype analysis in chronic myelogenous leukaemia (CML) by Peter Nowell and David Hungerford in 1960 (Nowell 2002). They described the presence of an unusually short chromosome 22 in all CML tumour cells analyzed, and the absence in the normal cells from the same patients. This observation suggested that this mutation was a somatic mutation that occurred in one cell in the bone marrow, which gave it a selective advantage to expand as a clone. Nowell postulated that a tumour develops by a Darwinian evolutionary process, where cells with mutations conferring a growth advantage are selected and expanded (Nowell 1976; Greaves 2002). In 1954 Peter Armitage and Richard Doll analyzed human cancer incidence over the age, and showed that chances of cancer increased in older people (Armitage and Doll 1954).
The concept that cancer might be contagious also recurs throughout the past 300 years.In the 17th and 18th centuries, physicians Daniel Sennert and Zacutus Lusitanus supported the hypothesis that cancer was contagious. In fact in 1779 a hospital in Paris was directed to move the cancer patients from the city (Cassileth 1983; Kenneth 2003).
1.2.1 Exogenous and endogenous factors
In 1844 the Italian physician Domenico Antonio Rigoni-Stern noted that cancer of the cervix was frequent among married ladies, rare among unmarried ladies and absent in Italians nuns. In contrast, breast cancer was more frequent among nuns (Greaves 2000). These observations led to the hypothesis that cervical cancer was sexually transmitted, and we now know that the cause is a papilloma virus (Hausen 2002).In 1908 Wilhelm E and Olaf B, transferred the leukemia in chicken by tissue filterates (Wyke 2003). In 1911, Peyton Rous demonstrated that viruses were the cause of solid tumours (Sarcoma) in chickens but it took many decades before his data were accepted (Dulbecco 1976). The notion that viruses can cause cancer was a discovery that brought back the fear that cancer was a contagious disease.
There are many exogenous and endogenous risk factors that affect the tumor suppressor genes and oncogenes (Todorova 2006). Tumour viruses (Bishop 1980), chemical carcinogens (Loeb et al. 2000), natural chemicals, (Ames et al. 1990), herbicides (Glickman et al. 2004), physical carcinogens like radiation (Upton 1978) are exogenous factors while inherited genetic defects, immune system (Rosenthal 1998) and hormonal factors (Rodney 2001) are among endogenous risk factors.
Although tumour cells are generally described as independent evolving units, recent results suggest that tumour cells are able to stimulate stromal cells to produce growth factors that increase tumour proliferation (heterotypic stimulation) (Kinzler and Vogestein 1998; Skibe and Fuseing 1998; Iyengar et al. 2003). It has been demonstrated that cells involved in the immune response to tumours may produce factors such as inflammatory chemokines that may also promote the tumour proliferation (Pollard 2004; Wyckoff et al. 2004)
1.2.2 Two hit hypothesis
Retinoblastoma is a tumour that becomes manifested early in life. Retinoblastoma can be inherited or sporadic. According to the two hit hypothesis in the inherited form a single mutation in the Retinoblastoma (Rb) gene is present in the germ line which gives the genetic predisposition to develop cancer, but a second mutation in the normal Rb allele which occurs in the retinoblast must be acquired to develop cancer (Knudson 2001). In the sporadic form the two mutations in the Rb alleles occur in the somatic cells. Although the epidemiological and molecular observations have consolidated the multistage theory of cancer, the number of mutations and in which sequential order they have to be acquired to develop cancer is still an open question (Hanahan and Weinberg 2001; Hahn and Weinberg 2002b).
1.2.3 Oncogenes
Early experiments involving transforming retroviruses and the transfer of genes from tumour cells into established rodent cells allowed the identification of several cancer causing genes called oncogenes. The result of these experiments suggested that cancer could be induced by the mutation of one proto-oncogene. However, the rodent cells used as recipient in the gene transfer experiments were not normal, but were immortalized, thus acquiring the ability to proliferate indefinitely. When the normal rodent cells were used, the transfer of a single oncogene failed to induce transformation, while the transfer of two oncogenes resulted in transformation. Human cells require more mutations than rodent cells and that there are differences also between cell types within the same species (Rangarajan et al. 2004).
1.3 Cancer Hallmarks
Despite the enormous variety of tumours affecting different types of tissues in animals and humans, research over the past 50 years has revealed that all malignant cancers share the same essential alterations (Hanahan D and Weinberg RA 2000).
These hallmarks include:
Immortalization
Evasion from programmed cell death (apoptosis)
Independence from growth stimulation
Resistance to growth inhibition
Angiogenesis
Invasion and metastasis
Genetic instability.
These hallmarks are briefly described below.
1.3.1 Immortalization
Telomeres contain DNA sequence repeats and protein. The repeat sequence consists of hexameric motifs such as GGGTTA in humans, extended for 10 –20 kilobases. The 3’ end has a 100-400 nucleotide over-hang (Mathon and LIoyd 2001). Telomeric DNA is generated by an enzyme called Telomerase Reverse Transcriptase (TERT) which has two subunits, RNA and catalytic protein subunit. This RNA binds the telomeres DNA ends thus acting as template for telomere elongation. The chromosome ends are protected by several proteins: TRF-1, TRF-2, and POT–1 (Mathon and LIoyd 2001; Hahn and Weinberg 2002a). Several experiments have shown that senescence is activated when the telomeres are shortened down to 5 kb and that senescence is triggered by the shortest telomere present in the cell (Hemann et al. 2001).
Many reports have suggested that the replicative senescence is not activated by the erosion of the double strand repetitive sequence, but by the degradation of the 3’end single strand overhang, resulting in loss of protective capping (Stewart et al. 2003). Telomere length is maintained by the activation of telomerase or by an alternative mechanism called alternative lengthening of telomeres (ALT), where the telomeres are regenerated through recombination-based inter chromosomal exchange of sequence information (Bryan et al.1997; Dunham et al. 2000). In the normal cell telomerase is transiently expressed, since it can be detected only in S phase, but in neoplastic cells its expression is increased and is detectable throughout the cell cycle (Mathon and Lloyd 2001). In tumour cells the senescence and crisis barriers are avoided by the activation of telomerase regenerating the telomeres and by the inactivation of tumour suppressor and pro-apoptotic genes (Hanahan and Weinberg 2000; Hahn and Weinberg 2000b).
1.3.2 Apoptosis.
The sensors detect the intra- and extra-cellular signals. The intracellular signals include DNA damage, hypoxia and oncogene overexpression (Evan and Littlewood 1998). The extracellular signals monitor the cell-cell and cell-matrix homeostasis (Aoshiba et al. 1997; Prince et al. 2002; Alberts et al. 2002a). The signals detected by the sensor are mainly conveyed to the mitochondria, where a series of cytoplasmatic proteins of the Bcl2 family control the release of cytochrome C from the mitochondria (Alberts et al. 2002a). The release of cytochrome C activates an array of intracellular proteases called caspases causing protein and DNA degradation (Hanahan and Weinberg 2000). The caspases can be directly activated by extracellular proteins such as FAS ligand, which binds to the death receptor FAS (Houston and O’ Connell 2004).
Once the caspase cascade is triggered it cannot be inactivated (Alberts et al. 2002a). It has been reported that the tumour suppressor p53 can trigger the caspase cascade by the overexpression of the Bax protein, a member of the Bcl2 family, which in turn increases cytochrome C release thus inducing apoptosis (Hanahan and Weinberg 2000). In CTVT it is likely that expression of c-myc is up-regulated, due to insertion of a LINE-1 element as discussed later. Ectopic c-mycexpression can promote tumour growth and survival, as seen, for instance, in immunoglobulin gene c-myc chromosome rearrangements in Burkitt's lymphoma (Hemann et al. 2005).
1.3.3. Independence from growth stimulation
1.3.3.1. Growth factors
Thus the proliferation of a cell is dictated by the needs of the cells around it (Hanahan and Weinberg 2000). In contrast, a tumour cell escapes from the external dependence to become an autonomous evolving unit, by producing its own growth signals.
1.3.3.2 Growth factor receptors
Another mechanism selected by tumour cells is the overexpressions of growth factor receptors, which induce the tumour cells to become sensitive to concentrations of growth factor that normally, do not trigger proliferation (Hanahan and Weinberg 2000). Proliferation can also be induced by a mechanism independent of the growth factor, for example the alteration of the cytoplasmic tail of growth factor receptor causes self-activation of the receptor, which therefore becomes independent from the external microenvironment (Alberts et al 2002b).
1.3.4 Resistance of growth inhibition
Like growth signals, the anti-proliferative signals derive from soluble factors or surface proteins that are produced by neighbouring cells, or are induced by components of the extracellular matrix (Hanahan and Weinberg 2000; Alberts et al. 2002d). These external inhibitory signals activate different intracellular pathways that regulate the cell cycle (Alberts et al. 2002c).
The Rb protein and its related proteins, p107 and p130 play a key role in controlling this transition (Weinberg 1995). The association of Rb with the transcription factor E2F inhibits the transcription of genes involved in the G1-S progression (Alberts et al. 2002c). The hyper-phosphorylation of the Rb protein induces the dissociation with E2F, therefore allowing progression to S phase (Alberts et al. 2002c). Normally complexes of cyclin and cyclin dependent kinase (CDK) induce the phosphorylation of the Rb protein (Alberts et al. 2002c). Many tumours can avoid the antigrowth signals by altering Rb activity or the proteins involved in Rb phosphorylation (Mittnacht 2005).
1.3.5 Angiogenesis
Although the majority of the new vessels in adult tissues are derived by sprouting from existing vessels, many evidences indicate that progenitor endothelial cells are derived from the bone morrow contributing to the vessel growth (Zhang et al. 2000; Contreras et al. 2003; Nishimura and Asahara 2005; Religa et al. 2005). Although endothelial cells are highly proliferative in response to several angiogenic factors, they have long half-lives up to several years (Carmeliet 2003). In order to adapt the vascular system to the tissue's requirements, several mechanisms regulate the process of angiogenesis (Carmeliet 2003). A key molecule involved in the angiogenesis process is the vascular endothelial growth factor (VEGF) (Carmeliet 2003). In addition it has been demonstrated that tumours can activate or inactivate pro- and anti-angiogenic factors respectively present in the extracellular matrix by producing several proteases (Gately et al. 1997; Harlozinska 2005).
1.3.6 Metastasis
In cancer during tumour progression, some tumour cells acquire the ability to migrate and form new colonies at secondary sites and these cells then make new tumour cells (Hanahan and Weinberg 2000). It has been estimated that 90 % of mortality associated with cancer is due to metastasis (Sporn 1996). Results show that few cells in the primary tumour acquire the ability to grow in the secondary sites and that the tendency to metastasise is acquired in the early steps of tumour progression (Van’t Veer and Weigelt 2003).
Progressive alteration of normal tissue homeostasis by tumour and stromal cells, allow tumour cells to move throughout degraded matrix, and to invade surrounding tissues (Hanahan and Weinberg 2000). Tumour cells are also aided to migrate by soluble factors (chemotaxis) and bound adhesion molecules (haptotaxis) (Nguyen 2004). In order to invade new organs, circulating tumour cells need to stop and exit the systemic circulation. In an unspecific manner, the extravasation may be due to the fact that large arteries progressively narrow in to arterioles and then capillaries and tumour cells can be trapped in this small vessel, thus allowing the migration in the new organ (Nguyen 2004). Although the exact mechanism behind the tumour homing is not completely understood, recent results suggest that the selective homing of cancer cells may be due to three mechanisms: 1) presence in the target tissue of specific growth factors or appropriate extra-cellular matrix that favour the selective tumour growth, 2) presence in the target organ vessel endothelium of specific adhesive proteins that interact with the tumour cells, favouring the tumour invasion, 3) production of a chemotaxis soluble factor by the target tissue that attract the tumour cells ( Fidler 2003).
1.3.7 Genetic instability
Over the past 25 years numerous genetic alterations have been described in human and animal tumours. These genetic alterations can affect the DNA sequence and the chromosomes (Lengauer et al. 1998). The mutations of DNA include: substitution, deletion, translocation and insertion and they can affect one or more nucleotides. The necessity to transmit genetic information faithfully between generations demands genetic stability (Eisen and Hanawalt 1999)
In normal conditions the genome is affected by spontaneous mutations caused by physiological DNA instability and by imprecision of the DNA polymerase proofreading activity during the DNA replication (Alberts et al. 2002e). In eukaryotic cells, several enzymes have been described with DNA polymerization activity, and five are the most important DNA polymerases involved in DNA replication and repair, alpha, beta, gamma delta and epsilon. To date the only polymerase involved in mitochondrial DNA replication is polymerase gamma. In vitro studies on the fidelity of DNA duplication has shown that the nucleotide mis incorporation rate varies among polymerases, with one in 5000 bases for beta and one in 10 000 000 for delta and epsilon polymerases (Umar and Kunkel 1996; Loeb and Loab 2000). To avoid non-complementary nucleotide incorporation, polymerase delta, gamma and epsilon contain a proofreading activity (Kunkel and Alexander 1986). Normally DNA replication is carried out by delta polymerase, but recent reports show that in some tumours this priority is shifted in favour of less accurate polymerases, thus increasing the mutation rate (Loeb and Loeb 2000). Environmental agents such as ultraviolet light, ionizing radiations and toxic substances in the dietary uptake can induce mutations (Loeb and Loeb 2000).
1.3.7a Single Base Excision Repair
When a mutation effects on a single nucleotide then base excision repair take place. BER employs enzymes called DNA glycosylases, which are specific in removing a specific mutated base (Krokan et al 2000).
1.3.7b Nucleotide excision repair
The nucleotide excision repair (NER) system is able to repair DNA damage induced by UV. In contrast to BER, the NER system recognizes altered nucleotides by scanning the DNA for a conformational alteration (bulky lesion) (Wood 1996).
1.3.7c Mismatch repair
The mismatch repair (MMR) pathway includes a series of proteins that are involved in correcting errors that escape the DNA polymerase proofreading activity during DNA replication. They are also involved in suppressing recombination between non-identical sequences both in mitosis and meiosis (Kolodner and Marsischky 1999). Unlike BER and NER, MMR does not act on damaged or mutated sequences, but it targets only the newly synthesized DNA strand.
Inactivation of the MMR system produces microsatellite instability (MSI) (Atkin 2001).
1.3.7d. Homologous recombination
Homologous recombination repairs double strand breaks by using an intact and homologous DNA molecule as a template. In eukaryotes several proteins are involved in the homologous recombination process (Kanaar et al. 1998; Haber 2000).
1.3.7e. Non-Homologous End Joining
Non-Homologous End Joining (NHEJ) is the more important repairing mechanism when there is break in DNA double strand and it is very important mechanism in mammals (Khanna and Jackson 2001). During the NHEJ process small deletions are generated. Given that majority of the mammalians genome is composed of non-coding regions, the probability that in normal situations the NHEJ process induces mutation in genes is low (Alberts et al 2002e). However, if there are multiple break points NHEJ increases the occurrence of illegitimate recombination
(Rothkamm et al 2001).
1.3.7f Chromosome Instability (CIN)
The cell reproduces by a series of events that allow DNA replication and cell division in a process known as the cell cycle. In order to check the correct order of events that take place in the cell cycle, a complex cell-cycle control system has evolved (Alberts et al 2002c). This system checks normal cell cycle progression by a series of stage-specific sensors known as checkpoints that are able to induce the arrest of the uncompleted stage until it is completed.
The two fundamental processes in the cell cycle are the duplication and the division of the chromosomes, which take place during the Synthesis (S) and Mitosis (M) phase respectively. To prevent the possibility that two daughter cells have non-identical genomes, there are two checkpoints known as DNA replication and DNA damage checkpoints before mitosis, and one known as spindle-attachment checkpoint during mitosis (Alberts et al 2002c).
Chromosome instability (CIN) is also associated with structural alteration of chromosomes, which include reciprocal and non-reciprocal translocations, amplifications, deletions and insertions (Cairns 2005). Structural chromosome instability, resulting from DNA breaks and rearrangements, is due to alteration of cell cycle checkpoints, DNA damage response and telomere integrity (Gollin 2005). Structural alterations may results in altered gene expression or produce fusion or chimeric proteins with dysregulated or new properties (Greaves and Wiemels 2003). Studies have shown that a large proportion of human tumours with chromosome instability have a high rate of loss of heterozygosity (Rajagopalan and Lengauer 2004). Therefore it has been argued that chromosome instability could accelerate the rate of inactivation or activation of tumour suppressor genes or oncogenes respectively (Rajagopalan and engauer 2004).
CIN-associated genes can be classified on the basis of the mutations (Michor et al. 2005). Class I genes of CIN, e.g Mitotic Arrest Deficient gene (MAD-2 ) boost up CIN in case one allele is mutated or deleted. Class II genes of CIN e.g. Human Budding Uninhibited by Benzimidazoles (hBUB-1) gene boost up CIN if mutation is in one allele in a dominant negative fashion. Both Class I and Class II genes are required at the spindle assembly checkpoint (Amon 1999; Hoyt 2001). Class III genes of CIN e.g. Breast cancer gene BRCA1 and another Breast cancer gene BRCA2 boost up CIN if both alleles are mutated. BRCA genes have very important role at checkpoint and it is involved in DNA repairing and recombination (Yarden et al. 2002).
1.4 Evolutionary Dynamics of Tumour Development
According to clonal evolution theory, cancer is the result of somatic mutations selected during tumour evolution (Nowell 1976). It has been argued that tumour cells cannot acquire the mutations needed for tumour progression at a physiological mutation rate, but that the tumour cell must acquire an increased mutation rate (Cairns 1998; Loeb and Loeb 2000).
In order to induce cancer the mutations must affect a variety of genes that restrain somatic conflict (Frank and Nowak 2004). These genes are known as cancer related genes and can be subdivided in three categories: Gatekeeper, Caretaker, and Landscaper (Michor et al 2004). Gatekeeper mutations increase the cellular proliferation rate by the alteration of oncogenes, tumour suppressor genes and apoptotic genes (Michor et al 2004). Caretaker mutations increase genome instability by inactivating genes involved in maintaining genome integrity (Lengauer et al 1998). Landscaper mutations increase tumour proliferation by affecting genes involved in regulating the external cellular microenvironment (Bissel and Radisky 2001).
While mutations affecting oncogenes behave in a dominant way, because only one mutated allele can induce a tumour phenotype, mutations affecting tumour suppressor genes can be neutral if the normal allele compensates the mutant allele, disadvantageous if the mutant allele triggers apoptosis, and advantageous if the mutated allele is inactivated and the second allele is insufficient to balance the wild type allele (Michor et al. 2004). In small compartments the inactivation of the two alleles of a tumour suppressor gene, is unlikely, unless the mutation rate is increased by genetic instability (Nowak et al. 2005). Loss of heterozygosity increases with chromosome instability (Michor et al. 2004).
1.5 Tumours of Feline and Canine included in this study
1.5.1Mammary Tumours
Mammary gland tumours are most frequent in dogs (Moulton 1990) while in cats it is third in prevalence, after haemopoietic and skin tumours (Misdorp et al. 1999). The average age of peak prevalence of tumours in cats is approximately 9.3 years (Roccabianca et al. 2006). Mammary tumours can also affect male cats and dogs, with the average age for them being 12.8 years (Rutterman et al. 2000). Siamese has twice the risk in comparison to other breeds of cat (Weijer et al. 1972). Same predisposition was observed with our data, that all five cases collected in this study were belonging to Siamese breed. Mammary tumours are more prevalent in Pakistan and all the cats and dogs were between 5-11 years old. This suggests that there are more chances of mammary tumours in older cats and dogs. Mammary tumours included in this study were 23% all 22 tumours studied.
1.5.2 Canine Transmissible Venereal Tumours (CTVT)
Canine transmissible venereal tumours first reported by Blaine in 1810 (Blaine, 1810) is a transmissible cancer in dogs. Studies found that CTVT was transmitted by transplantation of living cells (Novinski 1876), confirming it as a transmissible cancer. CTVT is of clonal origin, originating from a founder dog 11,000 years ago (Katzir et al. 1985; Murgia et al. 2006; Rebbeck et al. 2009; Murchison et al. 2014). It is one of only two transmissible cancers known (Murchison 2008) and is spread by allogeneic transfer of cells between dogs, usually during coitus. It manifests as a tumour, associated with the external genitalia of both male and female dogs, although tumours can also arise in the mouth, nose or skin. It is purported to be of histiocytic origin (Mozos et al. 1996; Mukaratirwa and Gruys 2003), and usually remains rather localised, except for rare cases of metastatic spread.
Recorded cases of metastasis include involvement of the lymph nodes (Higgins 1966), skin (Dass 1986) and eye (Barron et al. 1963), among others. Experimental transplants of CTVT tumours into subcutaneous sites in experimental dogs are characterized by progressive and regressive phases. This is seen as a rapid volume increase, followed by tumour shrinkage, and eventually complete regression accompanied by serum-transferable immunity to reinfection (DeMonbreun 1934). In this project we collected 6 samples for BRCA1 and COX-2 studies in different tumours while 16 more samples for Department of Veterinary Medicine, University of Cambridge, UK. Although the prevalent rate of CTVT is in second number, many attentions were paid to collect CTVTs. For BRCA1 and COX-2 studies the 28% were CTVT out of 22 different tumours.
1.5.3 Perianal adenomas/Adenocarcinomas
There are many glands present around the anus of dogs. These are sebaceous and non-secretary glands, while anal sac glands are positioned at 4 and 8 o clock to the anus and secrete their secretions into the lumen of theanal track (Yang Hai-Jie et al. 2008). Perianal adenomas are more frequent than adenocarcinomas (malignant form). In this study 3 tumours were collected which were 14% of total canine tumours collected. These tumours are mostly common in medium to older age dogs.
1.5.4 Granuloma
Granuloma is also called as lick granuloma in dogs it is a type of skin cancer It typically results from the dog’s urge to lick the lower portion of one of her or his legs. This study reported 9% of total tumours included in this study.
1.5.5 Oral Tumours (Squamous cell Carcinoma)
Oral tumours are 4th common cancers in canines. Male dogs have 2.4 times greater risk of developing oral tumours than female dogs (Dorn et al. 1968). This study reported 9% oral tumours in a period of 2 years.
1.5.6 Lymphoma
Lymphoma is the second most prevalent intra –ocular tumours of dogs. Basic cause of lymphoma in dogs is unknown but genetic (chromosomal segregation), environmental and infectious factors such as retroviruses play vital role in developments of this cancer (Fighera et al. 2002). This study reported 9% Lymphomas of total collected tumours.
1.6 Rationale behind selection of genes
1.6.1 BRCA1 gene
BRCA1 gene is tumour suppressor gene, it is involved in repairing the DNA double strands breaks and in case of failure it leads the cells towards apoptosis (Starita. 2003). BRCA1 forms BRCA1 Genome Surveillance Complex (BASC) when it combines with different types of tumour suppressor genes, DNA damage sensors and signal transducers (Wang et al. 2000). It is involved in Ubiqutination, transcription regulation (Friedenson 2007; Friedenson 2008). In humans BRCA1 was first identified at chromosome 17 (Hall et al. 1990) and it was isolated in 1994 (Miki et al. 1994). It is present at 17q21 with a length of 100 Kb. In canine it is located on chromosome 9. BRCA1 has 22 exons in canines and felines; it encodes a protein of 1882 amino acids in canine and 1871 amino acids in feline. Many scientists from different research showed that women who have famililal mutations in the BRCA1 or BRCA2 (BRCA1/2) genes have increased risk of breast cancer (Struewing et al. 1997).
Fig 1: BRCA1 mechanism in DNA repairing. http://www.publichealthunited.org/leading-by-example-angelina-jolie-and-the-brca1-gene-mutation/
1.6.2 Cyclooxygenase-2 Enzyme (Prostaglandins, COX-2).
Cyclooxygenase-2 enzyme (Cox-2) is also called as Prostaglandins Endoperoxide synthase (PTGS). It is involved in the synthesis of prostaglandins which act as biological mediators in many body functions. It was first isolated from prostate gland that’s why it is called as Prostanglandin. Cyclooxygenase enzymes have two types, cyclooxygenas-1 and cyclooxygenase-2. Cyclooxygenase-1 is constitutively produced in the cell while cyclooxygenas-2 is inducible and it is constitutively produced only in kidneys, seminal vesicles and central nervous system. Its high expression has been recorded in many different types of tumours, it has been involved in anti-apoptosis, cell proliferation, tumour angiogenesis, cell invasion and immune suppression activities. In canine COX-2 is present on chromosome 7 having 604 amino acids and 10 axons. This correlation of cyclooxygenase-2 in cancer development suggests using new therapeutics against it. Studies have shown cycoloxygenase-2 high expression in number of different tumours (León-A 2008), such as intestinal, pancreatic, ovarian, prostatic, nasal cavity, oral cavity and mammary tumours of dogs (McEntee et al. 2002; Mohammed et al. 2004; Borzacchiello et al. 2007; Eplattenier et al. 2007; Mullins et al. 2004; Pireset al. 2010; Dore et al. 2003).
Fig 2:COX-2 mechanism of actionhttps://www.google.com.pk/search?q=cox+2+mechanism+of+action.
1.6.3 DLADQA1 (MHCII gene) (Additional work performed at Department of Veterinary Medicine, University of Cambridge, UK).
The Major Histocompatibility Complex (MHC) is a cell-surface protein mediating immune recognition through its interactions with T cells (Fig 3). There are three classes of MHC molecules in mammals - the classical MHC-I and II, and non-classical MHC-III Table 1). MHC-I interacts with CD8+ cytotoxic T cells, whilst MHC-II binds to CD4+ helper T cells. MHC molecules mediate antigen presentation to T cells. MHC-I typically presents self- peptides, whilst MHC-II presents foreign peptides. MHC molecules are extremely variable and polymorphic across the population, with a huge number of alleles at each MHC locus.
This allows MHC molecules themselves to behave as antigens in transplant rejection, with the graft MHC peptide recognized as non-self by the recipient, and thus rejected. It would be expected that CTVT, an allergenic graft, should be rejected for two reasons: host MHC will present tumour antigens as foreign non-self to the host immune system, and tumour MHC will present a mismatch to the host immune system as a foreign antigen itself (Fig 3). This project focuses on the DLADQA1 locus (Wagner et al. 2002), a classical MHC-II gene on dog chromosome 12. There is high level of MHC allelic variability in any population (Niskanen et al. 2013).
Fig 3:MHC is involved in graft rejection.
This rejection (represented by the red arrow) occurs according to two principles. Firstly, host T cells may recognize the host MHC presenting a foreign peptide that should activate an immune response. Secondly, host T cells would also be able to recognize the tumour MHC presenting any peptide as foreign, since it is not self-MHC. It is thus surprising that CTVT is able to persist as an allogeneic graft. MHC expression was previously characterised molecularly by Murgia et al through RT-PCR of a MHC-I (DLA88) and MHC-II (DLADRB1) gene (Murgia et al. 2006). They found that there was downregulation of expression of both these MHC genes. DLA88 showed low levels of tumour-specific expression, whilst there was no detectable tumour expression of DLADRB1. Murgia et al. also performed MHC genotyping for a number of CTVT samples and confirmed that all CTVTs shared the same haplotype (Murgia et al. 2006). They identified two clusters at the DLADQA1 locus, with some CTVTs appearing to be haploid the locus, whilst others remained diploid. This is in contrast to evidence that suggests the DLADQA1 locus had undergone a copy-neutral loss of heterozygosity (LOH) (Murchison et al. 2014).
1.6.4 Technologies used in this research work.
Different technologies are being used in cancer research such as PCR, flow cytometry, immunohistochemistry (IHC), in- situ hybridization (FISH, CSH) and microarray for diagnosis (Pawanet al. 2010). Here, I used Real time PCR for gene expressional analysis of BRCA1 and COX-2 and DLADQA1 (MHCII). Histopathology (Hematoxyline and Eosin staining) was performed for the diagnosis of tumours. CTVT diagnostics qPCR was also performed to measure the allele’s quantity of LINE-myc gene and CDKN2A gene. Conventional PCR measures at End-Point, while Real-Time PCR collects data during the PCR shows the data and quality of data during exponential growth phase also it has increase dynamic range of detection, it is very sensitive and no need for post PCR processing. Immunohistochemistry was performed to find out the expression of MHCII antigens in CTVTs. The serum protein electrophoresis and serum biochemistry was also measured. Western blotting was performed to detect antibodies in CTVTs (protein expression). It is a very good technique to measure the gene expression at protein level in fluidic material of cells. We performed capillary electrophoresis to find the mutations/SNPs in our genes of interest (BRCA1, COX-2 and DLADQA1). Genetic analyzer was used to find the sequence variations in our genes of interests. Other methods used for sequence variation studies, like SSCP, DGCG and HPLC miss the mutations (Rassi 2009). So the sequencing by capillary electrophoresis was the best option for this study.
Availability: Items available for loan: UVAS Library [Call number: 2250-T] (1).
5.
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: ; 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).
6.
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: ; 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).
7.
Development Of Dna Based Diagnosis Of Babesia Canis In Dogs And Its Specificity With Peripheral Blood Smear Microscopy
by Muhammad Awais (2009-VA-539) | Dr. WasimShehzad | Dr. M. YasirZahoor | Dr. M. Imran Rashid.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Babesia canis (B. canis) is an intra-erythrocytic parasite and it is responsible for canine babesiosis. Mainly ticks are responsible for its transmission to vertebrate host. Currently, there are three sub-species of B. canis has been identified i.e. B. caniscanis(B. c. canis), B. canis vogeli (B. c. vogeli)and B. canis rossi (B. c. rossi).Clinical presentation and severity of infection are markedly different for each sub-specie. Routinely used methods for the diagnosis of canine babesiosis include clinical presentation, host/vector specificity, blood smear microscopy and Immunological testing (IFAT and ELISA).
In this study, blood smear microscopy and molecular based technique, PCR, were evaluated. This study has comparative as well as developmental nature.Peripheral blood smear microscopy is cost effective and quick method of diagnosis. But the limitations associated with microscopy include low parasitaemia in chronic and asymptomatic infection, morphological similarity of B. canis with other species of Plasmodium and Theileria. Furthermore, personal expertise in morphology based identification and slide preparation may also hamper the precise identification. These limitations may lead to misdiagnose the infection.
PCR based method, developed in this study, found to be more specific and sensitive than conventional microscopy. Fifty blood samples were collected from September, 2014 to November, 2014. These samples were screened microscopically as well as with PCR. B. canis-like bodies were identified in forty-two (42) samples. But all (50) samples were found positive for B. canis¬via PCR. Sequencing results following PCR have shown that identified sub-specie is similar withB. c. vogeli(99%).
This study clearly depict the likelihood of misdiagnosis with microscopy due to morphological similarities with other piroplasms. Therefore, PCR based methodology was found highly specific for B. canis and produced high throughput. Furthermore, sub-species differentiation is only possible with molecular based approach. As the described approach is found highly specific and sensitive than other conventional methods, therefore, it may aid in the development of clear canine babesiosis geographical distributional picture. Which ultimately help in the development of control and treatment strategies which may eradicate babesiosis.
Availability: Items available for loan: UVAS Library [Call number: 2287-T] (1).
8.
Seroprevalence And Molecular Detection Of Brucellosis In Hospitalized Patients With Clinical Manifestations Of Brucellosis
by Riffat Yousaf (2008-VA-342) | Dr. Wasim Shehzad | Prof. Dr.Tahir Yaqub | Dr. Haroon Akbar.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Brucella species are host specific facultative intracellular pathogens which cause brucellosis in both animals and humans. Brucellosis is one of the most common zoonotic diseases worldwide. In Pakistan, the incidence of brucellosis is increasing day by day due to lack of awareness of this deadly malady. It is transmitted from infected animals to humans who are in close contact with infected vaginal secretions, feces, blood, aborted fetus, or by consumption of unpasteurized milk and dairy products. Infection due to B. melitensis and B. abortus are mostly prevalent for brucellosis in human.
Total 218 blood samples were collected in gel vacutainer tubes from hospitalized patients who were clinically manifested with brucellosis. Out of 218, 12 RBPT positive blood samples were collected in EDTA containing vacutainer tubes separately. Serum was isolated from all blood samples (without EDTA). These serum samples were first screened by Rose Bengal Plate Test (RBPT). DNA was extracted from all positive RBPT blood and serum samples and randomly selected negative RBPT serum samples. All extracted DNA (≤10ng/µL) were subjected to Brucella genus and two species specific (B. abortus and B. melitensis) Quantitative Real Time Polymerase Chain Reaction (qRT-PCR) assay. Furthermore, few selected extracted DNA (≥20ng/µL) from blood and serum samples were examined by genus and Multiplex specie specific PCR. The PCR products were electrophoresed on 2.5% agarose gel. Then selected products were sequenced by ABI 3130 XL sequencer. The data were analyzed by SPSS software using Chi square test.
The present study helped to diagnose accurately and precisely brucellosis in clinical manifested patients, which is further helpful for devising the strategies to control this disease.
Availability: Items available for loan: UVAS Library [Call number: 2286-T] (1).
9.
Genetic Characterization Of Pakistani Wild Quails Using Mitochondrial Coi Gene
by Wajiha Shakil (2012-VA-817) | Dr. Ali Raza Awan | Dr. Muhammad Yasir Zahoor | Prof. Dr. Tahir Yaqub.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: A latest taxonomic tool termed as DNA barcoding is being used to genetically characterize animals. DNA barcoding is helpful in determining evolutionary relationship among species. Being a small sized genome and agile enough to show rapid mutation, mtDNA has been used as a pertinent marker of molecular biodiversity. DNA barcode works as an efficient tool in food manufacturing industry, diet investigation, forensics analysis, preventing unlawful trade and felonious poaching. The aim of this study was to develop DNA barcode for genetic characterization of Pakistani wild quail. Pakistani wild quail is important due to its demand for eggs, meat production, experimental purposes and gaming as well. Japanese quail was also included in this study because this quail is excessively produced in Pakistani farms. Japanese quail is present throughout the year and is comparatively bigger in size than wild quail. It has longer lifespan; farmers can easily breed this species in farms. It is suitable in poultry due to better meat yield.
COI gene (500bp) was used as a molecular marker for identification at species level. DNA was extracted from blood samples of ten wild quails (Coturnix coturnix and fifteen japanese quails (Coturnix japonica). Reported bird universal primers were used to amplify COI region from the extracted mtDNA samples using PCR. Amplicon were then sequenced by Sanger sequencing method (Sanger et al. 1977). Forward and reverse DNA Sequences were aligned with the reference sequence using nucleotide BLAST on NCBI to observe the dissimilarity among the sequences. Consensus sequences generated were used to construct their phylogenetic tree to see their evolutionary relationship with other bird species. Japanese quail which is thought to be domesticated from Japan, its Pakistani population showed close relation with sequences
Summary
90
generated in Japan for this particular species. Pakistani wild quail species showed its closest linkage with C. coturnix.
In conclusion, COI barcode proved as an authentic tool for species identification and phylogenetic inference of Pakistani wild and farm grown quails. Wild quail species has been characterized using partial COI gene sequences. This study has provided a specific genetic marker which can differentiate Japanese quail from wild quail at molecular level as most of the time both species are confused with each other. It can be helpful to the farmers and bird fanciers because they can select the birds of their choice correctly. This is the first study reporting DNA barcode of this Pakistani quail species. It would help researchers to study about phylogenetic and taxonomic status and also assist quail fanciers and quail farmers to unaffectedly identify their species of interest in farming. Identification of quail species is also important for conservation of biodiversity as it helps in preservation and identification of endangered species by generating their barcodes from even minimal evidence available. Availability: Items available for loan: UVAS Library [Call number: 2311-T] (1).
10.
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: ; 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).
11.
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: ; 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).
12.
Polymorphism Of The Slc11a1 Gene Associated With Resistance To Bovine Tuberculosis.
by Qamar Raza Qadri (2009-VA-569) | Dr. Asif Nadeem | Dr. Tahir Yaqoob | Dr. Abu Saeed Hashmi.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Bovine Tuberculosis (bTB), caused by Mycobacterium Tuberculosis is a health threat to
livestock. Information on genetic resistance or susceptibility because of polymorphisms of
candidate genes could be used in making selection decisions. Solute carrier family 11 (protoncoupled
divalent metal ion transporters), member 1 gene (SLC11A1), is a known candidate gene
which is associated with natural resistance to infection by Mycobaterium spp in buffalo.
Polymorphism in this gene can be studied for breeding disease resistance animals. Blood samples
were collected from Nili Ravi buffalo breed of Pakistan. DNA was extracted by organic method.
Primers were designed using Primer3 software. Amplification of gene was done by Polymerase
Chain Reaction. PCR products were sequenced bi-directionally on ABI 3130XL Genetic
analyzer. Sequence alignment was performed for polymorphism identification. The analysis of
identified polymorphism has been done by CHROMAS software. Sequences were aligned by
BLAST tool of NCBI. The results of analysis showed that no polymorphisms were identified in
exonic region of gene. This might be due to less sample size. Genetics play important role in
fighting against pathogens. Identifying the genes involved can lead to marker-assisted selection
strategies. Availability: Items available for loan: UVAS Library [Call number: 2332-T] (1).
13.
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: ; 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).
14.
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: ; 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).
15.
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: ; 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).
16.
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: ; 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).
17.
Mutational Analysis Of Hepatitis C Virus Ns4b Gene Encoding Protein
by Faiza Nisar Bukhari (2013-VA-12) | Dr. Muhammad Imran | Dr. M.Yasir Zahoor | Dr. Abu Saeed Hashmi.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Hepatitis C virus (HCV) infection has an estimated global prevalence of 2%–3% with approximately 270 million infected people worldwide of which 10 million belongs to Pakistan. HCV is a positive-sense single-stranded RNA virus from the family of Flaviviridea. Its genome encodes 10 proteins including a 261 amino acids protein NS4B. NS4B is a non-structural protein that performs hyperphosphorylation of NS5A, transactivates interleukin 8 promotor, suppresses HCV translation and modulates the ER stress response. Mutation in NS4B encoding gene may be targeted to develop an effective vaccine against HCV before virus invasion in host immune system.
Serum was collected from 20 patients who are infected with HCV. RNA was isolated from these samples to reverse-transcribe it into cDNA. This cDNA were PCR-amplified and amplicons were detected by UV light after their separation through agrose gel electrophoresis followed by seguencing of NS4B encoding gene.
Variations in HCV NS4B gene was analyzed by using Mega 6 software and sequence 2 blast analysis NCBI tool. Protein modeling was performed with phyre 2, DIANNA tool and I-Tasser online server and quality of model and antigenicity was checked by Vaxajen V 2.0 and Epijen softwares. Conservative analysis and epitope mapping were performed by using Immune Epitope Database (IEDB).
A systematic approach was employed for the prediction of potential epitopes in NS4B protein. Vaxijen V 2.0 was used to determine overall antigenicity of NS4B using cut off value of 0.4. Values above this threshold level show probable antigens in the protein. The topology of NS4B was determined by TMHMM server with the help of membrane topology data. Regions outside membrane and transmembrane were eradicated for epitopes prediction. T cell epitopes propred was used with proteosome cleavage site filter of 4% threshold. From the following data analysis 12 to 15 possible T-cell epitopes can be predicted; only these T cell epitopes were included in conservation analysis.
The present study provides information about mutational changes in HCV protein NS4B and thus preliminary data for the development of vaccine against HCV. This can also lead to future prospects for diagnosis, treatment and prognosis of HCV.
Hepatitis C virus (HCV) infection has an estimated global prevalence of 2%–3% with approximately 270 million infected people worldwide of which 10 million belongs to Pakistan. HCV is a positive-sense single-stranded RNA virus from the family of Flaviviridea. Its genome encodes 10 proteins including a 261 amino acids protein NS4B. NS4B is a non-structural protein that performs hyperphosphorylation of NS5A, transactivates interleukin 8 promotor, suppresses HCV translation and modulates the ER stress response. Mutation in NS4B encoding gene may be targeted to develop an effective vaccine against HCV before virus invasion in host immune system.
Serum was collected from 20 patients who are infected with HCV. RNA was isolated from these samples to reverse-transcribe it into cDNA. This cDNA were PCR-amplified and amplicons were detected by UV light after their separation through agrose gel electrophoresis followed by seguencing of NS4B encoding gene.
Variations in HCV NS4B gene was analyzed by using Mega 6 software and sequence 2 blast analysis NCBI tool. Protein modeling was performed with phyre 2, DIANNA tool and I-Tasser online server and quality of model and antigenicity was checked by Vaxajen V 2.0 and Epijen softwares. Conservative analysis and epitope mapping were performed by using Immune Epitope Database (IEDB).
A systematic approach was employed for the prediction of potential epitopes in NS4B protein. Vaxijen V 2.0 was used to determine overall antigenicity of NS4B using cut off value of 0.4. Values above this threshold level show probable antigens in the protein. The topology of NS4B was determined by TMHMM server with the help of membrane topology data. Regions outside membrane and transmembrane were eradicated for epitopes prediction. T cell epitopes propred was used with proteosome cleavage site filter of 4% threshold. From the following data analysis 12 to 15 possible T-cell epitopes can be predicted; only these T cell epitopes were included in conservation analysis.
The present study provides information about mutational changes in HCV protein NS4B and thus preliminary data for the development of vaccine against HCV. This can also lead to future prospects for diagnosis, treatment and prognosis of HCV.
Availability: Items available for loan: UVAS Library [Call number: 2339-T] (1).
18.
Study Of Genetic Polymorphism In Exon 7 And 9 Of Glucosidase Beta Acid (Gba) Gene In Gaucher Diseased Patients From Punjab, Pakistan
by Ayesha Khalid (2013-VA-07) | Dr. M. Yasir Zahoor | Dr. Sehrish Firyal | Mr. Tariq Mahmood.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Gaucher disease (GD) is an inborn metabolic disease transmitted through recessive
pattern of inheritance and it is a pan-ethnic disease. It is the most common lysosomal storage
disease caused by the deficiency of glucocerebrosidase (GCase), a lysosomal enzyme use in the
degradation of macromolecules into simpler molecules.
Glucosidase beta acid (GBA) gene encode glucocerebrosidase enzyme and mutations in
this gene is responsible for glucocerebrosidase deficiency which results in an accumulation of
unbroken glycolipids in those organs rich in monocyte-phagocyte immune system elements i.e.
spleen, liver, bone marrow and leads to histological changes. GBA is located on chromosome
1q21 consisting of 11 exons and 10 introns having 7.8kb length. It is divided into three types (I,
II and III) on the basis of neurological involvement. More than 300 mutations have been reported
in GBA and cause the GD.
The present study was performed in order to characterize GBA gene in GD patients from Punjab.
Blood samples of 10 patients,enrolled in Children Hospital, Lahore, were taken from DNA
repository of Molecular and Genomic Lab at IBBT, UVAS Lahore. The DNA was extracted
using organic method. Next step was the amplification of extracted DNA using PCR. After it, the
PCR product is purified and this purified PCR product was sent for sequencing. Sequencing of
exon 4, 7 and 9 was done using dideoxy sequencing method. After applying different
bioinformatics tool, it was found that there was no muttaion in these exons but a heterozygotic
variation G>A was found in intron 8. This finging will help in demonstration of molecular
pathogenesis of Gaucher disease. Availability: Items available for loan: UVAS Library [Call number: 2338-T] (1).
19.
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: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Theses submitted with blank cd. Availability: Items available for loan: UVAS Library [Call number: 2337-T] (1).
20.
Antiviral Effect Of Human Saliva Against Avian Influenza Virus Strain H9n2
by Maryam Riaz (2008-VA-340) | Prof. Dr. Tahir Yaqub | Dr. Sehrish Firyal | Prof. Dr. Kamran Ashraf.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Saliva is an important body fluid that contains a complex array of proteins, peptides and various substances that help in maintaining the health of the oral cavity. Saliva exhibits a broad-spectrum of antiviral activity against enveloped viruses as it disrupts the viral membrane. Influenza is a common virus that has been diagnosed in humans and avian species due to AIV. This study has demonstrated the naturally occurring antiviral activity of human saliva against the H9N2 influenza virus that serves as a serious threat to poultry and has been shown to possess high zoonotic potential which can cause a new pandemic.
In this study saliva samples from healthy individuals were taken and the natural antiviral ability of saliva was observed against AIV (Pk-UDL/01/08 H9N2) of calculated EID50 106.66. Inoculum prepared from saliva and H9N2 virus was injected in 9 days old embryonated eggs using CAS route and incubated at 37°C for 48 hours. A negative control (only saliva) and positive control (only virus inoculum) was also determined in the current study. The antiviral activity of saliva was observed through haemagglutination test. The HA test of harvested fluid showed that human saliva indeed possesses antiviral activity against H9N2 virus and can be used as a natural antiviral agent in medicine.
Furthermore, the genomic DNA was extracted from the blood samples. HTN3 gene responsible for histatin production, was amplified using gene specific oligonucleotides. The obtained HTN3 gene sequences were analyzed using Chromas software. The sequence alignment showed 99% similarity to the available sequences in NCBI database and 100% similarity to each individual sample. To conclude, this study has demonstrated that human saliva possesses antiviral activity against H9N2 virus. The nucleotide sequence analysis from each sample
CHAPTER 6
SUMMARY
Summary
47
showed no particular change which shows that antiviral activity of glycoproteins present in saliva does not vary at a genetic level. This innate antiviral activity can open a new frontier when it comes to combating viral infections that have grown resistant to conventional drugs in both human and animal subjects. Availability: Items available for loan: UVAS Library [Call number: 2336-T] (1).
21.
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: ; 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).
22.
Mutational Analysis of CaSR in Calcium Nephrolithiasis Affected Pakistani Families
by Asad Tufail (2013-VA-558) | Dr. Muhammad Yasir Zahoor.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Thesis submitted with blank cd. Availability: Items available for loan: UVAS Library [Call number: 2343-T] (1).
23.
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: ; 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).
24.
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: ; 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).
25.
Molecular Characterization Of Local Donkey Based On Mitochodrial D-Loop Analysis
by Shakeel Earnest (2012-VA-597) | Dr. Muhammad YasirZahoor | Prof. Dr. TahirYaqub | Mr. Tariq Mahmood .
Material type: ; Literary form:
not fiction
Publisher: 2014Dissertation note: Research on control region or mitochondrial d-loop is of special interest in all mammalian species because they the displacement loop hyper-variable region of mtDNA (D-loop), which is of 1200 bp, is very important for genetic variations. It is also important for amtDNA in Nuclear Mitochondrial sequences (NUMTs) in vertebrates species so that it rarely occurring NUMTs. The control region in equine consist of two highly variable regions (HVR1 and HVR2), 4 conserved blocks (CSB), and variable repeats of 8 bp motifs (Cothran, et al. 2013).
The displacement loop hyper-variable region of mtDNA (D-loop) of Pak- local donkeys are very similar to genome of other species; however there are considerable differences in the mtDNAevolutionary rate for different taxonomic groups.
The phylogenetic tree based on consensus sequences of 12 Asian Donkeys breeds available on NCBI and sequences of local Pakistani donkey breeds showed their genetic relationship among each other. The clade was consisting of Pakistani local donkey breeds i.e. 3-SF, 7-SF,10-SF,11-SF,12-SF,13-SF, K-3,K-6,K-11,K-13,K-16,K18,K-19 and K-20 showing their high relatedness.K-8 and 8-SF have more mutation rate in the sequence and have more diversity from other individuals.
The second major branch was furher divided in two sub branches i.e. Donkey family representing Equusburchellichapmani (JX312729), Equusburchelliquagga (JX312733), Equusgreyvi(NC020432) and Equus zebra (JX312718) clustered together. The second sub branch was consisting of other donkey breeds i.e. Equushemionusonager (JX312730), Equushemionus(NC016061) and Equushemionuskulan (NC018782) grouped together while two Pakistani local donkey breeds i.e. SF-8 and K-8 were clusterd with Equusasinussomalicus (AP012271) and Equusasinus (X97337).
The d-loop sequence of Human (Homo sapiens) was taken as out group and it clearly differed and separated from rest of phylogenetic tree i.e. camel, bovine, ovine, caprine and other mammals.
The phylogenetic tree constructed under this study not only confirmed the status of Pak- local donkey breeds but also confirmed the genetic relationship among other mammalian species, thus reconfirming the already established biological classification.
Availability: Items available for loan: UVAS Library [Call number: 2362-T] (1).
26.
Mutation Anlysis Of DTNBP1 Gene In Pakistani Patients With Schizophrenia Disorder
by Hafiza Sidrah Yasin (2013-VA-11) | Dr. Asif Nadeem | Dr. Maryam Javed | Dr. Abu Saeed Hashmi.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Schizophrenia (SCZ) disorder is a mental complex, heterogeneous and chronic neurodegenerative disorder with a cumulative prevalence of 1%. SCZ is an idiopathic disorder of the cortex and hippocampus. Environmental as well as genetic factors contribute to its complex pathogenesis. A functional repeat polymorphism in the Dystrobrevin Binding Protein 1 (DTNBP1) gene promoter conveys susceptibility for SCZ disorder. The DTNBP1 has been implicated in rare autosomal dominant forms of SCZ disorder because of mutations associated with severe disease progression and a typical physical signs and symptoms, indicative of neurodegeneration. Mutation in DTNBP1 gene has association with change in dysbindin protein which leads to change in abnormal neurotransmitter trafficking which leads to decrease in neuronal size, brain atrophy and reduced glutamate release in schizophrenia disorder. A systematic approach was applied to proceed the present study in order to identify the single nucleotides polymorphisms in schizophrenic patients. Blood samples (n=40) were collected from schizophrenia disorder patients. DNA was extracted by organic method. Primers were designed using Primer3 software. The amplification of gene was done by Polymerase Chain Reaction. PCR products were sequenced bi-directionally on ABI 3130XL Genetic analyzer. Sequence alignment was performed for polymorphism identification. The analysis of identified polymorphism was done by CHROMAS software. Sequence was aligned by Blast tool of NCBI. Difference between allele and genotype frequency of studied gene was evaluated and analyzed by using “SNPator”. The present study provides information about the susceptibility and genetic basis of the individual towards this disease and identified polymorphisms provides the opportunity to diagnose the disease earlier on the basis of particular SNPs in Pakistani patients. Availability: Items available for loan: UVAS Library [Call number: 2382-T] (1).
27.
Genetic Identification And Molecular Classification Of Sub-Family Phasianinae Of Pakistani Bird Species Through Dna Barcoding
by Maryem Hussain (2008-VA-349) | Dr. Ali Raza Awan | Dr. Sehrish Firyal | Dr. Muhammad Tayyab.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: DNA barcoding is a precise technique that uses molecular genetics tools for accurate identification, categorizing, relating and separating the phylogenies of species. Being a small sized genome and agile enough to show rapid mutation, mtDNA has been used as a pertinent marker of molecular biodiversity.The aim of this study was to develop DNA barcode for genetic characterization and classification of Sub-family Phasianinaeof Pakistani bird species. Theyhave not been genetically identified yet in Pakistan. It includes birds like domestic chicken(Gallus gallusdomesticus), aseel chicken(Gallus gallusdomesticus strain),blue peafowl(Pavo cristatus), green peafowl (Pavo muticus), white peafowl (Pavo cristatus leuticus), Kalij pheasant (Lophura leucomelanos),monal pheasant (Lophophorus impejanus),koklass pheasant(Purcrasia macrolopha), ring necked pheasant (Phasianus colchicus), Tragopan (Tragopan melanocepals) andred junglefowl (Gallus gallus). These birds are considered an important part of an ecosystembecause they play a significant role in seed dissemination, pollination of plants and disease spread which are the basic constituents of an ecosystem. They are used for food, hunting and entertainment purposes.
Mitochondrial geneCytochrome c oxidase subunit 1 (CO1)of 500bps was used as a marker for identification at specie level.Genomic DNA was extracted by each blood and tissue sample of eleven bird species (33 samples). Amplification of CO1 gene was a done by using a universal set of primers (BIRDF1 and BIRDR1)containing region of almost 750 bps (Hebert et al. 2003).Amplicons were purified and sequenced Sanger sequencing method (Sanger et al. 1977). Forward and reverse sequences were analyzed using softwaresEMBOSS merger,ClustalW, BioEdit and nBLAST. Phylogenetic analysis of selected bird species was done. Each sequence was aligned
with its reference sequences of CO1 gene present on NCBI. Every nucleotide position which did not align with the reference sequence was studied to identify SNPs. Fixation index (FST) were used to measure species diversity within a same sub population relative to that found in the entire population. Consensus sequences (500bps) generated was used to construct their phylogenetic tree to see their evolutionary relationship with other bird species. All species showed their closest linkage with their respective species. Pakistani population of peafowl and chicken species showed the close relation with same sequences generated in China. Tranopans showed its closest linkage with T. temminckii.
In conclusion, seven species ofPhasianinaesub-family of Pakistani bird species was genetically characterized first time in Pakistan by using CO1 as a barcode. It proves that DNA barcoding is an efficient and accurate molecular tool for species identifica¬tion and phylogenetic implication. This study leads to establish a DNA Data Bank that helped scientists to investigate the biodiversity, taxonomic classification, specie identification, in forensic purposes and to study the genetic and phenotypic evolution of these species. DNA barcoding through CO1 gene works as a functional tool for detectingmeat mislabeling and preventing illegitimate trade. This study has established foundations for molecular biologists to study taxonomic uncertainties at sub species level using SNP based identifying marker. It helps in preservation and identification of endangered species by generating their barcodes from even minimal evidence available.
Availability: Items available for loan: UVAS Library [Call number: 2376-T] (1).
28.
Molecular Characterization of Pakistani Common Leopard
by Muhammad Usman Ijaz (2012-VA-908) | Dr. Sehrish Firyal | Dr. Ali Raza Awan | Dr. Muhammad Tayyab.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: CD not available. Availability: Items available for loan: UVAS Library [Call number: 2379-T] (1).
29.
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: ; 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).
30.
Mutational Analysis Of Hcv Gene Encoding E1 Glycoprotein
by Muhammad Saad Junjua (2013-VA-893) | Dr. Muhammad Imran | Dr. Wasim Shehzad | Dr. Abu Saeed Hashmi.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Hepatitis C virus (HCV) is a positive single stranded RNA virus that belongs to Flaviviridae family and causes liver related issues like hepatocellular carcinoma, cirrhosis and chronic liver disease. HCV is affecting people worldwide; more than 170 million peoples have been affected so far and the number is increasing day by day. Its prevalence in Pakistan is about 3% to 6%. There is lot of variation in its genome and it is classified into 6 major genotypes and these genotypes are further classified into many subtypes. Size of HCV is about 9500 bps which only encodes single polyprotein. This 3000 to 3300 amino acids polyprotein is processed by cellular and viral proteases to generate 10 polypeptides consisting of 4 structural (Core, E1, E2 and P7) and 6 non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A, NS5B). The most immunogenic gene from all the genes is E1. It involves in the interaction with the host cell and easily escapes from the immune system of host due to the presence of hypervariable regions in E1 gene.
To isolate the E1 gene, RNA extraction was done using the kit method. RNA was converted to cDNA which was then amplified in two rounds of PCR using nested primers from HCV core region. After confirming the presence of HCV RNA in serum samples, PCR amplification of HCV E1 gene was carried out using gene specific nested primers. Amplified E1 gene products were Sanger sequenced and aligned with standard sequence to find out genetic variations. E1 gene sequences were converted to protein sequences for which secondary protein structures were made and analyzed. No noticeable change was seen in these secondary protein structures. The protein sequences were also analyzed for the presence of B-cell and T-cell epitopes; two T-cell epitopes (QAFTFRPRR, FLVGQAFTF) were found which may inform the development of a proper vaccine against HCV. Availability: Items available for loan: UVAS Library [Call number: 2399-T] (1).
31.
Identification Of Single Nucleotide Polymorphism In Toll Like Receptor 4 Gene And Its Association With Mastitis In Sahiwal Cows
by Hafiz Kamran Rizwan Ullah (2013-VA-557) | Dr. Sehrish Firyal | Dr. Muhammad Wasim | Prof. Dr. Habib Ur Rehman.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Several factors militate against realizing the milk production potential of cows. Mastitis is one of the shocking maladies of milch animals causing high production losses to livestock industry in Pakistan and elsewhere in the world. Mastitis has been familiar as one of the most inexpensively important diseases affecting dairy animal’s worldwide.
Susceptibility and resistance to mastitis is a complex trait and influenced by genetic variation of the immunity genes of animals. Among these variations, the polymorphisms in Toll-like receptor 4 gene (TLR4) play important role in the immune response to mastitis. Polymorphism in exon 3 of TLR4 gene is associated with mastitis susceptibility and resistance. It is a potential candidate gene for screening of the mastitis susceptible and resistant dairy cows.
The present study was designed for the identification of polymorphism in TLR4gene associated with mastitis. Blood samples from 20 Sahiwal cows having clinical and subclinical mastitis were sampled. Blood sample of 10 normal Sahiwal cows was also collected. DNA was extracted. Specific primers for amplification of TLR4 gene were designed from NCBI.
TLR4gene was amplified and sequenced to get the desire sequence of this gene. Comparative analysis of the resulted sequences using NCBI BLAST was done.
Availability: Items available for loan: UVAS Library [Call number: 2392-T] (1).
32.
Genetic Effect Of Interferon Gamma On Bovine Resistance Against Mycobecterium Bovis
by Syed Ahmed Raza Rizvi (2012-VA-819) | Dr. Maryam Javed | Dr. Tanveer Hussain | Dr. Muhammad Tayyab.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Bovine tuberculosis is a disease caused by the species included in the Mycobacterium tuberculosis complex. IFN-GAMMA are a family of conserved innate immune recognition receptors that trigger adaptive immune responses. IFN-GAMMA 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 IFN-GAMMA 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 IFN-GAMMA 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 IFNG mentioned in table. The Eight SNPs identified in the coding region of INTERFERON GAMMA 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, and P8 C >T. The one SNP found in the current research is in compliance with the (Sun et al. 2012) research on INTERFERON GAMMA hence Nine SNPs found in the current research are novel in Nili Ravi buffalo. Research on IFN-GAMMA hence Seven 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, among them four were transitions and four were transversion .
Population genetic analysis and allelic distribution at all loci was analyzed using
Summary
57
POPGENE 32 software indicated that at [P3=0.354539>0.05] , [P5=0.365524>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= 0.000032< 0.05], [P2=0.038766< 0.05] and [P7=000394< 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: 2419-T] (1).
33.
Lactoferrin Gene Polymorphism in Dairy Cattle
by Syeda Iqra Aiman Bukhari (2009-VA-556) | Dr. Sehrish Firyal | Dr. Ali Raza Awan | Dr. Muhammad Tayyab.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Several factors militate against realizing the milk production potential of cows. Mastitis is the most costly and the prevalent production-limiting disease of dairy animals in Pakistan and elsewhere in the world. It is accompanied by elevated Somatic cell count (SCC) in the milk and estimated genetic correlation between SCC and mastitis ranges between 0.53-0.77. Susceptibility and resistance to mastitis is a complex trait and influenced by genetic variation of animals. Among these variations, the polymorphism in Lactoferrin gene (LTF) plays an important role in the immune response to mastitis.
Polymorphism in intron 6 of LTF gene is associated with mastitis susceptibility and resistance. It is a potential candidate gene for imparting resistance mastitis in dairy cows.
The present study was designed for the identification of polymorphism in LTF gene associated with mastitis. Milk and blood samples were collected from 20 Sahiwal cows having clinical and subclinical mastitis. SCC of milk samples was performed using serial dilutions. 10 normal Sahiwal cows as control were included in present study. DNA was extracted from blood using organic extraction and kit method followed by DNA quantification. Amplification of LTF gene was designed by using already reported primers obtained from NCBI.
LTF gene was amplified and sequenced to get the full length sequence of this gene. Comparative analysis of the resulted sequences using NCBI BLAST was done.
Outcomes:
The results obtained from polymorphisms in LTF gene can play an important role for selection of mastitis resistant and susceptible dairy cows. This can be useful in selective breeding of cattle for enhanced immune response, as a tool to improve inherent animal health, which ultimately can lay the foundations to contain the magnitude of economic loss due to mastitis.
Develop a biological response modifier that will promote a sustained immunity of the mammary teat and protect the gland from invading pathogens. Availability: Items available for loan: UVAS Library [Call number: 2416-T] (1).
34.
Polymorphism Study Of Calcium-Sensing Receptor Gene (Casr)In Calcium Nephrolithiasis Affected Families
by Hafza Ammara (2013-VA-865) | Dr. Muhammad YasirZahoor | Dr. Asif Nadeem | Ms. Huma Mujahid.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Nephrolithiasis is a multi-factorial kidney stone disease resulting from the combined influence of epidemiological, biochemical and genetic risk factors. Calcium-sensing receptorprotien is plasma membrane G protein-coupled receptors that regulate secretion of parathyroid hormoneand calcium re-absorption by kidney tubular cells. This protienis able to sense small changes in circulating calcium concentration and, once activated, it inhibits parathyroid hormone secretion and renal tubule calcium re-absorption. The CaSR gene protein islocated on chromosome 3q13 is one of the candidate gene explaining individual predispositions to calcium nephrolithiasis. CaSR gene is a predecessor for nephrolithiasis due to its role in calcium re-absorption. CaSRgene has seven exons and several mutations have been reported globally related to calcium nephrolithiasis.
Twenty families affected with calcium nephrolithiasis having at least two affected individuals have been enrolled for this study. Ten families have already been analyzed for exon 3 & 4 in the laboratory. DNA has been extracted through inorganic extraction method from the blood of newly enrolled families. Primers have been designed for exon 5, 6 and 7 through Primer3 software. These exons have been sequenced using the BigDye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer/ABI) and have been read in an automated sequencer, ABI Prism model 3730 (Perkin Elmer).
We also screend the coding exon of CLDN14 genewhich is a membrane protein that regulates paracellular passage of ions and small solutesat epithelial tight junction.The overexpression of claudin-14 in the thick ascending limb of loop of henleof the kidney generates a renal phenotype characteristic with hypomagnesemiaand hypercalciuria that leads to the development of calcium nephrolithiasis.
All of the sequences have been evaluated by using Clustal-W programs, Chromas and Bioedit software for mutational analysis.Sequence analysis of CaSR gene revealed one novel splice mutationC>G at position 63722 at exon 5 in one affected family.This variation is found in the intronic region of the gene.We found one missense mutation Q536R at exon six in three different affected families. And one synonymous single nucleotide polymorphism(SNP) C>G found at exon 7at rs2036400 in six different affected families.These SNPs showsa significant association of CaSRgene with nephrolithiasis. It will help to determine the risk factor and role of CaSR gene in inheritance of calcium nephrolithiasis. And it will also be used for genetic screening and prenatal diagnosis.
Availability: Items available for loan: UVAS Library [Call number: 2426-T] (1).
35.
Genetic Polymorphism Of Prss12 Gene Responsible For Cognitive Dysfunction And Its Homology Analysis With Canine
by Hafsa Amjad (2014-VA-776) | Dr. Muhammad Yasir Zahoor | Dr. Muhammad Imran | Mr. Shahid Abass.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: Neurotrypsin a multi domain serine protease predominantly expressed in brain is considered to be involved in cognition by the establishment and maintenance of synapses in mammals. Mutations in PRSS12 gene have been reported for cognitive disability in Algerian family.
In present study, DNA of 10 enrolled non-relative cognitive dysfunctioned patients was extracted through organic method. The normal individual samples of siblings and parents of relevant families was also included in this study as control. This amplification exon 7 of PRSS12 was done after designing primer by using Primer3 software. Exons was sequenced by using BigDye Terminator Cycle Sequencing Ready Kit(Perkin Elmer/ABI) and read in automated sequener, ABI Prism model 3730 (Perkin Elmer). No significant mutation was identified in affected individuals.
Computational comparative sequence analysis tools were used for the nucleotide and amino acid sequences to predict the homology in PRSS12 gene among mammals of well-developed cognition. PROSITE domain database search was performed to determine domain organization and Phyre software was used to develop secondary structural features and 3D protein models and ReptroX for multiple sequence alignment of tertiary structures. Using the generated alignments highly conserved regions in primary and secondary structures of neurotrypsin in mammals were identified. Phylogenetic analysis indicated highest similarity of human PRSS12 with non-human primates (chimpanzee, orangutan and monkey) followed by Catecians, Felis, and Canine evolving from the same ancestor. The predicted domain architecture shows the neurotrypsin consisting of kringle domain, four scavenger receptor cysteine-rich
CHAPTER 6
SUMMARY
Summary
68
domains and a serine protease domain named trypsin. Whereas mouse consists of only three scavenger receptor cysteine-rich domain. Prediction and comparison of domains in mammals indicated that primates and catecians protein domains have high similarity with humans. Computational analysis by using animal models can aid in evolutionary studies and. understanding the role of neurotrypsin in cognition. Availability: Items available for loan: UVAS Library [Call number: 2498-T] (1).
36.
Nucleotide Sequence Variation In Heat Shock Protein 70-1 Gene Of Capra Aegagrus Blythi
by Fehmeeda Fatima (2014-VA-775) | Dr. Asif Nadeem | Dr. Maryam Javed | Dr. Abu Saeed Hashmi.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: Heat shock protein 70 (HSP 70) plays a vital role in survival of an organism by providing
cytoprotection against various kinds of stresses. Among all the HSPs present in the cell, the
ubiquitous HSP 70 proteins are the most abundant and temperature sensitive. Considering the
importance of HSP70-1 gene in conferring thermotolerance, present study has been designed to
characterize this gene in Sindh ibex which is a wild goat species of Pakistan. The
characterization of HSP70 gene might be helpful for deriving phylogenetic relationship among
different species and identifying new functions among the related species. Blood/meat samples
(n=25) were collected from Kirthar national park, Sindh. Standard DNA extraction method was
used for DNA extraction. PCR primers were designed by Primer3 software and amplification of
gene was done by Polymerase Chain Reaction. PCR product was sequenced bi-directionally by
Big DyeTM Terminator on ABI 3130XL Genetic Analyzer. Multiple sequence alignment was
performed for polymorphism identification. Genetic diversity was calculated by using DnaSP
v.5.0. Phylogenetic analysis using the MEGA v.6.0 software package was performed and
neighbor joining and UPGM trees were constructed. The results indicated that Sindh ibex
HSP70.1 gene was highly similar to of domestic goat, sheep, cattle, buffalo, camel and horse
which indicates their origin from a common ancestor. The results of this data might be helpful in
designing effective conservation strategies for Sindh ibex. Availability: Items available for loan: UVAS Library [Call number: 2524-T] (1).
37.
Development Of Dna Based Diagnosis Of Theileriosis In Cattle And Its Specificity With Blood Smear Microscopy
by Uzma Sarwar (2014-VA-777) | Dr. Wasim Shehzad | Dr. M. Yasir Zahoor | Dr. Ali Ahmed Sheikh.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: Theileria annulata and Theileria parva are intra-erythrocytic parasites which are responsible for
causing tropical theileriosis and East Coast fever in cattle respectively. This parasite is
transmitted by ticks to vertebrate host i.e. cattle. Currently used diagnostic methods for diagnosis
of bovine theileriosis are clinical symptoms, peripheral blood smear microscopy and serological
tests (IFAT and ELISA).
Current study was conducted to compare the specificity and sensitivity of blood smear
microscopy and PCR techniques to diagnose bovine theileriosis. This study is comparative as
well as developmental in nature. Although peripheral blood smears microscopy is cost effective
and quick method of diagnosis in case of high or moderate parasitaemia in blood. But the
limitations associated with microscopy include false negative diagnosis in case of low
parasitaemia in chronic and asymptomatic infection, morphological similarity of Theileria with
other species of Plasmodium and Babesia. These limitations may lead to misdiagnose the
infection due to which disease may remain unnoticed. PCR based method, developed in this
study, and is found to be more specific and sensitive than conventional microscopy. Fifty blood
samples were collected from September, 2015 to November, 2015. These samples were screened
microscopically as well as with PCR for presence of Theileria. Nine samples were found to be
positive microscopically but 18 samples were found positive by PCR. The results obtained from
the study clearly show that PCR is more reliable, precise and sensitive assay for diagnosis of
bovine theileriosis then microscopy. It is hoped that proposed method to diagnose Theileria will
help to nullify the problems associated with microscopy. This will ultimately facilitate in the
formulation of effective treatment control and vaccine development strategies. Availability: Items available for loan: UVAS Library [Call number: 2547-T] (1).
38.
Molecular Exploration Of Zbed6 Gene For Growth Trait In Lohi Sheep
by Usman Sagheer (2014-VA-03) | Dr. Maryam Javed | Dr. Akhtar Ali | Prof. Dr. Tahir Yaqub.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: ZBED6 gene is a central transcription factor. It is as a repressor of IGF2 (insulin-like growth factor II) interpretation in skeletal muscle myogenesis and development. It is essentially included in organism development, signaling, cell to cell collaboration, hepatic fibrosis, clathrin intervened endocytosis and tight intersection signaling falls. Chromatin immune precipitation (ChIP) sequencing utilizing C2C12 cells recognized around 2,500 ZBED6 binding locations in the genome, and the derived accord theme gave an immaculate match with the set up tying site in IGF2. Silencing of ZBED6 in myoblast cells influences IGF2 expression, wound healing, cell proliferation and myotube arrangement. Genes connected with ZBED6 binding sites demonstrated a very huge advancement for certain Gene Ontology groupings, including improvement and transcriptional regulation.
Forty two blood samples were collected. DNA extraction was done by using organic extraction method. Primers for PCR amplification designed using Primer3 software. PCR products were sequenced and then analyzed by using BioEdit software. Expasy translational tool for translation and POPGENE 32 software for analysis of population genetics at all the loci were used. Using this software the overall allele frequency, heterozygosity, probability using Chi-square test and Likelihood ratio test and Hardy-Weinberg equilibrium, genotype distribution at all SNP position, summary of genetic variation statistics for all loci and association were calculated. After this, for the association one way ANOVA was performed. Single nucleotide polymorphism within ZBED6 could be potential candidate gene to be serving as genetic marker for the selection of animals with higher tendencies towards weight gain. Availability: Items available for loan: UVAS Library [Call number: 2539-T] (1).
39.
Molecular Exploration Of Zinc Finger Bed-Type Containing 6 Gene For Growth Trait In Beetal Goat
by Kanwal Rashid (2014-VA-496) | Dr. Maryam Javed | Dr. Asif Nadeem | Dr. Abu Saeed Hashmi.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: Zinc finger, BED-type containing 6 (ZBED6), is a novel transcription factor.It acts as a repressor of IGF2 transcription in skeletal muscle myogenesis and development. it is mainly involved in organism development, signaling, cell to cell interaction, hepatic fibrosis, clathrin mediated endocytosis and tight junction signaling cascades. Chromatin immunoprecipitation (ChIP) sequencing using C2C12 cells identified about 2,500 ZBED6 binding sites in the genome, and the deduced consensus motif gave a perfect match with the established binding site in Igf2. Silencing of Zbed6 in myoblast cells affect Igf2 expression, cell proliferation, wound healing, and myotube formation. Genes associated with ZBED6 binding sites showed a highly significant enrichment for certain Gene Ontology classifications, including development and transcriptional regulation.Functional and signaling assays of BED6 gene indicate its probable role in controlling growth traits in Goat. Blood samples (n = 40) were collected. Inorganic method of DNA extraction used. Primers for PCR amplification will be designed using Primer3 software. PCR products will be sequenced bi-directionally on ABI 3130XL Genetic analyzer. The results of sequencing were analyzed using CHROMAS software. Sequence alignment tools (blast 2)were used for SNPs identification. Difference between allele and genotype frequency of studied gene evaluated by chi square test, likelihood test and analysis was done by POPGENE and one way ANOVA.Novel Variations identified which have probable implementation in selection of superior goats with higher tendencies towards weight gain. Availability: Items available for loan: UVAS Library [Call number: 2554-T] (1).
40.
Molecular Phylogeny And Diversity Analysis Of Bovidae (Boselaphus Tragocamelus, Antilope Cervicapra) And Cervidae (Axis Axis, Axis Porcinus) In Pakistan
by Ghulam Abbas (2011-VA-748) | Dr. Asif Nadeem | Prof. Dr. Mansoor Ellahi Babar | Dr. Muhammad Tayyab.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: Many species of mammals have declined within the past two centuries due to human
caused disturbances and the unsustainable use of natural resources. Molecular methods have an
important role in phylogeny and diversity analysis. The present study was designed for diversity
analysis of Boselaphus tragocamelus & Antelope cervicapra (Bovidae) and Axis axis & Axis
porcinus (Cervidae) family in Pakistan. A total of 25 samples from each of the four species were
collected from different parks, zoos and natural habitats. DNA was extracted, PCR primers were
designed and cytochrome-b, cytochrome-c gene and d-loop regions were amplified by PCR.
PCR products were sequenced bi-directionally by Big DyeTM Terminator. Bioinformatics tools,
Blast 2 sequences, Clustal-W, MEGA-6, Bioconductor in “R” were applied for analysis. The
clustering of the samples indicates that each species contains less within-population genetic
variability. Same pattern was observed when sequence of three genes was combined and MDS
plot was constructed. Phylogenetic analysis of the gene sequences revealed that each species
comprised a clade that is clearly distinct from the clade comprised of other species of deer
selected for this study. Finding of this study indicated that these species of deer have significant
genetic variations among-species that differentiate them from each other. This is the first report
from our region. The information of selected species of deer is prerequisite for designing
effective strategy in future conservation practices. However further genomic investigations
should be carried out at larger scale. Availability: Items available for loan: UVAS Library [Call number: 2560-T] (1).
41.
Identification Of Genetic Variants In The Low Density Lipoprotein Receptor Gene Causing Familial Hypercholesterolemia And Its Sequence Homology With Mus Musculus
by Ameer Hassan (2014-VA-504) | Dr. Wasim Shehzad | Dr. Muhammad Yasir Zahoor | Dr. Muhammad Tayyab.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: Familial hypercholesterolemia (FH) is caused by mutations in the genes coding for the low-density lipoprotein receptor (LDLR), apolipoprotein B-100, or proprotein convertase subtilisin/kexin type 9 (PCSK9). In this study, a molecular analysis of LDLR gene was performed in 20 unrelated patients from Pakistan. All patients were clinically diagnosed with definite or possible hypercholesterolemia according to a uniform protocol and internationally accepted WHO criteria. Preferable study was made to highlight the Genetic variation in Exon 4 of LDLR gene associated with defective catabolism of cholesterol effecting lipid metabolism which results in Familial Hypercholesterolemia. The extraction of genomic DNA was done from all selected blood samples. By selecting primers they were synthesized and optimized on extracted DNA samples. PCR product was sequenced and aligned. Mutations in the LDLR gene and its sequenced homology with Mus musculus were analyzed. We didn’t found any polymorphisms in the LDLR gene exon 4. So we concluded that there is no association between SNPs and increased levels of cholesterol in Pakistani population. More research should be carried out in Pakistan by increasing the sample size and considering the other regions of LDLR gene. This study will help the early detection and treatment of such cases and may ultimately reduce the incidence of mortality due to myocardial infarction. Apart from diagnosis, we also suggest it will be a potential therapeutic strategy to manage FH. Availability: Items available for loan: UVAS Library [Call number: 2538-T] (1).
42.
Molecular Investigation Of Low Density Lipoprotein Receptor Gene Causing Familial Hypercholesterolemia And Its Evolutionary Relationship With Pan Troglodytes
by Rida Zainab (2014-VA-808) | Dr. Maryam Javed | Dr. Asif Nadeem | Prof. Dr. Tahir Yaqub.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: Familial Hypercholesterolemia (FH) phenotype is related to improper metabolism of low density lipoproteins due to mutations in Low-density lipoprotein receptor (LDLR) gene with increased risk of ischemic heart disease. Genetic variants in LDLR gene are associated with defective catabolism of cholesterol effecting lipid metabolism which results in familial hypercholesterolemia. It occurs in both forms: Homozygous Familial Hypercholesterolemia and Heterozygous Familial Hypercholesterolemia.
Patients having high cholesterol were identified by observing the values of their serum lipid profile test reports. Their detailed history was taken and blood samples from the identified patients of familial hypercholesterolemia were collected. DNA extraction was done by Organic method. Primers were synthesized and PCR was conducted using optimized recipe and conditions. PCR products were sequenced.
Sequenced data was analyzed using Chromas or BioEdit software. BLAST was performed and sequences were aligned individually by comparing it to the reference sequence. This showed difference in any specific position of a mutated sequence against the reference sequence. CLUSTALW aligned all the sequences together in one time. Sequences were compared with reference sequence to detect the presence of any mutation or SNPs.
SNPs were identified manually and the peaks were observed in order to determine if the genotype is heterozygous or homozygous. Statistical Analysis was done and any amino acid change due to the observed SNPs was determined by using Expasy Translate Tool. It was found that both the SNPs showed amino acid changes. In the end, homology analysis was done which showed that Homo sapiens had their LDLR gene closest to that of Gorilla gorilla gorilla. Availability: Items available for loan: UVAS Library [Call number: 2551-T] (1).
43.
Polymorphism Analysis Within Tata-Box Of Bovine Lactoferrin Gene And Its Association With Mastitis In Sahiwal Cows
by Kashmala Haroon (2014-VA-04) | Dr. Sehrish Firyal | Dr. Immad Rashid | Dr. Muhammad Tayyab.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: Mastitis is one of the most important diseases in dairy cows throughout the world, and is responsible for significant economic losses to the dairy industry especially in Pakistan. Several factors are responsible for this disease and about 20% bovines are suffering with this disease. Mastitis susceptibility and resistance is influenced by genetic variation of animals. Variations to polymorphisms in LF gene assume critical part of the immune response to mastitis. Polymorphism within LF gene may influence immune response to the mastitis in bovines. Recent study shows that promoter region of LF gene is highly polymorphic among bovines.
Present study was planned to identify polymorphism analysis within TATA-box of bovine LF gene and its association with mastitis. Multiple blood samples were collected from Sahiwal cows having clinical and sub-clinical mastitis. 10 samples were collected as a control. DNA extraction was done by organic extraction method and then quantification was done by Nanodrop. Amplification and sequencing was performed to get desire sequence of the gene. Comparative study of obtained sequence results were analyzed by using NCBI blast. Bioinformatics analysis was done with the help CLUSTAL W and BioEdit softwares.
Two novels and one reported SNPs were discovered within TATA-box of LF gene that might be having strong genetic association with mastitis in Sahiwal cows. This gene is strong candidate gene to differentiate between mastitis susceptible and resistant Sahiwal cows.
Availability: Items available for loan: UVAS Library [Call number: 2584-T] (1).
44.
Sequence Analysis Of Mitochondrial Atpase 8/6 Gene Variants In Sindh Ibex (Capra Aegagrus Blythi)
by Javeria Zafar (2014-VA-222) | Dr. Asif Nadeem | Dr. Maryam Javed | Prof. Dr. Abu Saeed Hashmi.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: ATPase 8/6 gene plays a vital role in survival of an organism by generating energy in the form of ATP synthase. Considering the importance of ATPase8/6 gene in energy generating, present study has been designed to characterize this gene in Sindh ibex. The characterization of ATPase8/6 gene might be helpful for deriving phylogenetic relationship among different species and identifying new functions among the related species. Tissue/blood samples (n=15) were collected from Kirthar National Park, Sindh. Standard DNA extraction method was used for DNA extraction. PCR primers were designed by Primer3 software and amplification of gene was done by Polymerase Chain Reaction. PCR product was sequenced bi-directionally by Big Dye TM Terminator on ABI 3130XL Genetic Analyzer. Multiple sequence alignment was performed for polymorphism identification. Genetic diversity was calculated by using DNAsp. Phylogenetic analysis using the MEGA6 software package and an equally weighted maximum parsimony analysis was performed using the close-neighbor-interchange algorithm. The results indicated that Sindh ibex ATPase8/6 gene was highly similar to Capra caucasica. The results of this data might be helpful in designing effective conservation strategies of different species of wild animal. Availability: Items available for loan: UVAS Library [Call number: 2587-T] (1).
45.
Development Of Novel Mtdna Metabarcodes For The Species Differentiation Of Class Aves
by Syeda Rida Mehak Sherazi (2010-VA-477) | Dr. Muhammad Imran | Dr. M. Yasir Zahoor | Mr. Shahid Abbas.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: The Folmer COI mtDNA universal primers that are considered standard for DNA barcoding of life contain so many mismatches against the target sequences of vertebrate origin that they often end in failure to amplify many of vertebrate DNA extractions. This discrepancy favors for the selection and designing of new metabarcode primers that can be used to identify all individuals of vertebrates or at least all individuals represented in a class of Vertebrata such as Class Aves. The current study embarks on such an endeavor. In this study development of new mtDNA metabarcode (16SrRNA) that can be used as universal primers to amplify almost all species of Class Aves for different forensic and molecular biodiversity analyses.
Blood/feather/tissue samples were collected from Class Aves (one specimen from every order reported to be present in Pakistan). DNA was extracted from the collected specimens through standard organic method, qualified and quantified and then PCR-amplified using novel universal primers selected from aligned mtDNA sequences originating from all Aves mitochondrial DNA genomes submitted to different online sequence databases such as NCBI nucleotide database. The sensitivity of PCR also be assessed using a range of DNA concentrations. The amplified products were sequenced on ABI Genetic Analyzer following Sanger’s dideoxy method of sequencing. The correctness of obtained mtDNA sequences were examined visually in Chromas Lite 2.1 software and then alignment of these sequences were performed against highly similar DNA sequences in NCBI nucleotide databases using BLAST in order to identify origin of unknown mtDNA sequences. With the help of sequencing and phylogenetic studies specificity of the universal primer set confirmed and presented as a novel metabarcode (16SrRNA) for species level identification of large number of Avian species
In summary, we present universal method for species classification of Aves using a targeted parallel sequencing approach. Both sequencing and phylogenetic studies experiments confirm
Summary
82
specificity of universal primer set. Although promising results were obtained with current settings, rapid improvement of bench top instruments will further develop method with less hands-on, fewer sequencing errors and lower detection limit. So, in future, this barcode can be used for species identification in various fields of study such as meat adulteration, illegal trade, food mislabeling and molecular estimation of biodiversity. Availability: Items available for loan: UVAS Library [Call number: 2617-T] (1).
46.
Identification And Expression Analysis Of Genes Involved In Obsessive Compulsive Disorder In Pakistani Population
by Javeria (2008-VA-627) | Prof. Dr. Masroor Ellahi Babar | Dr. Muhammad Wasim | Prof. Dr. Muhammad Abdullah.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: The background of this study is that WHO reports that psychiatry disorders affect worldwide 0.8 to 2% population. Anxiety illnesses are a class of illness associated with unreasonable and disturbing sensation of fear and tension. There are several types of anxiety disorders, such as panic disorder, agoraphobia, specific phobia, social phobia, OCD. Obsessive-compulsive disorder is a chronic disabling condition. OCD is characterized by repetitive, intrusive thoughts, images, and impulses and by repetitive, ritualistic physical or mental acts performed to reduce the attendant anxiety. The severity of OCD depends on the amount obsessions and compulsions. The Yale-Brown Obsessive Compulsive Scale (Y-BOCS) is a reliable and consistent scoring system that can be used to categorize OCD. The major genes involve in OCD are SLC6A4, BDNF, SLC1A1 and COMT genes.
The study was enrolled patients treated for OCD. Blood samples have been collected from the patients. DNA extracted from fresh blood. Primers were designed. Then DNA amplification have done by Bio-Rad thermal cycler. Then gel electrophoresis was done for PCR product quantification. PCR products precipitated and sequenced. SNPs were identified. Real-time quantitative RT-PCR was performed for each sample with TaqMan Universal PCR mastermix which showed down regulation of COMT gene in OCD patients in Pakistani population.
The aim of this study was SNP identification in Pakistani Population in Obsessive Compulsive disorder and to analyze the gene expression of COMT gene involved in OCD in Pakistani Population.
Availability: Items available for loan: UVAS Library [Call number: 2620-T] (1).
47.
Development Of Novel MtDNA Metabarcodes For Species Differentiation Of Class Reptilia
by Imran Tariq (2014-VA-505) | Dr. Muhammad Imran | Dr. Wasim Shehzad | Dr. Saadat Ali.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: The FoImer COI: mtDNA. universal primers that are considered standard for DNA barcoding of life contain so many =matches against the target sequences of vertebrate men tat they often end in failure to amplify many of vertebrate DNA eurections. This disaepancy fawn foe the seaman and designing of new metabarcode panes that can be used m ideally all inditdrals of vertebrees or at least all individuals represented in a class of Vertebrate such as Cass Reprilia. The current study was embadang on such an endeavor
The proposed study was develop new m5DNA membarc ode that may be used as universal Kilns; to amplify almost all species of Class Repalia for different formic and molectdr biodivesity analyses.
Blood and tissue samples were collected from Class Repdha (at :east 24 species from every ceder reported to be present in Pakistan) DNA was extracted from the collected specimen through stacdasd organic method. qualified and =meted and then PCR-amplified using novel universal primers selected from aligned =DNA sequences origtadng from all repdlian mitochondria DNA pnomes submitted to diens online sequence databases such as NCB: micleotide database. Tne sensitiviry. of PCR was assessed using a range of DNA come:madam. The amplified products were sequenced on A131 Genetic Analyzed following Sarge's dideacy method of sequencing.
The correctness of obtained croDNA sequences were examined visually in Chromes Lite 2.1 software and then alipmmt of these sequences were per: waxed agitinc highly similar DNA sequences in NCBI nu6eonde databases using BLAST in order to identify the coigin of la-noun =DNA sequences
sequencing everimeas and phyla...net< studies was confirm the specificity of the universal primer set developed and present a novel metabarcode for species level identification of large number of reptelian species. So, In future this barcode can be used for species identification in various fields of study such as illegal trade and molecular estimation of boidiversity.
Availability: Items available for loan: UVAS Library [Call number: 2628-T] (1).
48.
Homology & Polymorphism Analysis Of Cc2d1a Gene In Human And Canine For Cognitive Function
by Hafiz Qamar Abbas (2014-VA-214) | Dr. Muhammad Yasir Zahoor | Dr. Wasim Shehzad | Dr. Saadat Ali.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: Cognitive disability is a group of genetically heterogeneous abnormality that leads to variable degrees of cognition deficits. It has been shown that inherited disorders can be caused by mutations in large number of different genes and there is evidence for the presence of as yet unknown genes in a significant proportion of patients. This disease can affect 1-3% of overall population and higher in consanguineous families. We aimed to identifying the homology and polymorphism of the gene CC2D1A between human and canines. The present research work was carried out in four phases. The first phase was including enrolment of 10 affected non relevant families with disease history and consent was taken on consent forms as approved by IRB, UVAS. Secondly DNA extraction was done by using standard lab protocols. Thirdly amplification of the selected domains of selected gene (CC2D1A) was done through PCR amplification after designing primers of the selected domains. Sequencing of the amplified products has to be done through Sanger method and mutation analysis was conducted for variants We found two new asynonymous mutation one is deletion of c. 1664_1664delA which lead to the change in the normal function of protein (88%) and other is heterozygous mutation c.1921A/T that result in amino acid change from R to W (12%). Whereas homology analysis shows that deletion region is partially conserved as it code different amino acid but some key domains are conserved. This homology shows that deletion in this region can change the protein expression which can relate to unconscious condition like behavioral or mental retardation. This will be helpful in providing genetic counseling services to indigenous population for intellectual disability cases. Availability: Items available for loan: UVAS Library [Call number: 2627-T] (1).
49.
Molecular Characterization Of Canine Babesiosis In Ticks And Dogs
by Tahira Sarwar (2014-VA-523) | Dr. Wasim Shehzad | Dr. M. Yasir Zahoor | Dr. Ali Ahmed Sheikh.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: Babesia canis is an intra-erythrocytic parasite which cause canine babesiosis in both animals and humans. Currently, there are three sub-species of Babesia canis has been identified i.e Babesia canis canis , Babesia canis vogeli and Babesia canis rossi. Currently used diagnostic methods are clinical symptoms, peripheral blood smear microscopy and serological tests (IFAT and ELISA).Current study was conducted to compare the specificity and sensitivity of blood smear microscopy and PCR techniques to diagnose canine babesiosis. This study is comparative as well as developmental in nature. Although peripheral blood smear microscopy is cost effective and quick method of diagnosis in case of high or moderate parasitaemia in blood. But the limitations associated with microscopy include false negative diagnosis in case of low parasitaemia in chronic and asymptomatic infection,morphological similarity of Babesia with other species of Plasmodium and Theileria these limitations may lead to misdiagnose the infection due to which disease may remain unnoticed.Total 50 samples comprising of 25 blood samples and 25 ticks were collected randomly from infected dogs from June, 2015 to November, 2015. These samples were screened microscopically as well as with PCR. Out of 50 samples of dogs and ticks, 18 samples found to be positive for the Babesia canis. 11 samples are Babesia canis vogeli and 07 samples are Babesia canis canis were to be identified in positive samples of dogs and ticks.The results obtained from the study clearly show that PCR is more reliable, precise and sensitive assay for diagnosis of canine babesiosis then microscopy. It is hoped that proposed method to diagnose babesiosis will help to nullify the problems associated with microscopy. This will ultimately facilitate in the formulation of effective treatment control and vaccine development strategies which may eradicate babesiosis. Availability: Items available for loan: UVAS Library [Call number: 2642-T] (1).
50.
Genetic Identification And Characterization Of Pakistani Birds Of Perdicinae Subfamily (Partridge) Through Dna Barcoding Method
by Asim Iqbal Jutt (2013-VA-557) | Dr. Ali Raza Awan | Dr. Muhammad Wasim | Prof. Dr. Kamran Ashraf.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: Pakistani birds of Perdicinae sub family are cage and game birds. Birds includes Altectoris chukar, Ammoperdix heyi, Ammoperdix griseogularis, Francolinus francolinus and Francolins pondicerianus. Traditional methods of identification were based on the phenotypical characterization of birds, which may lead to incorrect identification, so there was need to explore their characters at DNA level for accurate identification and to establish a DNA reference.
Birds of sub-family Perdicinae have not been genetically characterized in Pakistan. A new precise method “DNA barcoding” was applied using COI gene of mDNA for authentic identification and classification of these birds. Blood and tissue samples of five species (fifteen samples) were obtained. DNA of each sample was extracted by organic method. Amplification of CO1 gene was done by using a universal set of primers BIRDF1, BIRDR1. Sequence of 450bp were analyzed using bioinformatics softwares. Each sample was aligned with its reference sequence of COI gene available on NCBI. Every nucleotide position which did not align with the reference sequence was studied to identify SNPs. A common phylogenetic tree of all partridges showed that they have common ancestor about 0.7 million year ago, F.francolinus, F.pondicerianus and A.heyi shared a common clade whereas A.chukar made a separate clade from the ancestor. A.heyi and F.pondicerianus showed closed resemblance. It has been proved that DNA barcoding is an efficient and accurate molecular tool for species identification and phylogenetic implication. This study established a DNA Data Bank that helped scientists to investigate the biodiversity, taxonomic classification, species identification and also established foundations for molecular biologists to study taxonomic uncertainties at sub species level using SNP based identifying marker. Availability: Items available for loan: UVAS Library [Call number: 2714-T] (1).
