Genetic Characterization Of Pakistani Wild Quails Using Mitochondrial Coi Gene
Material type: Book ; Literary form:
Publisher: 2015 Dissertation note: A latest taxonomic tool termed as DNA barcoding is being used to genetically characterize animals. DNA barcoding is helpful in determining evolutionary relationship among species. Being a small sized genome and agile enough to show rapid mutation, mtDNA has been used as a pertinent marker of molecular biodiversity. DNA barcode works as an efficient tool in food manufacturing industry, diet investigation, forensics analysis, preventing unlawful trade and felonious poaching. The aim of this study was to develop DNA barcode for genetic characterization of Pakistani wild quail. Pakistani wild quail is important due to its demand for eggs, meat production, experimental purposes and gaming as well. Japanese quail was also included in this study because this quail is excessively produced in Pakistani farms. Japanese quail is present throughout the year and is comparatively bigger in size than wild quail. It has longer lifespan; farmers can easily breed this species in farms. It is suitable in poultry due to better meat yield.
COI gene (500bp) was used as a molecular marker for identification at species level. DNA was extracted from blood samples of ten wild quails (Coturnix coturnix and fifteen japanese quails (Coturnix japonica). Reported bird universal primers were used to amplify COI region from the extracted mtDNA samples using PCR. Amplicon were then sequenced by Sanger sequencing method (Sanger et al. 1977). Forward and reverse DNA Sequences were aligned with the reference sequence using nucleotide BLAST on NCBI to observe the dissimilarity among the sequences. Consensus sequences generated were used to construct their phylogenetic tree to see their evolutionary relationship with other bird species. Japanese quail which is thought to be domesticated from Japan, its Pakistani population showed close relation with sequences
generated in Japan for this particular species. Pakistani wild quail species showed its closest linkage with C. coturnix.
In conclusion, COI barcode proved as an authentic tool for species identification and phylogenetic inference of Pakistani wild and farm grown quails. Wild quail species has been characterized using partial COI gene sequences. This study has provided a specific genetic marker which can differentiate Japanese quail from wild quail at molecular level as most of the time both species are confused with each other. It can be helpful to the farmers and bird fanciers because they can select the birds of their choice correctly. This is the first study reporting DNA barcode of this Pakistani quail species. It would help researchers to study about phylogenetic and taxonomic status and also assist quail fanciers and quail farmers to unaffectedly identify their species of interest in farming. Identification of quail species is also important for conservation of biodiversity as it helps in preservation and identification of endangered species by generating their barcodes from even minimal evidence available.
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Mutation Analysis Of Alpha-Synuclein Gene In Patients With Parkinson Disease
Material type: Book ; Literary form:
Publisher: 2015 Dissertation note: Parkinson disease is a complex, heterogeneous and chronic neurodegenerative disorder with a cumulative prevalence of greater than one per thousand, caused by neuronal loss, mainly affecting dopaminergic neurons of the substantia nigra. Parkinson disease is an idiopathic disorder of the extra pyramidal system characterized by tremors. Genetic factors contribute to its complex pathogenesis. A functional repeat polymorphism in the α-synuclein (SNCA) gene promoter conveys susceptibility for Parkinson disease. The α-synuclein (SNCA) has been implicated in rare autosomal dominant forms of Parkinson disease. The mutations in α-synuclein were associated with severe disease progression and a typical physical signs, indicative of neuro degeneration extending beyond the substantia nigra. Mutation in α-synuclein gene may have association with dopaminergic neuronal loss in Parkinson disease. Blood samples were collected from Parkinson disease patients. DNA was extracted by organic method. Primers were designed using Primer3 software. Amplification of gene was done by Polymerase Chain Reaction. PCR products were sequenced bi-directionally on ABI 3130XL Genetic analyzer. Sequence alignment was performed for polymorphism identification. The analysis of identified polymorphism has been done by CHROMAS software. Sequences were aligned by BLAST tool of NCBI. The results of analysis showed that no mutation found in exonic region of α-synuclein (SNCA) gene in Pakistani individuals selected for this study. Any change in exonic region of α-synuclein (SNCA) gene is a rare cause of sporadic and familial Parkinson disease in different populations.
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Identification Of Genetic Variations In Toll Like Receptor 1(Tlr-1) Gene To Evaluate Its Potential For Enhanced Resistance To Bovine Tuberculosis
Material type: Book ; Format:
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Publisher: 2015 Dissertation note: Bovine tuberculosis is a disease caused by the species included in the Mycobacterium tuberculosis complex. Toll-like receptors (TLRs) are a family of conserved innate immune recognition receptors that trigger adaptive immune responses. TLR1 play an important role in host defense against mycobacteria, especially by mediating the response to mycobacterial triacylated lipopeptides.
The objective of this study is the identification of single nucleotide polymorphisms within the coding region of TLR1 gene to evaluate its potential for enhanced the resistance to bovine tuberculosis in Nili-Ravi buffalo breed. Fifty blood samples of Nili-Ravi breed were collected from UVAS Pattoki Campus, Research Farm B and Buffalo Research Institute (BRI) Pattoki. Inorganic method was used for DNA extraction, for amplification of the coding region of TLR1 gene PCR (Polymerase Chain Reaction) was used using specially designed primers and the PCR products were sequenced through Sanger’s Chain Termination method. For the analysis and alignment of sequencing the results obtained after sequencing were analyzed and aligned using the CLUSTAL W and BLAST software. After all these analysis Ten SNPs were identified in the coding region of TLR1 mentioned in table.
The Ten SNPs identified in the coding region of TOLL LIKE RECEPTOR 1 were in this order P1 C>T, P2 T>C, P3 T>C, P4 T>C, P5 T>C, P6 C >T, P7 T>C, P8 C >T, P9 A>G and P10 A>G. The one SNP found in the current research is in compliance with the (Sun et al. 2012) research on TOLL LIKE RECEPTOR 1 hence Nine SNPs found in the current research are novel in Nili Ravi buffalo. The SNPs in the exonic region that is P1 C>T, P2 T>C, P3 T>C, P4 T>C, P5 T>C, P6 C >T, P7 T>C, P8 C >T, P9 A>G and P10 A>G were all transitions i.e. the conversion of purines to purines.
Population genetic analysis and allelic distribution at all loci was analyzed using POPGENE 32 software indicated that at [P3=0.243009> 0.05] followed the assumptions of the Hardy-Weinberg equilibrium indicating that the alleles were randomly distributed throughout the population, no migration had occurred, no bottlenecks happened and population remained large in numbers.This Non-significant and obeying HWE, so can be potential marker for genetic selection.At [P1= 0.040418< 0.05], [P2=0.033603< 0.05], [P4=0.000649< 0.05], [P5=0.000262< 0.05], [P7=0.015112< 0.05] and [P9=0.000111< 0.05] the probability value below 0.05 indicated that population at these polymorphic sites was not obeying Hardy-Weinberg equilibrium. This indicated that at these positions alleles were not equally distributed in population. It can be concluded from my research that the SNPs identified in the current research may also hold potential for marker-assisted breeding programs, with the aim of breeding more BTB-resistant animals and herds within both the national farms and the private sector.
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Comparison Of Antifungal Activity Of Human Salivary Histatin Between Diabetic And Nondiabetic Individuals
Material type: Book ; Literary form:
Publisher: 2015 Dissertation note: Histatins are antimicrobial proteins found in human saliva. These proteins have also been
observed to have the ability to aid in wound healing in various organisms. The genes HTN1 and
HTN3 have been studied to govern these proteins. Histatin proteins have a vast array of
antimicrobial properties. While a fungus, Candida albicans or C. albicans is a part of the human
normal gut flora, it is a threat to people who have a compromised immune system. An
overgrowth of the fungi belonging to the Candida family leads to candidiasis in humans, and oral
candidiasis has been reported to a large extent namely in diabetic patients. The antifungal
activity of histatin proteins laid the basis of the current research work.
In this study, the antifungal activity of saliva from a total of 64 healthy and diabetic
human samples against Candida albicans has been evaluated. The samples of both healthy and
diabetic human samples belong from different age ranges: 15-25, 25-35, 35-45 and 45-55 years
in order to change in antifungal activity with respect to age of an individual. Antifungal activity
was observed through both agar well and agar disk diffusion methods, with agar disk diffusion
methods showing positive results. According to the outcomes of this study at least 120μL of
healthy saliva sample is required to create a zone of inhibition. Saliva from diabetic individuals
showed no antifungal results.
This occurrence led to the next part of this study involving amplification of HTN3 gene.
The nucleotide sequences of both healthy and diabetic individuals were compared together and
showed that the absence of antifungal activity in diabetic individuals might have reasons other
than a genetic one, according to this study. The results observed from the present study indicate
that healthy human saliva possesses antifungal activity against Candida albicans. In accordance
to these results, the naturally occurring antimicrobial activity of histatin proteins present in
human saliva can have immense use in the field of medicine.
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Antiviral Effect Of Human Saliva Against Avian Influenza Virus Strain H9n2
Material type: Book ; Literary form:
Publisher: 2015 Dissertation note: Saliva is an important body fluid that contains a complex array of proteins, peptides and various substances that help in maintaining the health of the oral cavity. Saliva exhibits a broad-spectrum of antiviral activity against enveloped viruses as it disrupts the viral membrane. Influenza is a common virus that has been diagnosed in humans and avian species due to AIV. This study has demonstrated the naturally occurring antiviral activity of human saliva against the H9N2 influenza virus that serves as a serious threat to poultry and has been shown to possess high zoonotic potential which can cause a new pandemic.
In this study saliva samples from healthy individuals were taken and the natural antiviral ability of saliva was observed against AIV (Pk-UDL/01/08 H9N2) of calculated EID50 106.66. Inoculum prepared from saliva and H9N2 virus was injected in 9 days old embryonated eggs using CAS route and incubated at 37°C for 48 hours. A negative control (only saliva) and positive control (only virus inoculum) was also determined in the current study. The antiviral activity of saliva was observed through haemagglutination test. The HA test of harvested fluid showed that human saliva indeed possesses antiviral activity against H9N2 virus and can be used as a natural antiviral agent in medicine.
Furthermore, the genomic DNA was extracted from the blood samples. HTN3 gene responsible for histatin production, was amplified using gene specific oligonucleotides. The obtained HTN3 gene sequences were analyzed using Chromas software. The sequence alignment showed 99% similarity to the available sequences in NCBI database and 100% similarity to each individual sample. To conclude, this study has demonstrated that human saliva possesses antiviral activity against H9N2 virus. The nucleotide sequence analysis from each sample
showed no particular change which shows that antiviral activity of glycoproteins present in saliva does not vary at a genetic level. This innate antiviral activity can open a new frontier when it comes to combating viral infections that have grown resistant to conventional drugs in both human and animal subjects.
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Study Of Wound Healing Effects Relating To Topical Application Of Human Saliva On Rabbits
Material type: Book ; Literary form:
Publisher: 2015 Dissertation note: Histatin proteins present in human saliva have been observed to show natural antibacterial and antifungal properties, as well as play a role in wound healing. These naturally occurring proteins can serve as effective agents when combating microbial infections of vulnerable wounds that have become drug resistant, without inducing negative side effects in the host. Focusing on these proteins can create a new outlook with regards to wound-healing medicine for both humans and animals.
Subjects of this study were 30 fully grown adult male rabbits weighing 2.0 to 3.4 kg and ranging from 8 to 16 months in age. They were acclimatized for two weeks in stainless steel cages and fed commercial diets, vegetables, crushed wheat and corn all over the whole experiment. Out of all 30 rabbits 24 rabbits were experimental on which saliva was applied, three were negative control to check natural wound healing, and three were positive control on which wound healing medicine was applied.
The 24 experimental rabbits were further divided into four groups with each group consisting of 6 rabbits to check the effect of age on wound healing. The age groups of human samples were divided as 15-25, 25-35, 35-45 and 45-55 (Verma et al. 2013). Saliva of human individuals belonging from these four age groups was applied on the wounds of experimental group. Furthermore, all age groups contained saliva from both gender i.e. each age group consisted of 3 male and 3 female saliva samples.
Furthermore, DNA was extracted from blood samples of the same individuals from whom saliva samples were procured. HTN1 gene which is responsible for the production of salivary histatin protein was amplified using specific primers and PCR optimization.
The results of this study demonstrated the wound healing properties of histatin proteins present in saliva and thus, providing a basis of using the natural ability of human saliva to act as a major component in the future of medicine for wound healing and preventing wound infections in both human and animals.
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