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Study Of Genetic Polymorphism In Exon 7 And 9 Of Glucosidase Beta Acid (Gba) Gene In Gaucher Diseased Patients From Punjab, Pakistan

by Ayesha Khalid (2013-VA-07) | Dr. M. Yasir Zahoor | Dr. Sehrish Firyal | Mr. Tariq Mahmood.

Material type: book Book; Literary form: not fiction Publisher: 2015Dissertation note: Gaucher disease (GD) is an inborn metabolic disease transmitted through recessive pattern of inheritance and it is a pan-ethnic disease. It is the most common lysosomal storage disease caused by the deficiency of glucocerebrosidase (GCase), a lysosomal enzyme use in the degradation of macromolecules into simpler molecules. Glucosidase beta acid (GBA) gene encode glucocerebrosidase enzyme and mutations in this gene is responsible for glucocerebrosidase deficiency which results in an accumulation of unbroken glycolipids in those organs rich in monocyte-phagocyte immune system elements i.e. spleen, liver, bone marrow and leads to histological changes. GBA is located on chromosome 1q21 consisting of 11 exons and 10 introns having 7.8kb length. It is divided into three types (I, II and III) on the basis of neurological involvement. More than 300 mutations have been reported in GBA and cause the GD. The present study was performed in order to characterize GBA gene in GD patients from Punjab. Blood samples of 10 patients,enrolled in Children Hospital, Lahore, were taken from DNA repository of Molecular and Genomic Lab at IBBT, UVAS Lahore. The DNA was extracted using organic method. Next step was the amplification of extracted DNA using PCR. After it, the PCR product is purified and this purified PCR product was sent for sequencing. Sequencing of exon 4, 7 and 9 was done using dideoxy sequencing method. After applying different bioinformatics tool, it was found that there was no muttaion in these exons but a heterozygotic variation G>A was found in intron 8. This finging will help in demonstration of molecular pathogenesis of Gaucher disease. Availability: Items available for loan: UVAS Library [Call number: 2338-T] (1).

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2.
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Antiviral Effect Of Human Saliva Against Avian Influenza Virus Strain H9n2

by Maryam Riaz (2008-VA-340) | Prof. Dr. Tahir Yaqub | Dr. Sehrish Firyal | Prof. Dr. Kamran Ashraf.

Material type: book Book; Literary form: not fiction Publisher: 2015Dissertation note: Saliva is an important body fluid that contains a complex array of proteins, peptides and various substances that help in maintaining the health of the oral cavity. Saliva exhibits a broad-spectrum of antiviral activity against enveloped viruses as it disrupts the viral membrane. Influenza is a common virus that has been diagnosed in humans and avian species due to AIV. This study has demonstrated the naturally occurring antiviral activity of human saliva against the H9N2 influenza virus that serves as a serious threat to poultry and has been shown to possess high zoonotic potential which can cause a new pandemic. In this study saliva samples from healthy individuals were taken and the natural antiviral ability of saliva was observed against AIV (Pk-UDL/01/08 H9N2) of calculated EID50 106.66. Inoculum prepared from saliva and H9N2 virus was injected in 9 days old embryonated eggs using CAS route and incubated at 37°C for 48 hours. A negative control (only saliva) and positive control (only virus inoculum) was also determined in the current study. The antiviral activity of saliva was observed through haemagglutination test. The HA test of harvested fluid showed that human saliva indeed possesses antiviral activity against H9N2 virus and can be used as a natural antiviral agent in medicine. Furthermore, the genomic DNA was extracted from the blood samples. HTN3 gene responsible for histatin production, was amplified using gene specific oligonucleotides. The obtained HTN3 gene sequences were analyzed using Chromas software. The sequence alignment showed 99% similarity to the available sequences in NCBI database and 100% similarity to each individual sample. To conclude, this study has demonstrated that human saliva possesses antiviral activity against H9N2 virus. The nucleotide sequence analysis from each sample CHAPTER 6 SUMMARY Summary 47 showed no particular change which shows that antiviral activity of glycoproteins present in saliva does not vary at a genetic level. This innate antiviral activity can open a new frontier when it comes to combating viral infections that have grown resistant to conventional drugs in both human and animal subjects. Availability: Items available for loan: UVAS Library [Call number: 2336-T] (1).

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Genetic Identification And Molecular Classification Of Sub-Family Phasianinae Of Pakistani Bird Species Through Dna Barcoding

by Maryem Hussain (2008-VA-349) | Dr. Ali Raza Awan | Dr. Sehrish Firyal | Dr. Muhammad Tayyab.

Material type: book Book; Literary form: not fiction Publisher: 2015Dissertation note: DNA barcoding is a precise technique that uses molecular genetics tools for accurate identification, categorizing, relating and separating the phylogenies of species. Being a small sized genome and agile enough to show rapid mutation, mtDNA has been used as a pertinent marker of molecular biodiversity.The aim of this study was to develop DNA barcode for genetic characterization and classification of Sub-family Phasianinaeof Pakistani bird species. Theyhave not been genetically identified yet in Pakistan. It includes birds like domestic chicken(Gallus gallusdomesticus), aseel chicken(Gallus gallusdomesticus strain),blue peafowl(Pavo cristatus), green peafowl (Pavo muticus), white peafowl (Pavo cristatus leuticus), Kalij pheasant (Lophura leucomelanos),monal pheasant (Lophophorus impejanus),koklass pheasant(Purcrasia macrolopha), ring necked pheasant (Phasianus colchicus), Tragopan (Tragopan melanocepals) andred junglefowl (Gallus gallus). These birds are considered an important part of an ecosystembecause they play a significant role in seed dissemination, pollination of plants and disease spread which are the basic constituents of an ecosystem. They are used for food, hunting and entertainment purposes. Mitochondrial geneCytochrome c oxidase subunit 1 (CO1)of 500bps was used as a marker for identification at specie level.Genomic DNA was extracted by each blood and tissue sample of eleven bird species (33 samples). Amplification of CO1 gene was a done by using a universal set of primers (BIRDF1 and BIRDR1)containing region of almost 750 bps (Hebert et al. 2003).Amplicons were purified and sequenced Sanger sequencing method (Sanger et al. 1977). Forward and reverse sequences were analyzed using softwaresEMBOSS merger,ClustalW, BioEdit and nBLAST. Phylogenetic analysis of selected bird species was done. Each sequence was aligned with its reference sequences of CO1 gene present on NCBI. Every nucleotide position which did not align with the reference sequence was studied to identify SNPs. Fixation index (FST) were used to measure species diversity within a same sub population relative to that found in the entire population. Consensus sequences (500bps) generated was used to construct their phylogenetic tree to see their evolutionary relationship with other bird species. All species showed their closest linkage with their respective species. Pakistani population of peafowl and chicken species showed the close relation with same sequences generated in China. Tranopans showed its closest linkage with T. temminckii. In conclusion, seven species ofPhasianinaesub-family of Pakistani bird species was genetically characterized first time in Pakistan by using CO1 as a barcode. It proves that DNA barcoding is an efficient and accurate molecular tool for species identifica¬tion and phylogenetic implication. This study leads to establish a DNA Data Bank that helped scientists to investigate the biodiversity, taxonomic classification, specie identification, in forensic purposes and to study the genetic and phenotypic evolution of these species. DNA barcoding through CO1 gene works as a functional tool for detectingmeat mislabeling and preventing illegitimate trade. This study has established foundations for molecular biologists to study taxonomic uncertainties at sub species level using SNP based identifying marker. It helps in preservation and identification of endangered species by generating their barcodes from even minimal evidence available. Availability: Items available for loan: UVAS Library [Call number: 2376-T] (1).

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Molecular Characterization of Pakistani Common Leopard

by Muhammad Usman Ijaz (2012-VA-908) | Dr. Sehrish Firyal | Dr. Ali Raza Awan | Dr. Muhammad Tayyab.

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

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Identification Of Single Nucleotide Polymorphism In Toll Like Receptor 4 Gene And Its Association With Mastitis In Sahiwal Cows

by Hafiz Kamran Rizwan Ullah (2013-VA-557) | Dr. Sehrish Firyal | Dr. Muhammad Wasim | Prof. Dr. Habib Ur Rehman.

Material type: book Book; Literary form: not fiction Publisher: 2015Dissertation note: Several factors militate against realizing the milk production potential of cows. Mastitis is one of the shocking maladies of milch animals causing high production losses to livestock industry in Pakistan and elsewhere in the world. Mastitis has been familiar as one of the most inexpensively important diseases affecting dairy animal’s worldwide. Susceptibility and resistance to mastitis is a complex trait and influenced by genetic variation of the immunity genes of animals. Among these variations, the polymorphisms in Toll-like receptor 4 gene (TLR4) play important role in the immune response to mastitis. Polymorphism in exon 3 of TLR4 gene is associated with mastitis susceptibility and resistance. It is a potential candidate gene for screening of the mastitis susceptible and resistant dairy cows. The present study was designed for the identification of polymorphism in TLR4gene associated with mastitis. Blood samples from 20 Sahiwal cows having clinical and subclinical mastitis were sampled. Blood sample of 10 normal Sahiwal cows was also collected. DNA was extracted. Specific primers for amplification of TLR4 gene were designed from NCBI. TLR4gene was amplified and sequenced to get the desire sequence of this gene. Comparative analysis of the resulted sequences using NCBI BLAST was done. Availability: Items available for loan: UVAS Library [Call number: 2392-T] (1).

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