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1. 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).

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

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

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

3. 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: book Book; 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).

4. 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: book Book; 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).

5. 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: book Book; 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).

6. Development Of Novel Mtdna Metabarcodes For The Species Differentiation Of Class Amphibia

by Rehmatullah (2011-VA-365) | Dr. Muhammad Imran | Dr. M. Yasir Zahoor | Dr. Amjad Riaz.

Material type: book Book Publisher: 2017Dissertation 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 Amphibia. 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 Amphibia for different forensic and molecular biodiversity analyses. Tissue samples were collected from order Urodela of Class Amphibia (Toads , Bull frog and skittering frogs sample were collected from Punjab, Pakistan). DNA was extracted from the collected specimens through standard organic method, qualified and quantified and then PCRamplified using novel universal primers selected from aligned mtDNA sequences originating from order Urodela 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 Summary 67 presented as a novel metabarcode (16SrRNA) for species level identification of large number of Amphibian species. In summary, we present universal method for species classification of Amphibia using a targeted parallel sequencing approach. Both sequencing and phylogenetic studies experiments confirm 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: 2874-T] (1).

7. Mutational Analysis Of Atp7b Gene Responsible For Wilson’s Disease And Its Homology Analysis In Primates And Mouse

by Amama Ghaffar (2011-VA-375) | Dr. M. Yasir Zahoor | Prof. Dr. Huma Arshad Cheema | Dr. M. Imran | Dr. Amjad Riaz.

Material type: book Book; Literary form: not fiction Publisher: 2017Dissertation note: Copper being an essential element to carry out different cellular processes normally is maintained through proper regulation mechanisms to avoid its accumulation in the body. ATP7B gene that codes for ATP dependent P type ATP7B protein controls the regulation of copper in the body. It is required for the proper delivery of copper to apoceruloplasmin and its excretion through bile in the form of feces. Therefore, mutation occurring in the ATP7B gene can cause excessive cellular copper accumulation which results into Wilson’s disease. Variation in ATP7B gene related to copper transportation leads to Wilson’s disease and transmitted in generation through recessive pattern of inheritance. For this study blood samples of fifteen Wilson’s disease affected patients along with normal individuals of the same family were collected from Children's Hospital & Institute of Child Health, Lahore. DNA was extracted from blood through organic extraction method followed by DNA quantification. Amplification of exons 8, 13, 14 and 18 of ATP7B gene was performed after designing specific primers for these specific regions. Sequencing of amplified products was done through dideoxy chain termination method. A disease causing mutation of ATP7B gene c.3155 C>T; p1052 Proline (CCC) to Leucine (CTC) has been mapped on exon 14 in family with Wilson’s disease. This mutation can be used for genetic testing, prenatal diagnosis and genetic counseling. No mutation was found in exons 8, 13 and 18 which mean that further study needs to be done to find more local mutation(s) that can be used for fast direct genetic testing of Wilson’s disease patients or the carriers with heterozygotic conditions who can develop this disease at any age of their life. Results 87 MUSCLE and Clustal Omega were used for homology analysis of ATP7B gene nucleotide and protein sequences that revealed Gorilla to be closest to human regarding coding sequences, while Clustal Omega output file showed all the species varied highly in their protein structure homologies. Through the prediction of secondary structure homologies it was seen that marmoset was closest to humans. This study helped in providing prenatal diagnosis and genetic screening services in the country. It has facilitated in selecting animal models for further study and research on ATP7B gene and molecular pathogenesis of the Wilson’s disease leading to prevention and cure of disease. Availability: Items available for loan: UVAS Library [Call number: 2892-T] (1).



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