1.
Single Nucleotide Polymorphisms In Pouifi Gene And Its Association With Milk Production Traits In Pakistani Cattle
by Sadia Munir | Dr. Asif Nadeem | Dr. Abu Saeed | Dr. Muhammad Wasim.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 2011Dissertation note: In farm animals, the primary focus of selection has been to improve milk yield. Milk field is a polygenic trait and a few potential candidate genes have been recognized.
Association studies have shown that POUlFl is related to many production traits in
domestic animals and is one of those candidate genes that are involved in milk
production. POUl F 1 encodes a pituitary-specific transcription factor. It is well
established that growth hormone (GH) released from pituitary gland plays an essential
role in growth, mammary gland development and lactation process. The bovine
POUlFl gene is of 15952 bp length having 6 exons. The genetic characterization of
the POUlFl gene to identify the SNPs as genetic markers and validation of these
potential markers by associating them with milk production traits has been performed.
A total 35 samples from Sahiwal and 30 from Holstein-Friesian cattle breeds were
sequenced for all 6 exonic portions of the POUlFl by using 6 sets of primers. A total
15 polymorphic sites in Sahiwal and 14 in Holstein-Friesian were identified from
these sequences. Out of total 15 SNPs identified in Sahiwal, 12 were in intronic
region and 3 were in exonic. Out of 14 SNPs identified in Holstein-Friesian, 10 were
in intronic and 4 were in exonic region. The sequences of the amplified POUl F 1 gene
fragments were aligned with the help of BLAST for SNPs identification. This study is
first step in finding some confirmed markers for milk yield in Sahiwal and Holstein-
Friesian cattle breed that can be used in future for selection and breeding programmes.
Availability: Items available for loan: UVAS Library [Call number: 1373,T] (1).
2.
Srudy Of Gamma-Aminobutyric Acid A Receptor Delta Subnuit Gene Mutations Involved In Generalized Epilepsy With Febrile Seizures Plus (GEFS+) Patients in Punjab
by Iram Javed | Dr. Muhammad Wasim | Dr. Abu Saeed Hashmi | Dr. Asif Nadeem.
Material type: ; Format:
print
Publisher: 2012Dissertation note: World health organization (WHO) reports that neurological disorders affect one billion people worldwide, including 50 million affected by epilepsy. Epilepsy is a common neurological disorder characterized by recurrent, periodic, spontaneous and unprovoked seizures. Generalized epilepsy with febrile seizure plus (GEFS+) is an autosomal dominant disorder and a heterogeneous familial condition in which family members express febrile seizures initially, and then show multiple phenotypes of myoclonic epilepsy including partial or absence seizures and generalized tonic conic seizures. Molecular genetics techniques have identified various GEFS+ associated mutations in many genes i.e. sodium channel genes (SCN2A, SCN1A, and SCN1B) and some GABA receptor genes (GABRG2 and GABRD). GABAA receptors are the principal intermediaries of fast inhibitory neurotransmission in the eNS and have been frequently reported to playa significant role in a number of seizures. GABRD gene encodes the delta (8) subunit and is usually located in extrasynaptic GABAA receptors. The present study was aimed to investigate coding regions of GABRD gene for analyzing the mutations involved in epilepsy. Blood samples of unrelated true representative ofGEFS+ were collected from psychiatry departments of different hospitals of Lahore. DNA were extracted with the standard protocol and amplifications of the GABRD regions were done with specially designed primers. Later on, sequencing of target fragments was carried out. Sequences were analyzed through BioEdit software and then aligned with the help of custalW2 software. Out of 14 GEFS+ patients, only 3 were identified with a novel heterozygous transition mutation in intron 5. Further study, with much larger sample number, is required to revise the effects of this polymorphism and accurately identifying the associated factors. There is a need to explore the other gene mutations causing epilepsy in local population of Punjab and Pakistan that will ultimately help to develop genetic counseling strategies, gene therapies and prenatal diagnostic procedures for the population of Pakistan.
Availability: Items available for loan: UVAS Library [Call number: 1394,T] (1).
3.
Molecular Approach For Sex Determination In Avian Species
by Sehrish Basheer | Dr. Asif Nadeem | Dr. Muhammad Wasim | Madam.
Material type: ; Format:
print
; Literary form:
drama
Publisher: 2011Dissertation note: To tell the difference between male and female birds takes keen observation. While not all bird species have easily visible gender differences. Almost all birds' species are sexually monomorphic so it is difficult to distinguish between male and female characters. There are some techniques for bird's sex identification. Surgical Sexing, in this an endoscope is passed into the body cavity and ovaries or testis can be observed. It is available for all types of birds. Surgical sexing is oldest and quickest method. But it cause stress to the bird and expensive technique. Fecal steroid analysis is another technique in which stool sample is analyzed for reproductive hormone presence. In this technique bird should be sexually mature for analysis. Bload Sexing is used to determine the male or female bird. This can be done at any age. Feather exing is very useful and economical method. Retrieve a feather pulp from growing feather and analyze the presence of male and female chromosome. Two chromosomes Z and Ware present in birds. ZZ in male and ZW in female. The method used in this study was based on the chromosome differences. Nine different species of birds which includes green parrot, budgerigar, .pigeon, quail, sparrow, chicken, peacock, duck and pheasants were analyzed and their sex is determined by molecular methods. DNA will be extracted from feathers and blood. The intronic egion of CHD 1- Wand CHD l-Z gene will be amplified by sex specific primer. PCR products were screened by agarose gel electrophoresis. The PCR products were show double and single bands on the agarose gel. The double bands indicate female bird because of ZW chromosome is present in females and single band indicates the male bird because of ZZ chromosomes in males.
Availability: Items available for loan: UVAS Library [Call number: 1400,T] (1).
4.
Molecular Characterization Of Cacna 1H Gene To Fine Out Association Of Polymorhism In Childhood Absence
by Qurrat-ul-Ain | Dr. Muhammad Wasim | Dr. Muhammad Asif | Mrs. Shagufta.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 2012Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1472,T] (1).
5.
Genetic Variability Of Sahiwal And Cholistani Cattle Breeds Of Pakistan Usin Mitochondrial D-Loop Sequences
by Sania Saeed | Mr. Tanveer Hussain | Dr. Abu Saeed | Dr. Muhammad Wasim.
Material type: ; Format:
print
; Literary form:
drama
Publisher: 2011Dissertation note: Pakistan is rich in cattle genetic resources. The phenotypic and genetic diversity of animal breeds in Pakistan is very vast. Efforts to manage and utilize these genetic resources efficiently are lacking due to lack of both awareness and weakness of Government institutions. The genetic data of dairy cattle breeds (Sahiwal and Cholistani) is not yet been studied for their genetic identification, conservation and to find the genetic diversity among them and it needs to be established. For this study the blood samples( 25 samples from each breed) were collected from their home tracts and livestock farms. Unrelated animals with typical phenotypic features known for Sahiwal and Cholistani cattle breeds were selected from their breeding areas and Government livestock farms. Blood samples from true representative individuals of Sahiwal breed were collected from Research Centre for the Conservation of Sahiwal Cattle (RCCSC), Jahangirabad, Khanewal, Semen Production Unit (SPU) Qadirabad and Barani Livestock Production & Research Institute (BLPRI), Kherimurat District Attock. Cholistani cattle samples were collected from Govt. Livestock Farm, Jugaitpeer, Bahawalpur. Sampling from siblings was avoided to minimize inbreed samples as it results in depleting of gene pool along with causing inbreeding depression.DNA was extracted and quantified with the standard protocol in Molecular Biology and Genomics Laboratory of Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Lahore. Specific primers were designed by using special software i.e. Primer Fox for mitochondrial D-loop region from NCBI accession no. NC_006853.1.Primers optimization was done after primer designing and afterwards, PCR amplification was performed. Then sequencing of target fragments was carried out using Prism ABI 3130L sequencer and Analyser.Sequences were alligned with the help of software blast2sequence and SNPs were detected. It was found that ratio of transition mutation was higher than transversions i.e. 41 transition and 10 transversions. Sequences were analyzed and compared with already reported sequence of Mitochondrial DNA of Bosindicuss, Bostaurus, Bubalusbubalis, Canis lupus familiaris, Caprahircus, Equuscaballusisolate, Ovisaries and Cameliusdromedaries sequencesavailable at NCBI. Single Nucleotide Polymorphisms (SNPs) were then detected. A phylogenetic tree constructed using MEGA 5.1 software revealed that Pakistani, European and Asian cattle are genetically same but different from Buffalo.This work is very helpful about breed characterization of two cattle breeds (Sahiwal and Cholistani) and developing understanding about genetic architecture of cattle breeds as present study conclude that 52 SNPs were present in Sahiwal and Cholistani breed of Pakistan.
Availability: Items available for loan: UVAS Library [Call number: 1498,T] (1).
6.
Mutation Screen Of "Gamma-Aminobutyric Acid (Gaba)-A Receptor, Gamma 2" In Punjab Population
by Muhammad Javed Iqbal | Dr. Muhammad Wasim | Dr. Abu Saeed | Dr. Ali Raza Awan.
Material type: ; Format:
print
; Literary form:
drama
Publisher: 2012Dissertation note: Epilepsy is a formidable form of neural disorder that can impose its long lasting effect on person's life and development. To date, it lacks any effective therapy and is multistep disease strengthened by an overwhelming number of genetic and epigenetic mechanisms that streamline epileptic attacks. This particular study encompasses two major types of epilepsy, CAE and GTCS by targeting a GABRG2 gene. Mutation analysis of the coding exons (exon 3, 5 and 9) was performed by direct sequencing of GABRG2 in order to sought out complex biological entities in both types of epilepsies. GABRG2 is a molecule that has recently been characterized as the culprit for epileptic seizures onset. GABRG2 encodes GABA receptor that is fundamental inhibitory neurotransmitter in mammalian brain and is a ligand-gated chloride channels. This ligand-receptor coupling results in the inward shuttling of chloride ions through the channels and this hyperpolarizes the neurons, which induce the inhibitory effect of neurotransmitters. Direct sequencing of candidate gene "GABRG2" traced out a single polymorphic site in the exon 3 of the CAE as well as GTCS cases. However, this single nucleotide alteration is more commonly identified in childhood absence epilepsy patients as compared to the generalized cases. Silent mutation was identified at locus 27909 C>T of 46.66% of the total screened or analyzed cases.
Availability: Items available for loan: UVAS Library [Call number: 1500,T] (1).
7.
Assessment Of Genetic Diversity In Balochi And Rakhshani Sheep Breeds Of Balochistan Using Microsatellite Dna
by Abdul Wajid | Dr. Muhammad Wasim | Dr. Abu Saeed | Mr. Tanveer Hussain.
Material type: ; Format:
print
Publisher: 2011Dissertation note: Pakistan being agriculture based country has a great potential in livestock sector, it plays an important role in the economy of the country. Pakistan is rich in Animal Genetics Resource (AnGR) and has various breeds of sheep but lacking genetic data of these breeds which need to established data for their genetic identification. Customarily, classification of breed was based on phenotypic traits. In some cases, recent genetic studies have found differences in the structure proposed. Molecular characterization is a prevailing tool to consider the genetic variation existed within and among breeds. Characterization and evaluation of genetic differences among these breeds is necessary for their effective and meaningful improvement and conservation. The advent of molecular techniques has led to an increase in the studies that focus on the genetic characterization of domestic breeds using genetic markers. Due to their reliability and availability, the microsatellites have become preferred method for the genome mapping. Microsatellites or STRs are the 2-6 nucleotide tandem repeats present in both coding and non coding regions of both prokaryotes and eukaryotes. Microsatellites are powerful tools in genome mapping, forensic DNA studies, paternity testing, population genetics and conservation/ management of biological resources.
The present study was conducted on the molecular diversity analysis of two sheep breeds Balochi and Rakhshani of Balochistan using 11 FAO recommended microsatellites markers. Blood samples of unrelated true representative animals of sheep breeds were selected from their breeding tracts and from different Government Livestock Farms in Balochistan province. DNA was extracted with the standard protocol and amplification of DNA done with selected markers in Molecular Biology and Genomics Laboratory in the Institute of Biochemistry and Biotechnology. PCR products were examined on non denaturing Polyacralamide Gel Electrophoresis (PAGE). Genotyping results vanalyzed through the software POPGENE VERSION 1.31 and "POWER STATE" for calculating the observed and expected number of alleles, expected and observed heterozygosity, homozygosity, F-statistics (FST, FIT, FIS), Polymorphic Information Content (PIC), matching probability power of discrimination and power of exclusion. This work provided the genetic data which is useful in breed identification and making effective breeding policies and conservational activities in future according to FAO global Farm Animal Genetic resource data.
Average observed heterozygosity, average observed homozygosity, observed number of allels (na) and expected number of alleles for all loci and population in this study were 0.6055, 0.3945, 6.3636 and 4.2805 respectively. Almost all of the microsatellite markers showed significant variations in both breeds of sheep.
This work provided the genetic data which will be helpful in breed identification and making effective breeding policies and conservational activities in future according to FAO global Farm Animal Genetic resource data. Moreover this study can become the basis for further research investigations in sheep breeds in Balochistan and Pakistan.
Availability: Items available for loan: UVAS Library [Call number: 1516,T] (1).
8.
Develoopment Of A Reliable Microsatellites Maarkers Panel For Parentage Analysis In Cattle Breeds Of Pakistan and Its Validatio Through Cytochrome B Gene Sequencing
by Tanveer Hussain | Prof. Dr. Masroor Ellahi Babar | Dr. Ahmad Ali | Dr. Muhammad Wasim.
Material type: ; Format:
print
Publisher: 2013Dissertation note: Pakistan posseses enormous Animal Genetic Resource (AnGR) with 36.9 millions of cattle population. The data on genetic fabric of these breed is yet to be documented for their genetic characterization and identification. This work reports first country wide microsatellite markers and cytochrome b gene based genetic characterization of 10 famous cattle breeds of Pakistan. A total of 352 blood samples from unrelated and phenotypically representative of ten native cattle breeds including Bos indicus; Sahiwal, Cholistani, Red Sindhi, Tharparker, Dhanni, Dajal, Lohai, Bhagnari, Achai and Bos indicus x Bos taurus; Nari Master, and an exotic Bos taurus; Holstein Friesian breeds were collected from their respective home tracts, institutional herds and private livestock farms located throughtout the country. These samples were subject to DNA extraction using inorganic method caliberated to same concentration in Molecular Biology and Genomics Laboratory of the Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Lahore Pakistan. A total of 21 microsatellite markers recommended by the programme for the global management of genetic resources (MoDAD) for breed characterization of Food and Agriculture Organization (FAO) of the United Nations and International Society for Animal Genetics (ISAG) were applied. Multiplex PCR were optimized for amplification and were genotyped using ABI Genetic Analyzer 3130 xl using LIZ as size standard. Genotyping results were analyzed using POPGENE and Arlequin ver 3.5 software. The observed and effective number of alleles ranged from 10 (INRA32) to 43 (TGLA126) and 2.3574 (CSSM66) to 15.0019 (BM6526) respectively in all breeds? The observed and expected heterozygosity estimates ranged from 0.0638 (INRA32) to 0.7101 (BM2113) and 0.6510 (INRA32) to 0.9347 (BM6526) respectively in the experimental samples. Mean values for observed and expected heterozygosity was 0.4943 ± 0.1647 and 0.8164 ± 0.0930 respectively. Mean values for Fis, Fit and Fst in all cattle breeds were calculated as 0.2819, 0.3864 and 0.1456 respectively. Average polymorphic information content (PIC) of all microsatellite loci was 0.81 indicating a high degree of informativeness of all microsatellite markers used. It implies that the same set of markers is equally good and could reliably be used for parentage confirmation in Pakistani cattle breeds. The data produced, also showed least degree of genetic difference between Red Sindhi and Tharparker breeds. This may due to mixing of the two breeds for being in close proximity of their home tracts.
Fragment mitochondrial cytochrome b gene was also amplified using specific primers through PCR of 130 individuals representing all selected breeds and sequencing was done using ABI Genetic Analyzer 3130 xl. The sequences were aligned and analyzed with CodonCode Alligner 4.0.4 software. The analysis revealed highly degree of sequence conservation in all the Pakistani cattle while documenting changes in only 9 nucleotides from 26 individuals whereas multiple nucleotide changes in 5 locations were shown by more than one individual in the data presented. One polymorphic site was found in nucleotide 318 (T?C) in several breeds of indicine cattle while 2 Lohani and 5 Nari Master individuals showed nucleotide changes specific to taurine cattle. Of all the changes found, only three of them caused changes in the amino acid sequence. The UPGMA tree using MEGA 5.1 showed a clear differentiation between taurine and indicine cattle, except for Nari Master Pakistani cattle showing mitochondrial taurine sequences because it's a cross between Bhagnari (Bos indicus) and Australian Draught Master (Bos taurrus). The estimates of divergence among breeds were also low for most breed pairs, except for Nari Master and Dhanni whereas the overall divergence within Bos indicus or within Bos taurus were also very low (0.002 and 0.003, respectively) but the differences between Bos indicus and Bos taurus were significantly higher (0.014) as should be the case.
These results of microsatellite markers have produced a set of information that can be recommended as a reliable marker panel for studies on genetic diversity analysis, parentage confirmation. The cytochrome b data on the other hand not only substantiated genetic diversity analyses but it also proved to be equally good for comparative Phylogenetic analysis of Pakistani cattle breeds and exotic breeds. This work provides most authenticated data and adds a great deal, to already existing information on Pakistani AnGR. This information coupled with prospective data using next generation genetic technologies will assist designing breed improvement focused breeding policies and conservation activities in future.
Availability: Items available for loan: UVAS Library [Call number: 1597,T] (1).
9.
Designing The Small Interference Rna Against Expression Of Coat Protein (Cp) Gene Of Potato Virus X (Pvx)
by Shafique Ahmed | Prof. Dr. Tahir Yaqub | Dr. Muhammad Wasim | Ms. Faiza.
Material type: ; Format:
print
; Literary form:
drama
Publisher: 2013Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1605,T] (1).
10.
An Insight Into Mutational Analysis Of B-Cell Lymphoma-2 (Bcl-2) Gene And Its Involvement In Pets Cancer
by Asma Irshad | Dr. Muhammad Wasim | Mr. Akhtar Ali | Ms. Shagufta Saeed.
Material type: ; Format:
print
Publisher: 2013Dissertation note: There are various type of tumors associated with dog (Canis familiaris) and cat (Feline catus) which are responsible for death of these pets. Bcl-2 proto-oncogene was firstly depicted as of the t(14;18) trans-location cut-off point inside human follicular B-cell lymphoma. The Bcl-2 protein is a core control device of planed cell death as well as is concerned within DNA transformation, cell-cycle and differentiation control. Bcl-2 expression within endothelial cells was described en route for enhance cancer metastasis. Mammary gland tumors are the mainly frequent neo-plasms happening into feminine dogs and cats and are malevolent inside more or less 50% of the cases. Bcl-2 expression is not merely interrelated through an enhanced expression but as well by means of an abridged aptitude on behalf of far-away immigration of mammary gland cancer cells. Metastasis to tissues like skin, nasal passage and oral cavity has also been reported in 5-6.9 percent of cases.
Various parameters, used in the present study were aimed to analyze coding regions of Bcl-2gene to study the mutations involved in cancers. Blood samples of unrelated true representative of cancers were collected from Pet center, University of Veterinary and Animal Sciences, Lahore. DNA was extracted with the standard protocol and amplification of the Bcl-2 gene was done with specially designed primers.
Later on, analysis of the results was done by sequencing of amplicons. Sequences were analyzed through BioEdit software and then aligned with reference sequence using clustalW2 software.
In the present study, analysis of mutations was done in Bcl-2gene isolated from Canis familiaris and Feline catus. But not a single nucleotide polymorphism was found in exon 1 and 2 of Bcl-2 gene isolated from blood of affected animals with different cancer types.
In the conclusion, we report that no mutations were observed in the Bcl-2 gene isolated from different affected pets. It may be due to limited number of samples and/or require extraction of DNA from tumor tissue. There is a need to explore the other gene mutations causing cancers in population of pets that will ultimately help to develop genetic counseling strategies, gene therapies and prenatal diagnostic procedures for the population of Pets.
Availability: Items available for loan: UVAS Library [Call number: 1612,T] (1).
11.
The Study Of Retinoblastoma (Rb) Gene Mutations Involved In Different Types Of Cancers In Dogs And Cats.
by Siddra Pervaiz | Dr. Muhammad Wasim | Dr. Muhammad | Dr. Muhammad Yasir Zahoor.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 2013Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1632,T] (1).
12.
Dna Quantification Of Blood Stains Recovered From Washed Fabrics
by Zeenat Salah-ud-din | Dr. Muhammad Yasir Zahoor | Dr. Muhammad | Dr. Muhammad Wasim.
Material type: ; Format:
print
; Literary form:
drama
Publisher: 2013Dissertation note: Bloodstains are extremely important forensic evidences for DNA profiling. Bloodstains
recovered at crime scene can help to identify and confirm the credentials of the victim. Criminal
can make all efforts to destroy and hide possible evidence at crime scene therefore these
bloodstains on the crime place can hardly provide any evidence against the culprit. The criminal
will try to take away or change clothes with the bloodstains of the victim. Once perpetrator has
removed the bloodstains, culprit will feel secure and satisfied. But as a suspect if clothes are
recovered with washed bloodstains and forensic evidence of DNA of the victim can be recovered
from the washed clothes and can provide strong evidence against suspect. Present study has been
planned to quantiate DNA from 10 different types of bloodstained fabrics with possible ways of
washing to hide the blood from fabrics. 100 ìl of preservative blood was used in creating blood
spot on fabric. After that dried bloodstained fabrics were hand washed with tap water and in
detergent for 5 and 10mins and air dried at room temperature (25°C- 30°C) in laboratory. DNA
was extracted through Chelex® 100 extraction procedure from these fabrics and analyzed on
Real time PCR kit i.e. Quantifiler™ Human DNA Quantification kit which is specific for Human
DNA. Research study showed there was no statistically significant difference in DNA
concentrations among different washing methods (p-value = 0.318). There was statistically
significant difference in DNA concentrations among different types of fabrics divided into two
categories (p-value = 0.001, 0.002). Research findings showed that cellulose fibers recovered
highest quantity of DNA, followed by artificial fibers and then protein fibers (cellulose
>artificial>protein fiber) despite their washing methods. Thus provides a lead in solving such
type of criminal cases.
Availability: Items available for loan: UVAS Library [Call number: 1663,T] (1).
13.
Deoxyribo Nucleic Acid Extraction & Qantification From Human Saliva Deposited On Fruits With Human Bite
by Shahid Nazir | Dr. Muhammad Wasim | Dr. Muhammad Yasir Zahoor | Ms. Faiza.
Material type: ; Format:
print
; Literary form:
drama
Publisher: 2013Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1717,T] (1).
14.
Comparison Of Locally Available Synthetic And Non-Synthetic Powders For Latent Fingerprint Development
by Arman Khan | Dr. Muhammad Wasim | Dr. Abu Saeed Hashmi | Dr. Wasi.
Material type: ; Format:
print
Publisher: 2013Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1718,T] (1).
15.
Estimation Of Caffeine In Decaffeinated Coffee And Tea Available In Pakistan
by Muhammad Abbas Sadiq | Dr. Muhammad Wasim | Dr. Abu Saeed Hashmi | Ms. Sehrish.
Material type: ; Format:
print
; Literary form:
drama
Publisher: 2013Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1719,T] (1).
16.
To Investigate The Morphology Of Lip Prints And Their Effectiveness In Individualization And Sex Determination
by Makhdoom Saad Waseem Ghouri | Dr. Muhammad Wasim | Dr. Abu Saeed | Prof. Dr. Tahir Yaqub.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 2013Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1720,T] (1).
17.
Estimation Of Cyanide In Different Speciis Of Apple Seed
by Zohra Bhatti | Dr. Muhammad Wasim | Dr. Abu Saeed Hashmi | Dr. Ali Raza.
Material type: ; Format:
print
Publisher: 2013Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1748,T] (1).
18.
Method Development And Estimation Of P-Phenylenediamine In Biological Sample.
by Muhammad Adnan Jamil | Dr. Muhammad Wasim | Dr. Abu Saeed Hashmi | Ms. Sehrish | Faculyt of Biosciences.
Material type: ; Format:
print
; Literary form:
drama
Publisher: 2013Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1754,T] (1).
19.
Estimation Of Various Adulterants In Milk Available In Local Market
by Farhan Tanveer | Dr. Muhammad Wasim | Dr. Abu Saeed | Ms. Sehrish Firyal.
Material type: ; Format:
print
; Literary form:
drama
Publisher: 2013Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1781,T] (1).
20.
Characterization And Use Of Phytase Producing Bacterial Isolates To Enhance The Nuteitive Value Of Poultry Feed
by Sohail Aslam | Prof, Dr. Ahmad anjum | Dr. Imran Altaf | Dr. Muhammad Wasim.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 2014Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1930,T] (1).
21.
Molecular Investigation Of K99 Enterotoxigenic Escherichia Coli
by Nida Javaid | Prof. Dr.Tahir yaqub | Dr. Muhammad wasim | DR.Abu Saeed.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 2014Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1967,T] (1).
22.
Forensic Utilization Of Synthetic And Non Synthetic Powders And Liquid Soaps For Development Of Fingerprints On Adhesive Sides
by Sobia nosheen | Dr. Muhammad Ashraf tahir | Dr. Abu saeed | Dr. Muhammad wasim.
Material type: ; Format:
print
Publisher: 2014Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1993,T] (1).
23.
Analysis Of Epidermal Growth Factor Receptor Gene Polymorphism Implicated In Tumors Including Oralsquamous Cell
by Noveen nawaz | Dr. Muhammad Wasim | Dr. Muhammad | Dr. Muhammad Yasir zahoor.
Material type: ; Format:
print
Publisher: 2014Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1998,T] (1).
24.
Detection And Analysis Of Traces Of Ignitable Liquids On Burnt Substeates
by Abdul basit | Akhtar ali | Dr. Abu Saeed | Dr. Muhammad Wasim.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 2014Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 2007,T] (1).
25.
Analysis Of Cyclin-Dependent Kinase Inhibitor P16 Polymorphism In Canin Tumors
by Hafiz muhammad farooq yaqub | Dr. Muhammad wasim | Dr. muhammad imran | Ms. Faiza.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 2014Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 2047,T] (1).
26.
Evaluation Of Different Methods Of Dna Extraction From Burnt Human Tissues And Generation Of Geneticprofiles For Identification
by Anum yousaf | Dr. Muhammad Wasim | Dr. Muhammad | Dr. Muhammad Tayyab.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 2014Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 2081,T] (1).
27.
Study Of Candidate Genes Involved In Milk Production Traits For Identification Of Probable Biomarkers In Nili-Ravi Buffalo
by Maryam Javed | Prof. Dr. Masroor Ellahi Babar | Dr. Abu Saeed | Dr. Muhammad Wasim.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 2014Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 2120,T] (1).
28.
Differential Expression And Mutation Analysis Of Heat Shock Proteins (Hsps) And Tumor Suppressor Gene (P53) In Differemt Cancer Types of Pakistani Dogs and Cats
by Rashid Saif | Dr. Muhammad Wasim | Dr. Ali Raza Awan | Dr. Muhammad.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 2014Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 2170,T] (1).
29.
Biochemical Evaluation Of Armoracia Rusticana And Raphanus Sativus On Alloxan Induced Diabetic Rats
by Nadia Rana (2012-VA-540) | Ms. Asma Waris | Dr. Abu Saeed Hashmi | Dr. Muhammad Wasim.
Material type: ; Literary form:
not fiction
Publisher: 2014Dissertation note: Diabetes mellitus (DM) is a metabolic disorder characterized by hyperglycemia in which the body does not produce or properly utilize insulin. It the reason of interruption in protein, carbohydrate and lipid metabolism and caused the complications such as nephropathy, microangiopathy and retinopathy. It is the most widespread endocrine disorder, affects more than 176 million people worldwide (WHO 2004).
Diabetes mellitus is generally classified into three types; type I, type II diabetics and gestational diabetes (Velho and Foguel, 2002). Type I diabetes mellitus is commonly occur among young people, it is also known as juvenile-onset diabetes or insulin dependent diabetes mellitus. Type I is the result of absolute deficiency of insulin that is commonly caused by the chronic autoimmune disease that results from complex interaction of both genetic and environmental factors (Pietropolo 2001). Type II diabetes mellitus is mostly occur in adults aged 40 years or above, it is commonly known as non-insulin diabetes mellitus characterized by too much hepatic glucose production, reduced insulin secretion from beta cells of pancreas, and peripheral tissues such as muscle adipose and liver become resistant to insulin (Ahmad 2006).
Association of hyperglycemia with long term damage, dysfunction as well as ultimate organs failure, mainly the heart, blood vessels, eyes, kidney and nerves has previously been recognized (Hung et al. 2005).
Dyslipidemia is another main reason of mortality and morbidity that results in development of cardiovascular complications (Reasner 2008). It is a main risk factor of diabetes and mostly result from prolonged hyperglycemia and insulin resistance in both (type I and type II) diabetic patients is called ‘diabetic dyslipidemia’ (Mooradian 2009). Hyperlipidemia and an increase in blood cholesterol and triglyceride are results from decrease in lipolysis which is caused by deficiency of insulin, eventually increases the risk of heart attack and atherosclerosis (Avramoglu et al. 2006). The risk of heart disease, stroke, kidney disease, retinopathy, neuropathy, ulceration and gangrene of extremities is increased with association of diabetes mellitus (Rotshteyn and Zito, 2004).
According to current statistics, diabetes mellitus is worse or greater in developing countries than the developed countries worldwide (Oputa 2002). So there is a great need to discover, design and test new drugs having dual therapeutic properties to control and cure both closely related critical diseases, diabetes and dyslipidemia and their mutually linked chronic complications (Bhandari et al. 2002).
In order to design and develop the drugs for the treatment, one of the best strategies is experimental animal models to understand pathophysiology of any disease (Rees and Alcolado, 2008; Chatzigeorgiou et al. 2009). For studying and testing anti-hyperglycemic agent, several animal models have been developed for the past few decades (Srinivasan and Ramarao, 2007). Chemical induction of experimental diabetes by alloxan is one of the most effective methods (Etuk 2010).
Alloxan is a widely used diabetogenic agent that induced the type I diabetes in animals but it also represent the end stage type II diabetes milletus: as there is severe deficiency of insulin in plasma, the end stage type II diabetes mellitus also adopts the characteristics of T1DM (Viana et al. 2004). Alloxan exerts its action by generating reactive oxygen species (ROS) along with cytosolic calcium raised in islet B of pancreas, when administered parenterally (Szkudelski 2001). Diabetic dyslipidemia is also acquired by the untreated alloxan induced diabetic animals (Alnoory et al. 2013).
Currently herbal remedies are in great demand due to side effects associated with therapeutic synthetic drugs (Mahmood et al. 2011). There are large numbers of plants that have shown effective hypoglycemic activity after laboratory testing, more than 1200 plants species are used in the treatment of diabetes mellitus worldwide (Eddouks et al. 2005).
It is believed that antioxidants present in the diet help to reduce certain diseases, vegetables are rich in these compounds (Astley 2003; Bazzano et al. 2002). There are large number of herbs, spices and other plant materials that have shown hypoglycemic and antioxidant properties, and are less harmful than synthetic drugs (Eidi et al. 2006). For the development of new pharmaceutical lead along with dietary supplement to already existing therapies, medicinal plants provide a valuable source of oral hypoglycemic compounds (Bailey and Day, 1989).
Raphanus sativus (radish) belong to the family Brassicaceae and it is an edible root vegetable (Lewis-Jones et al. 1982). Radishes contain high quantity of calcium, magnesium potassium, copper, ascorbic acid, folic acid, vitamin B6, and riboflavin and low amount of saturated fat and are very low Cholesterol (Nunes et al. 2011). Roots, seeds and leaves are the different parts of radishes (Raphanus sativus) that are used for medicinal purposes (Nadkarni et al. 1976). Radish roots are beneficial to protect the cell membranes against lipid peroxidation and also inhibit the changes in membrane caused by fat rich diet (Sipos et al. 2002).
Radishes (Raphanus sativus) have good hypoglycemic potential coupled with antidiabetic efficiency (Shukla et al. 2011). Due to hyperlipidemia the probabilities of cardiovascular disease increases in diabetic patient. Raphanus sativus (radish) is a traditional plant which is used to lower plasma lipid. It has the capability to lower the plasma triglyceride, cholesterol, and phospholipids in normal rats (Taniguchi et al. 2006).
Radishes are recommended as an alternative treatment for various diseases including hyperlipidemia, coronary heart diseases and cancer due to its high medicinal and nutritional value (Cetin et al. 2010). Phosphatase, catalase, sucrase, amylase, alcohol dehydrogenase and pyruvic carboxylase are the main enzymes that found in the radish roots (Singh et al. 2013). It is beneficially used in curing poor digestion and liver dysfunction (Lugasi et al. 2005), antioxidant activities (Wang et al. 2010), anti tumorigenic (Kim et al. 2011), anti-diabetic (Shukla et al. 2010). The leaves of radish are good source of protein (Singh and Singh, 2013).
Armoracia rusticana (Horseradish) belongs to the Brassicaceae family; it is a hardy perennial plant, mustard and cabbage are also including in this family. The roots of horseradish are rich in vitamin C and B1, iron, potassium, calcium and magnesium, phytoncide and essential oils; Allyl isothiocyanate a (volatile aglycone) which is released by a glycoside is identical with the essence of mustard plant (Istudor 1998). Root of horseradish smells pungent due to the allyl sulfide, a substance present in garlic and onion.
Armoracia rusticana is a source of many compounds that have been broadly studied for various health benefits (Lin et al. 2000). It contains several substances that have beneficial effects on peripheral blood flow. Its utilization normalizes the blood pressure and prevents the risk of thrombosis and sulfurous substances also improve the elasticity of cerebral and coronary blood vessels (Cirimbei et al. 2013). It has antibacterial properties due to allyl isothiocyanate present in volatile oils, especially mustard oil (Rosemary 1976).
The main component of the horseradish and the other vegetables from Brasicaceae family is sinigrin, degraded by the myrosinase enzyme complex to the allyl isothiocyanate (Wang et al. 2010). The enzyme horseradish peroxidase, is a heme-containing enzyme found in the plant that utilizes hydrogen peroxide to oxidise a extensive variety of organic and inorganic compounds, widely used in molecular biology and biochemistry (Bladha and Olssonb, 2011).
Availability: Items available for loan: UVAS Library [Call number: 2206,T] (1).
30.
Bioconversion Of Molasses To Glucose Oxidase Through Solid State Fermentation With Aspergillus Niger
by Wajeeha Zafar (2012-VA-574) | Dr.Abu Saeed Hashmi | Dr. Muhammad Tayyab | Dr. Muhammad Wasim.
Material type: ; Literary form:
not fiction
Publisher: 2014Dissertation note: Enzymes can be defined as soluble colloidal organic catalysts which are produced by living cells and are capableof acting independently of the cells. Glucose oxidase belongs to oxidoreductaseand is also called as glucose dehydrogenase. The glucose oxidase enzyme (GOX) oxidizes glucose to gluconic acid. In cells, it aids in breaking the sugar down into its metabolites. Glucose oxidasehas found several commercial applications including glucose removal from dried egg; improvement of color, flavor, and shelf life of food materials; oxygen removal from fruit juices, canned beverages. It has also been used in an automatic glucose assay kit in conjunction with catalase and chiefly in biosensorsfor the detection and estimation of glucose in industrial solutions and in body fluids such as blood and urine. It is often extracted from Aspergillusniger. GOX is a dimeric protein. The active site where glucose binds is in a deep pocket. This enzyme acts outside of cells, is covered with carbohydrate chains (Raba and Horacio 1995).
Aspergillus niger is the potential source for the production of glucose oxidase and is preferreddue to its high production ration of extracellular enzyme. The ability of Aspergillus niger toutilize a wide range of waste products as nutrition source makes it more economical source of the enzyme (Rajesh et al .2002).
The glucose oxidase fromA. nigerisalso an intracellularenzyme present in the mycelium of the organism. Aspergillus nigeris a filamentous fungus belonging to phylum Ascomycota. It Produces microscopic conidia on conidiophores that are produced asexually. Hyphae possess septa and are hyaline. They are supported at their base by foot cells from which conidiophores originate. It possesses long, double-walled, smooth and colorless to brown conidiophores.It is commonly foundin mesophilic environments such as soil, plants and enclosed air environments. It is capable of surviving in various environments, it is not only a xerophilic fungus, but is also a thermo tolerant organism. It is because of this property that it exhibits a high tolerance to freezing temperature(Schuster 2002).
Glucose oxidase was first isolated from mycelia ofA. nigerandPenicilliumglaucumby Müller.
A large number of microbes including bacteria and filamentous fungi have been used for the production of glucose oxidase. Glucose oxidase is produced at large scale using A. nigerand P. amagasakiense. Many bacteria are also involved in the production of this enzyme; some of these are Zymomonasmobilis, Micrococcus and Enterobacte(Yogananth et al. 2012).
Glucose oxidase (GOX) from Aspergillus niger is a well-characterised glycoprotein consisting of two identical 80-kDa subunits with two FAD co-enzymes bound. Both the DNA sequence and protein structure at 1.9 A have been determined that these identical subunits size vary from 70 to 80 KDa. It catalyzes the oxidation of D-glucose (C6H12O6) to D -gluconolactone (C6H10O6) and hydrogen peroxide. It is produced naturally in some fungi and insects where its catalytic product, hydrogen peroxide, acts as an anti-bacterial and anti-fungal agent (Ikram et al . 2014).
Glucose oxidase has a molecular weight of 160,000 a.m.u. (Tsugeet al .1975) and consists of two identical polypeptide chain subunits having nearly equal molecular weights linked by disulphide bonds (O'Malley and Weaver 1972) and it is highly specific for β-D-glucose (Bentley 1963). Each subunit of the glucose oxidase contains one mole of Fe and one mole of FAD (Flavin adenine dinucleotides) and it contains 74% protein, 16% natural sugar and 2% amino sugars (Tsugeet al. 1975). The Glucose oxidase enzyme in its purest form is pale-yellow powder. The molecular weight of GOX ranges from approximately 130 kDa to 175 KDa (Kalisz et al. 1997).
Gluconic acid, the oxidation product of glucose, is a mild neithercaustic nor corrosive, non-toxic and readily biodegradable organic acid of great interest for many applications. As a multifunctional carbonic acid belonging to the bulk chemicals and due to its physiological and chemical characteristics, gluconic acid itself, its salts (e.g. alkali metal salts, in especially sodium gluconate) and the gluconolactone form have found extensively versatile uses in the chemical, pharmaceutical, food, construction and other industries (Anastassiadis and Morgunov 2007 ).
This enzyme is present in all aerobic organisms and normally functions in conjunction with catalase (Coxon and Schaffer 1971). This enzyme is also used as an antioxidant (Berg et al. 1992). It is mainly available from microbial sources and is normally produced by aerobic fermentation of Aspergillus nigerand Penicillium species (Fiedurak1996; Lu et al. 1996; Plush et al .1996; Rando et al. 1997). It has high specificity for D-glucose (Kuly and Cenas 1983).
This enzymeis also widely used to produce gluconicacidthatGOXtogether with Horse Reddish peroxidase has a range of applications in the food industry for glucose determination. GOX is being used in the textile industry producing hydrogen peroxide for bleaching process. This enzyme is also used to determine capillary glucose in screening of gestational diabetes (Mesiggi et al. 1988). This enzyme is utilized to extend the shelf life of fish(Field et al.1986) andproduction of calcium gluconate, gluconic acid and its derivatives (Khurshid2009).
Solid state fermentation [SSF] has been recently considered as the most cheapest and more environmentally friendly relative to submergedliquid fermentation [SLF] in the production of value added industrial based products such as enzymes, bio fuels.Advantages of Solid State Fermentation over Submerged Fermentation isHigher volumetric productivity, usually simpler with lower energy requirements, Might be easier to meet aeration requirements, Resembles the natural habitat of some fungi and bacteria and Easier downstream processing (Mienda et al. 2011).
Molasses is a dark brown, almost black, moist granular sugar. Its distinctive molasses taste is due to its high content of minerals. Nutritively, it has high iron content (Draycott and Philip 2008).
Aspergillus niger is a fungus, one of the most common species of the genus Aspergillus.The genus Aspergillus isimportant economically, ecologically and medically (Nizamuddinet al.2008).
Glucose oxidase enzymewas producedthrough the microbial fermentation. For that purpose solid state fermentation wasdevelopedwithA.niger. Solid state fermentation was applied to utilize agricultural residue such as Molasses as substrate.
The current research work was focused on production ofextracellular Glucose Oxidase (GOX) fromAspergillusniger using industrial waste such as molasses as substrate by Solid state ( static ) fermentation.
Availability: Items available for loan: UVAS Library [Call number: 2219-T] (1).
31.
Detection And Analysis Of Improvised Explosive Devices Used In Terrorism Activities In Pakistan
by Arslan Nazar (2012-VA-630) | Dr. Sehrish Firyal | Dr. Muhammad Sarwar | Dr. Muhammad Wasim | FaizaMasood.
Material type: ; Literary form:
not fiction
Publisher: 2014Dissertation note: Defence and security organizations are in steadyrequirement of finding new options for the detection of explosives. Fundamental applied research in this area focuses on uncovering of highly energetic substances as well as home-basedexplosives that could be a weapon of mass devastation (Marshal and oxley, 2009 yinon and Zitrin 1996, Scubert and Rimiski-Korsakove, 2006). Current methods of detection for explosives or highly energetic materials are based on a wide variety of technologies that focus on either massexplosives or little portions of explosives. Mass explosives can be distinguishedin some way by imaging features,character of the explosives charge, wires and detonators or unswervingly by spotting the chemistry and dielectric characteristics of the explosive substance. Trace recognition method relay on revealing of vapours given off by the explosives or on explosive’sconstituent part that are set down on nearbyexteriors (national Academy of sciences Committee, 2004). Even though, numerous published material is available about methods of sensing of explosives present in air,water, clothing,soiletc and these put forward the benefit of providing traceconfines of sensation at ppb intensities (Caron et al,. 2010; Hilimi and Luonge). Inthe bulk of the criminal acts, sampling is done at the scene trailed by a sample preparationmove, to be shortly processed by a particular technique for analysis. Sampling and samples preparation are amidmajor, shortcomings in explosive uncovering in many cases frightening the physical condition and life of examiner and the responding officer.
Improvised explosive devices are widely used by military in wars and police to keep up regulation and command. Gush of terrorist activities and increase in criminal conduct have been a matter of great concern worldwide and particularly for Pakistan. There have been motiveless
annihilation of private and community properties as well as industrial centres, causing irretrievabledamages to state and local markets and imperillinghumanexistence(Shen et al. 2005). Potentially perilous explosives like dynamite, varied military explosives havingnitroglycerine (NG), Cyclotrimethylenetrinitramine commonly known as RDX, Cyclotetramethylenetetranitramine and alternativehome-produced low explosives and provocative devices are now currently promptly obtainable to scandalous and terrorists. The haphazard and deliberate uses of these explosives consist ofextortion of cash and taking vengeance, unlawful transportation of prohibited substances, assassinations, terrorist and delinquent activities in numerous regions of the country (Sharma and Lahiri 2005).
Recognition of detonating method, estimating the path ways taken by explosive transportation andarresting the anti-social charactersconnected with unstable materials and explosions is primary aim of explosive analysis. For this purpose various explosive substances and explosive remains are to be examined qualitatively and the ingredients are to be approximated quantitatively using primarily by thin layer chromatography (TLC). TLC is a technique employed for the screening of organic constituents at hand in the post blast samples. The identification of explosives containing alkylammonium nitrate is done by TLC. Secondly Gas chromatography with mass spectrometry (GC-MS) technique with the benefit of anelevated resolving supremacy is avitalapparatus for the analysis of chemical composition of explosives. The extremelyproficient GC analysis withcapillary columns authorizes the examination of explosive hydrocarbons, identical substances of nitroaromatics, hexogen (RDX) and the high explosive pentaerythritoltetranitrate (PETN) in a single run. The spectroscopic identification of explosive materials by FTIR is striking due to the intrinsicpotential of real-time detection, non-vicious analysis, and nominal sample preparation, thirdly the scanning electron microscope (SEM) produces an increased image of the sample based on the contact of an electron beam with the sample’s exterior. Finding of minutemasses of explosive remains play an important part in forces, inhabitant, and counter terrorism requests(Pacheco-Londono et al. 2005). To press on explosives sensor methods, present methods need to become affordable and transportable without disturbing the integrity of the devices. The uncovering of ordinary explosives as well as trinitrotoluene (TNT), RDX, HMX, 2,4,6 Trinitrophenyl-N-methylnitramine (TETRYL)Pentaerythritoltetranitrate (PENT), and NG were carried out using diverseprocedures(Sanchez et al. 2007).
Detection of explosives is anparticularly relevant analytical concern for law enforcement personals and for the environmental protection agencies. As the use of explosive substances have been increased by the terrorists, problems have increased for law enforcement and environment and security agencies regarding the detection ofexplosives residues in baggage, parcels vehicles, aeroplane, on travellers, etc. In bomb scene investigations, it is important to find debris that includes detection of explosive residues. Mobile and hand held explosives detectors, similar to those used for detecting hidden explosives, can be of great help in detecting such residues. Several methods i.e. GC/MS, SEM, FTIR were used in Punjab Forensic Science Agency (PFSA) to analyze residues of explosives. The detection of landmines is an acute, urgent worldwide problem that needs specific and effective detection methods (Yinon 2002). Keeping in mind the above said situations, the project was designed with following objectives
Availability: Items available for loan: UVAS Library [Call number: 2235-T] (1).
32.
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).
33.
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).
34.
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).
35.
Production Of Single Cell Protein By Arachniotus Ruber Using Remnants Of Carrot As Substrate And Its Biological Evaluation In Broiler Chicks
by Lutfullah Siddiqui (2012-Va-601) | Ms. Shagufta Saeed | Dr. Abu Saeed Hashmi | Dr. Muhammad Wasim.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: CD Error. Summary could not opened. Availability: Items available for loan: UVAS Library [Call number: 2371-T] (1).
36.
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).
37.
Polymorphism Analysis Of Exon 2,5 And 10 Of Bovine Lactoferrin Gene And Its Association Within Mastitis In Sahiwal Cows
by Sidra Mukhtar (2014-VA-223) | Dr. Sehrish Firyal | Dr. Sultan Ali | Dr. Muhammad Wasim | Dr. Muhammad Avais.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: A few factors militate against understanding the milk production capability of bovines. Mastitis is the most costly and the prevalent production-limiting disease of dairy animals in Pakistan and somewhere else on the world. Susceptibility and resistance to mastitis is a complex characteristic and impacted by hereditary variation of animals. Among these variations, the polymorphisms in LF assumes critical part in the immune responses to mastitis.
Susceptibility and resistant to mastitis is a complex trait and influenced by genetic variation of the immunity genes of the animals. Among these variations, polymorphism in Lactoferrin gene (LF) play important role in immune responses to mastitis. Polymorphism in exons 2, 5 and 10 of Lactoferrin gene are 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 LF gene associated with mastitis. Blood samples from 20 Sahiwal cows having clinical and subclinical mastitis were sampled. Blood samples of 10 normal Sahiwal cows were also collected. DNA was extracted. Specific primers for amplification of LF gene were designed by using Primer 3 software.
LF gene was amplified and sequenced to get the full length sequence of the gene. Comparative analysis of resulted sequences was done with the help of NCBI BLAST. Multiple sequence alignment was done by using CLUSTAL W and BioEdit softwares. Protein analysis was done with ExPasy translate tool and the development of 3D structure were using PYMOL software.
Availability: Items available for loan: UVAS Library [Call number: 2582-T] (1).
38.
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).
39.
Molecular And Serological Characterization Of Soilborne Francisella Tularensis
by Javed Muhammad (2010-VA-65) | Prof. Dr. Masood Rabbani | Prof. Dr. Khushi Muhammad | Dr. Muhammad Wasim.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: Tularemia, caused by Francisella tularensis (F. tularensis ), is a zoonotic disease transmitted through contact with infected animals and contaminated environment. The disease has been reported from many countries of the world but no study has been done in Pakistan. In the current project, a total of 2280 soil samples representing 456 villages of eight districts of Punjab province were collected from way-points having human-animal interaction, processed for genomic extraction and tested through real time PCR for presence or absence of F. tularensis. Association of risk factors was determined from data such as gender and age of animals, plough method, irrigation system, fertilizer type used, availability of veterinary services, level of farmer education, physical and chemical composition of the soil. Moreover, sero-prevalence against F. tularensis in cattle, buffaloes, sheep and goats was determined using ELISA. Seventy four soil samples (3.24 percent) were found positive for F. tularensis. Phylogenetic analysis showed 100 percent similarity index with F. tularensis sub specie holarctica reported from other regions like USA, Sweden, Spain, Turkey and Germany. Presence of F. tularensis in soil showed negative association with increase in number of human density (0.7159; 0.3834-0.2054). Prevalence of anti- F. tularensis ELISA antibodies were significantly higher (p<0.05) in large ruminants (cattle and buffalo) as compared to small ruminants (goat and sheep). Age and gender-wise analyses showed non-significant differences (p>0.05) between small and large ruminants. Whereas, rain-irrigation system (2.96: 1.35- 6.48), lack of veterinary services (4.77:1.26-18.03) and use of organic fertilizer (5.3: 11.38- 20.39) have positive association with prevalence of anti- F. tularensis ELISA antibodies in the serum. Sero-prevalence of Ft in the animals has significant association with quantity of clay in soil (p<0.05). A conventional PCR based test has also been optimized for
Summary
103
detection of F. tularensis using tul4 gene specific primers. Specificity of primer showed F. tularensis detection in soil DNA in the presence of other cross-reactive organism. Sensitivity was determined in two fold dilutions with detection limit of up to 320 pg/μL. Utilizing pET28a vector, a construct was prepared containing transformed tul4 gene (450bp) showing 100 percent sequence homology to query gene sequence. For manufacturing diagnostic assays especially in developing countries where availability of BSL-3 facilities and positive control reagents is an issue, provision of tul4 gene based constructs in vector can act as positive control and biosafe to use.
It is recommended that similar studies may be done in other parts of Pakistan to have spatial distribution of F. tularensis all over Pakistan. In future studies, other sources of transmission like water, ticks and rodents may be considered with soil for complete analysis. Transportation of whole genome of F. tularensis has been prohibited by Russian government, ATCC and CDC, WHO is working on designing a complete protocol for transportation of this bacteria or genome to other countries. Under such situation, conventional PCR optimization can be done for diagnosis of F. tularensis and pET28+tul4 constructs, developed in this study, can be used as a PCR positive control reagents. Availability: Items available for loan: UVAS Library [Call number: 2640-T] (1).
40.
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).
41.
Snp Genotyping Of Cacna1a Gene Implicated In Childhood Absence Epilepsy (Cae)
by Wajeeha Tariq (2010-VA-487) | Dr. Muhammad Wasim | Dr. Ali Raza Awan | Dr. Muhammad tayyab.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: Childhood absence epilepsy (CAE) is more pediatric epileptic syndrome. It is about 5 to 15% of all childhood epilepsies. CAE is polygenic and multifactorial syndrome. Many different genes other than CACNA1A gene are involved to cause the CAE collectively. Mutation in P/Q type alpha 1 A subunit channel (Cav2.1) gene CACNA1A, leading to the reduction of Cav2.1 activity in both neurons and in expression system. Reduction in Cav2.1 channel activity altered the neurotransmitter release at neocortical synapses. Molecular genetics techniques have identified various mutation in the genes of ion channels such (CACNA1A, CACNA1G, CACNA1H, CACNB4), sodium channel genes (SCN1A, SCN1B and SCN2A) and GABA receptor genes (GABRD and GABRG2). CACNA1A ion channels are the standard mediator of neurotransmission in Central nervous system (CNS) and mutations in this gene play significant role in the generation of absence seizures. Pore forming alpha 1 a (Cav2.1) channels encoded by CACNA1A gene and are usually located in presynaptic neuron.
Present study was aimed to examine coding regions of CACNA1A gene for analyzing the mutations involve in epilepsy.
Blood samples (n = 40) of true CAE representatives were collected from Children hospital Lahore. DNA was isolated from all blood samples through standard organic method. Amplification of CACNA1A gene exon 36 regions was done with specially designed primers.
Later on, results were analyzed through sequencing of target region. Sequenced samples were analyzed through BioEdit software and alignment was done through Clustal Omega software.
It has been identified that absence epileptic patients of Pakistan showed Mutation in exon 36 of CACNA1A gene at position 281258bp and 281285bp which alter the protein sequence. Due to frame shift mutation a stop codon was detected at position 1813 in protein sequence. So a truncated and loss of function Cav2.1 channel might be formed. In epileptic patients, mutation is responsible for the absence seizures.
In the conclusion, we can say that additional study with large number sample is required to amend the effects of these mutations and their associated factors are precisely and perfectly identified. Further, there is need to investigate the other gene variation causing epilepsy in the local population of Punjab Pakistan. This study will ultimately help to develop genetic counseling strategies, gene therapies and parental diagnostic procedures for the Pakistani population.
Availability: Items available for loan: UVAS Library [Call number: 2746-T] (1).
42.
Comparative Genomic Study of Motor Neuron Disease in Horses and Human
by Shakeela Daud (2011-VA-534) | Dr. Muhammad Wasim | Prof. Dr. Tahir Yaqub | Dr. Abu Saeed Hashmi.
Material type: ; Literary form:
not fiction
Publisher: 2017Dissertation note: cd not submitted Availability: Items available for loan: UVAS Library [Call number: 2810-T] (1).
43.
Molecular Characterization of Carbapenem Resistant Pseudomonas Aeruginosa Isolated from a Tertiary Case Hospital in Lahore
by Sarfraz Hussain (2015-VA-1326) | Dr. Muhammad Imran Najeeb | Prof. Dr. Aftab Ahmed Anjum | Dr. Muhammad Wasim.
Material type: ; Literary form:
not fiction
Publisher: 2017Dissertation note: Empty CD. Availability: Items available for loan: UVAS Library [Call number: 2834-T] (1).
44.
Mutation Detection Of Cacnb4 Gene Involved In Childhood Absence Epilepsy And Its Comparative Genomics In Mice
by Ayesha Amin (2015-VA-1048) | Dr. Muhammad Wasim | Dr. Sehrish Firyal | Prof. Dr. Aftab Ahmad Anjum.
Material type: ; Literary form:
not fiction
Publisher: 2017Dissertation note: Childhood absence epilepsy (CAE) is due to multiple factors affecting almost 9-12 years children. Depolarization of ion channel activates the channel. CACNB4 gene is affected by epileptic seizures. Disturbance in ion channel can affects different genes as CACNA1G, CACNA1H, CACNA1A, SCN1B, SCN1A,SCN2A and GABA receptor genes. CACNB4 gene has a major role in influencing epilepsy in human.
In present study,it is directed to analyze the mutations in epilepsy present in coding region of CACNB4 gene.
Collection of blood samples were from Children Hospital, Lahore, Punjab Pakistan from CAE patients of epilepsy. By using standard DNA extraction method, DNA was extracted from samples. Primers were designed for the amplification of exon 3 and 13 of CACNB4 gene.
Results were examined after sequencing the samples. BioEdit software was used to study the samples thoroughly. NCBI BLAST was used to align the sequences.
It is investigated that the sequences of CAE patients of epilepsy of CACNB4 gene has mutation at position position 258023bp which changes A>G. In protein sequence, the mutation is at position 413 which changes L (Leucine) to L (Leucine). This mutation has no effect because this is a synonymous mutationwhere the codon CUG is changes to CUA, both codes for same amino acid that is leucine, so no effect at all by this change in exon 13.
Three mutations are present in the intronic region of exon 13 first, second and third at positions 258184bp A deleted, 258289bp and 258191bp of CACNB4 gene respectively. These all mutations are present in intronic region so has no effect in phenotypes of individual.
In conclusion, maximum numbers of samples were needed to observe the effect of mutations and factors that causes epilepsy. This study will now help the researchers to investigate genetic therapies, strategies of genetic counseling and parental diagnosis for the population of Pakistan.
Availability: Items available for loan: UVAS Library [Call number: 2924-T] (1).
45.
Mutational Analysis Of Cacna1ggene Implicated In Childhood Absence Epilepsy And Its Comparative Genomics In Mice
by Fiza Idrees (2015-VA-803) | Dr. Muhammad Wasim | Dr. Ali Raza Awan | Prof. Dr. Aftab Ahmed Anjum.
Material type: ; Literary form:
not fiction
Publisher: 2017Dissertation note: Childhood absence epilepsy (CAE) is the subtype of Idiopathic generalized epilepsy (IGE). It accounts for 2-8% of patients with epilepsy. The frequency of CAE is more in girls than boys. The percentage of CAE in youngsters is 10-12%. In addition to CACNA1G, many other genes can be the possible cause of CAE. The pattern of inheritance of CAE is polygenic and complex. SNP might be a gain of function mutation in T- channel genes that results in increase T-type calcium channel activity. Ion channel genes and genes for GABA receptors are affected in epilepsy. By using various techniques of molecular genetics mutations have detected in genes of calcium channels (CACNA1H,CACNA1I, CACNA1A, CACNA1G and CACNB4), in genes of sodium channels like (SCN1B, SCN2A, SCN1A ) and genes for GABA receptor (GABRG2 and GABRD ). Gain of function mutation in CACNA1G gene and increased activity of α1G channels are the possible reason for abnormal SWD in absence epilepsy.
Aim of this study was to assess acknowledged and/or the novel mutations in CACNA1G gene obtained from local childhood epileptic patients.
Blood samples (n=20) were obtained from CAE patients. These samples were collected from children hospital Lahore. Organic method was used to extract DNA from these collected samples. Specific primers were designed for exon 13 and 17 and these exonic regions were amplified using PCR.
After PCR, sequencing of PCR products was performed and then sequencing results were analyzed using chromas lite software.
It has been observed that CACNA1G gene has two mutations in exon 17. It was noticed that protein sequence was altered and the positions of mutations were 38594bp and at 38635bp 38594bp and at 38635 bp. So SNP was detected and there was a gain of function mutation α1G channel activity. In conclusion, these mutations are responsible for absence seizures in CAE patients.
So, it can be concluded that to find out how individuals get affected by these mutations and what factors are involved in causing such mutation, a large scale study should be conducted.In addition, other genes involved in causing epilepsy should also be investigated in local Pakistani Punjab population. As a result of such studies, various diagnostic procedures, strategies for counseling and gene therapies can develop.
Availability: Items available for loan: UVAS Library [Call number: 2925-T] (1).
46.
Genetic Evolution And Development Of Recombinant Vaccine Against Newcastle Disease For Chicken In Pakistan
by Abdul Wajid (2009-VA-705) | Dr. Muhammad Wasim | Prof. Dr. Tahir Yaqub | Dr. Muhammad Tayyab.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 2017Dissertation note: Newcastle disease (ND) is one of the most contagious diseases of poultry worldwide. The
disease is endemic in Pakistan and recurrent outbreaks have been reported in commercial poultry
flocks, domestic pet and migratory birds since 1963 an inception of commercial poultry farming
in the country. Disease surveillance is necessary to determine the incidence of the disease as well
as to identify the etiological agent of the disease status in the region. The analysis of the field
data provides a clue for the higher authorities to take steps for the remedy of the devastating
outbreak. A virulent form of Newcastle disease virus caused an outbreak in the northern region
of Pakistan during the mid of 2011. The virus was identified as a virulent viscerotropic vvNDV
and characterize, belonging to the sub genotype VIIi. However, the virus of this genotype is still
circulating in the field though the intensity of the strain to succumb the chickens to cause
mortality does not exist. The particular thing in this genotype was its susceptibility to other avian
species like pheasants, peafowls, ducks turkeys, peacocks, sparrows and parakeets. As this
genotype is circulating since 2011 2016 and still spill over in these avian species. Thus for the
last five years (2011-16), 3500 healthy, diseased and dead chickens, pheasants, peacocks,
turkeys, peafowls, ducks, sparrows, exotic parakeets, rosy-faced parrots, pigeons, and partridges
from 750 different locations s were monitored. Samples were collected from the Northern region
of the country Punjab, Khyber Pakhtoonkhawa, Azad Kashmir, including Gilgit,Baltitssan and
from Southern region, Karachi, Hyderabad , Mirpursakro and other small cities where the poultry
farms are located. The samples were collected by the local veterinarians, poultry Assistants and
Animal health practitioners who assist during the surveillance program. Samples were also
collected from the farmers who brought their birds for inspection in the lab with the details of the
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farm. Mostly sampling was done where there was reports of NDV outbreak, tissues were
collected usually the trachea, spleen and brain, moreover, the pharyngeal and cloacal swabs not
only from the infected birds but also from the healthy birds were collected to assess the virus
shedding in the flock. Blood samples were also collected (1% of the birds at farm), for serum
collection to assess the immune status of the flock using Haemagglutination Inhibition (HI) test
and Enzyme linked immunosorbant assay (ELISA). The Survey Form meet the international
standard was filled for each farm for recording the information required to find the diagnostic
clue as well as the molecular characterization of the isolates. Pool of five pharyngeal swabs were
processed after the passage into 9-day old chicken embryonated eggs and confirming the positive
HA test and then confirmed by real time PCR (RT-PCR). In addition, sera were tested against
NDV by HI and ELISA tests. The targeted samples were sequenced by complete fusion gene and
whole genome using 22 pairs of overlapping primers. The observations indicated that the
commercial broiler industry is highly susceptible to virulent NDV and confirmed by data
available in the laboratory in the survey form. Contrary to that a little is known regarding the
maintenance and enzootic trends of vNDV infection level in domestic and wild birds. Poor
strategy of the use of vaccines and vaccination as well as the existence of virulent form of NDV
in the domestic and pet birds indicate a possibility of the root cause of the ND eruption in the
developing countries. A continuous isolation of virulent viruses of the panzootic Newcastle
disease virus of sub-genotype VIIi since (2011-2016 from commercial chickens and from various
other avian species in the country provide evidence for the existence of epidemiological links
intermingling of the strain among them. Therefore, to avoid the huge economical losses in the
commercial poultry the second largest industry in Pakistan, their close proximity should be
strictly avoided. The mass vaccination of the poultry flocks is in practice in all commercial
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poultry farms in Pakistan. However, the use and availability of a reliable and standard vaccine, as
well as the correct usage of vaccine dose of the live attenuated LaSota vaccine are the key factors
to improve their efficacy in the field. Minor outbreaks have been occurring in the field even
though a severe outbreak was occurred in 2011-12 collapsed the poultry industry with other pet
and wild birds. To minimize the continuity of these minor outbreaks in the field for long time
there is a need for more effective vaccine to control the particular genotype of the ND virus. In
the present study, DNA vaccine was developed using the SFR-55 NDV strain antigens, in the
form of fusion (F) and hemagglutinin-neuroaminidase (HN), namely pcDNA3.1-F and
pcDNA3.1-HN. In vitro expression of both genes construct was assessed by reversetranscriptase-
PCR (RT-PCR) and western blotting. In the trial an inactivated oil-based emulsion
vaccine was prepared using the field strain SFR-55 and compare with the commercial vaccine
LaSota strain commonly used by the poultry industry. Birds were divided into six groups, the
first two groups were immunized with pcDNA3.1-F and pcDNA3.1-HN alone respectively and
third group with was vaccinated with both antigens pcDNA3.1-F+HN. The other two groups
were immunized with inactivated (wvSFR-55) and LaSota vaccines as described above, the last
group was injected with empty vector as control. The birds were immunized twice at 14 and 21
days of age intramuscularly (DNA vaccine), subcutaneous and eye-drop by inactivated and
LaSota vaccines respectively. The birds were challenged with live virulent NDV strain using a
dose of 10,000 ELD50/0.1ml per chicken. Results indicate that Inactivated and LaSota vaccines
provided high protection (>80%), as compared to pcDNA3.1-F, pcDNA3.1-HN, pcDNA3.1-
F+HN gave 70%, 75% and 20% respectively. There was 100% mortality in control chickens.
The administration of two vectors expressing F and HN antigens induced high immune response,
and provide protection than when used separately. However, the groups immunized with
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pcDNA3.1-F, pcDNA3.1-F+HN and inactivated vaccine resulted in lower amount of virulent
virus shed after challenge when compared to the group immunized with standard LaSota. In
summary, the co-administration of both NDV glycoprotein antigens increased protection than
use separately. DNA-based vaccine can be used safely to reduce mortality and most importantly
lower the risk of virus transmission due to decreased level of virulent virus shedding. Availability: Items available for loan: UVAS Library [Call number: 2910-T] (1).
47.
Analysis Of Tp53 Gene Isolated From Oral Cancer Patients
by Amir Saleem (2013-VA-897) | Dr. Muhammad Wasim | Prof. Dr. Tahir Yaqub | Prof. Dr. Habib-ur-Rehman.
Material type: ; Literary form:
not fiction
Publisher: 2017Dissertation note: Cancer is a term used for disease in which abnormal cells divide without control and are able to invade other tissues. Cancer cells can spread to other parts of the body through blood and lymph system.
TP53 is one of the most important tumor suppressor genes, mutated in more than 50% of human malignancies. It controls DNA repair, cell cycle and apoptosis and therefore plays an essential role in keeping genetic constancy. TP53 gene is present on the short arm of chromosome number 17. In human it extends 19,200 bp in 11 exons.
Various parameters are used in the present study was aimed to investigate coding regions of TP53 gene for analyzing the mutations involved in oral cancer. Human OSCC samples (15) and normal tissue samples (15) were collected from Fatima Memorial Hospital Lahore. Samples were collected in tubes by oncologist containing ethanol and then brought to Molecular Biology and Biotechnology Lab of Institute of Biochemistry and Biotechnology, UVAS Lahore and frozen at -20°C before DNA extraction. Samples have been processed for research purpose. DNA was extracted from tissue by using Trizol Method and quantity was checked by nanodrop spectrophotometer. Two Primer sets were designed to amplify protein coding region of TP53 gene. After amplification through PCR, DNA Sequencing was done. Data interpretation was done by using several softwares like BLAST alignment tool, Bioedit, Clustal W2.
In this study it was tried to find out the mutations in TP53 but no any kind of mutations were identified. Because I conducted my research only on 15 samples. So in future to use this gene as a potentional biomarker we can increase our number of samples.
46
The need for today is to develop valid biomarkers, which can be incorporated in ongoing in vivo and in vitro clinical mechanistic and improve the diagnosis and prognosis of this dreadful disease.
In the conclusion, we must say that further study, with much larger sample number, is required to revise the effects of this polymorphism and accurately identifying the associated factors. There is a need to explore the other gene mutations causing oral cancer in local population of Punjab and Pakistan that will ultimately help to develop genetic counseling strategies, gene therapies and prenatal diagnostic procedures for the population of Pakistan. Availability: Items available for loan: UVAS Library [Call number: 2937-T] (1).
48.
Polymorphism Analysis Of Bovine Tumor Necrosis Factor Alpha Gene And Its Association With Mastitis In Nili Ravi Buffaloes
by Samia Tanveer (2011-VA-362) | Dr. Sehrish Firyal | Dr. Muhammad Wasim | Dr. Muhammad Nawaz.
Material type: ; Literary form:
not fiction
Publisher: 2017Dissertation note: Various number of factors cause hindrance in the milk production potential of buffalos. Mastitis is the costly and most prevalent disease causing production losses of dairy herds in Pakistan and elsewhere in the world. Susceptibility and resistance to mastitis is complex trait influenced by genetic variation of animals. Among these immunity gene variations, the polymorphism in tumor necrosis factor alpha gene (TNF-α) play important role in immune response to virus. Polymorphism in TNF-α gene is associated with mastitis susceptibility and resistance. It would be a potential candidate gene for imparting resistance mastitis in dairy buffalos. Blood sample were taken from the 20 Nili Ravi buffalos having clinical and subclinical mastitis. Extraction of DNA was done from frozen blood after thawing, using organic extraction method & also kit method followed by DNA quantification (i.e. gel electrophoresis and nanodrop). Total 5 primers were designed using Primer3 bioinformatics tool. All these primers were optimized using different protocols and a set recipe was obtained for each primer. The amplification of DNA samples was done one by one using all these five primers on optimized protocol. The amplicons obtained were subjected to agarose gel electrophoresis to check whether we have the required product or not using 100 kb ladder and then amplicones were send for the sequencing.
Summary
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The sequencing analysis of resulted amplicon sequence was done using Bioinformatics software Finch TV. Total of 6 mutations were found while 5 were same in all the samples whereas 6th mutation was found only in clinical samples. It is valuable in accomplishing genetic progress for resistance and to improve the immune response. This study will paved the way for animal breeder for selection of Nili Ravi mastitis resistant buffalos for breeding. TNF-α gene polymorphism based marker is now available for screening of resistant bulls as well. Availability: Items available for loan: UVAS Library [Call number: 2936-T] (1).
