51.
Molecular Exploration Of Zbed6 Gene For Growth Trait In Lohi Sheep
by Usman Sagheer (2014-VA-03) | Dr. Maryam Javed | Dr. Akhtar Ali | Prof. Dr. Tahir Yaqub.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: ZBED6 gene is a central transcription factor. It is as a repressor of IGF2 (insulin-like growth factor II) interpretation in skeletal muscle myogenesis and development. It is essentially included in organism development, signaling, cell to cell collaboration, hepatic fibrosis, clathrin intervened endocytosis and tight intersection signaling falls. Chromatin immune precipitation (ChIP) sequencing utilizing C2C12 cells recognized around 2,500 ZBED6 binding locations in the genome, and the derived accord theme gave an immaculate match with the set up tying site in IGF2. Silencing of ZBED6 in myoblast cells influences IGF2 expression, wound healing, cell proliferation and myotube arrangement. Genes connected with ZBED6 binding sites demonstrated a very huge advancement for certain Gene Ontology groupings, including improvement and transcriptional regulation.
Forty two blood samples were collected. DNA extraction was done by using organic extraction method. Primers for PCR amplification designed using Primer3 software. PCR products were sequenced and then analyzed by using BioEdit software. Expasy translational tool for translation and POPGENE 32 software for analysis of population genetics at all the loci were used. Using this software the overall allele frequency, heterozygosity, probability using Chi-square test and Likelihood ratio test and Hardy-Weinberg equilibrium, genotype distribution at all SNP position, summary of genetic variation statistics for all loci and association were calculated. After this, for the association one way ANOVA was performed. Single nucleotide polymorphism within ZBED6 could be potential candidate gene to be serving as genetic marker for the selection of animals with higher tendencies towards weight gain. Availability: Items available for loan: UVAS Library [Call number: 2539-T] (1).
52.
Molecular Investigation Of Low Density Lipoprotein Receptor Gene Causing Familial Hypercholesterolemia And Its Evolutionary Relationship With Pan Troglodytes
by Rida Zainab (2014-VA-808) | Dr. Maryam Javed | Dr. Asif Nadeem | Prof. Dr. Tahir Yaqub.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: Familial Hypercholesterolemia (FH) phenotype is related to improper metabolism of low density lipoproteins due to mutations in Low-density lipoprotein receptor (LDLR) gene with increased risk of ischemic heart disease. Genetic variants in LDLR gene are associated with defective catabolism of cholesterol effecting lipid metabolism which results in familial hypercholesterolemia. It occurs in both forms: Homozygous Familial Hypercholesterolemia and Heterozygous Familial Hypercholesterolemia.
Patients having high cholesterol were identified by observing the values of their serum lipid profile test reports. Their detailed history was taken and blood samples from the identified patients of familial hypercholesterolemia were collected. DNA extraction was done by Organic method. Primers were synthesized and PCR was conducted using optimized recipe and conditions. PCR products were sequenced.
Sequenced data was analyzed using Chromas or BioEdit software. BLAST was performed and sequences were aligned individually by comparing it to the reference sequence. This showed difference in any specific position of a mutated sequence against the reference sequence. CLUSTALW aligned all the sequences together in one time. Sequences were compared with reference sequence to detect the presence of any mutation or SNPs.
SNPs were identified manually and the peaks were observed in order to determine if the genotype is heterozygous or homozygous. Statistical Analysis was done and any amino acid change due to the observed SNPs was determined by using Expasy Translate Tool. It was found that both the SNPs showed amino acid changes. In the end, homology analysis was done which showed that Homo sapiens had their LDLR gene closest to that of Gorilla gorilla gorilla. Availability: Items available for loan: UVAS Library [Call number: 2551-T] (1).
53.
Prevalence Of H9n2 In Biotic And Abiotic Factors Post Avian Infleunza Outbreak In Different Districts Of Punjab
by Iqra Mahfooz (2010-VA-301) | Prof. Dr. Tahir Yaqub | Dr. Aamir Ghafoor | Dr. Maryam Javed.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: Avian influenza H9N2 is not only a potential threat to the poultry industry but it is also a disease of zoonotic importance. In the past it caused very high rate of mortality in the poultry and cause huge economic loses. Present investigations were aimed to find out the uncommon resting points of avian influenza H9N2 virus in the environment. This virus is very dangerous for the poultry industry of the country so it is important to find out the hiding places of virus so that we can stop or control the future outbreaks of virus in the poultry and minimize the economic loss of the country. To rule out the above condition a total of 150 biotic and 200 abiotic samples were collected. Refrigerated samples were processed in the Influenza laboratory. Virus isolation and propagation was done through egg inoculation technique. Presence of virus was confirmed by using Polymerase chain reaction (PCR). The biotic samples (11/150) 7.0 percent reacted positive to HA, HI and also confirmed by PCR. All the abiotic samples were found negative for any evidence of presence of avian influenza virus. This study helped us in understanding the natural reservoirs of avian influenza virus. This study design revealed the hibernation of H9N2 virus in the apparently healthy flock production of broilers. Availability: Items available for loan: UVAS Library [Call number: 2563-T] (1).
54.
Pathological Investigation And Molecular Detection Of Avian Pathogenic E.Coli Serogroups In Broiler Birds
by Muhammad Azeem Riaz (2008-VA-132) | Prof. Dr. Asim Aslam | Dr. Muti Ur Rehman Khan | Prof. Dr. Tahir Yaqub.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: The present study was designed to identify the serogroups present in field and to study their pathological effects in experimentally infected broiler chicks. The present study was attempted to scan the rfb gene clusters in APEC predominant serotypes O1, O2 and O78 strains and to develop Multiplex PCR method for serotyping of the O-antigens. The Multiplex PCR method was used for the identification of serotypes of APEC. The second part of the study was to study the pathological lesions caused by most prevalent serogroup in experimentally infected broiler chicks.
A total of 100 tissue samples (lungs and livers) were collected from colibacillosis suspected broiler birds. Streaking was done from these samples on three different media and it was found that 80% isolates were positive on MacConkey media, 60% were positive on EMB media and 40% were found pathogenic for E.coli on Congo red media.
The colonies which were of pink color on congo red media were considered as pathogenic. DNA was extracted from these colonies by boiling method by picking single colony from each petri plate. Extracted DNA was further used for PCR to confirm the three serogroups i.e O1, O2 and O78.
The PCR results showed that 8% isolated samples were found as pathogenic as O2 strain was found dominant among all. Only two genomic DNA samples were found of O1 serogroup After confirmation of serogroups inoculum of Avian pathogenic E.coli O2 strain was prepared to experimentally infect the broiler birds. Birds were infected at the age of day 7 via intratracheal route.
Following the experimental infection of birds, they were monitored for any pathological lesions which were not present significantly while some birds were off feed, reluctant to move, head down posture and were keeping themselves in isolation.
Summary
46
Postmortem of dead birds was performed and pathological lesions were noted. Livers were found to be congested, enlarged and white fibrinous layer over liver was present. Lungs were also affected with the disease and white layer was present on lungs too. Lungs were consolidated and congested.
Histopathology of lungs and livers was performed. It was noted that there was mononuclear cells infiltration and thin fibrinous layer over liver. Thickening of the liver capsule was noted due to infiltration of mononuclear cells and there was marked congestion in hepatic portal areas and the central vein. There was atrophy of adjoining hepatic cords due to greatly distended and congested sinusoids. Besides these changes, hepatocytes in various stages of degeneration along with hemorrhages, areas of congestion and fatty changes in a few places could be seen.
There was infiltration of heterophils, severe congestion, lymphocytes and macrophages in the wall of the bronchus as well as in the peribronchial alveoli. There was marked presence of granuloma in lungs. Some birds displayed thickening of the pleura and consolidated areas covered with yellowish fibrin in lungs.
The experimental infection of avian pathogenic E.coli confirmed the hypothesis that it causes pronounced histopathological lesions in broiler birds. Availability: Items available for loan: UVAS Library [Call number: 2591-T] (1).
55.
Isolation And Molecular Characterization Of Rotavirus From Calf Diarrhea And Preparation Of Vaccine
by Nadia Mukhtar (2008-VA-718) | Prof. Dr. Tahir Yaqub | Dr. Jawad Nazir | Prof. Dr. Asim Aslam.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: The main contribution of the thesis “Title” is threefold. First, rotavirus was isolated and identified from calf diarrhea samples from 10 districts in Punjab. Second, optimization of molecular diagnostics and genome sequencing was done of the positive bovine rotavirus isolates from Pakistan. And thirdly, the preparation as well as evaluation of killed vaccine against bovine rotavirus isolates was performed.
The above three objectives of this study were created due to the distribution of rotavirus all over the world as an enteric pathogen in both human as well as animal species. In developing countries where cases of malnutrition are very common in young children and animals, this virus has a special importance as an etiologic agent. It causes severe diarrhea, when accompanied with severe dehydration, leads to high rate of mortality. Among the rest of the infectious diseases present in calves, neonatal diarrhea is a dire threat as it has a major impact on economic viability. Calf diarrhea is the most important problem in dairy calves that causes more financial losses to the calf producers than any other. Although numerous etiological agents may be implicated, Rotaviral diarrhea is one of the main infections causing calves to scour between five to fourteen days of age.
The cattle and buffalo calves’ population in Pakistan is devastatingly affected by the neonatal calf diarrhea due to rotavirus outbreaks. Neonatal calf mortality varies from 8.7 to 64 per cent throughout the world accounting for 84 per cent of the total mortality in the first month of age and is particularly high in the third week. While vaccination is available for the disease, it is being imported in Pakistan from other countries. The importation of the said vaccine thus, leads
SUMMARY
117
to extra expenses for the farm managers. As mentioned above one of the aims of this study is to develop an effective vaccine against bovine rotavirus and cut down expenses for farm managers.
To fulfill the objectives proposed in this thesis, rectal swabs and fecal samples were collected from public/private sector buffalo and cattle farms from 10 districts of the Punjab: Lahore, Faisalabad, Okara, Sahiwal, Sargodha, Chakwal, Bhakkar, Bahawalnagar, Multan and Bahawalpur. The samples were selected on the basis of agro-ecological zones of the province. As sampling based on agro-ecological zones allow for better data collection for recording incidence rate of the disease. Samples (n=10) from each diarrheic and apparently healthy cattle and buffalo calves from all of the districts were collected. In this way a total of 200 samples from buffalo calves and 200 samples from cattle calves were collected for this study.
Antigen of bovine rotavirus was screened from calf feces through Direct Sandwich ELISA. Bovine rotavirus samples were further confirmed through the amplification of the VP4 and VP6 genes through Rt-PCR. Homology and phylogenetic analysis of the sequenced samples was also performed. The data gathered through this analysis was helpful in collecting important data regarding the similarities as well as differences of the bovine rotavirus strain present in Pakistani isolates when compared to local regions as well as international ones. The data is also valuable when it comes to production of effective vaccines again rotavirus. RNA viruses are known to mutate unpredictably and it is safe to assume that a particular vaccine might not work effectively against all strains of a particular virus. That’s why analysis of data pertaining to all possible BRV strains is important for creation of an effective vaccine of import quality in order to help the economy of Pakistan.
Rotavirus isolate, after adaptation on MDBK cell line, was further propagated to determine TCID50 for vaccine preparation purposes. Final dose of the vaccine was adjusted to
SUMMARY
118
approximately 3ml, containing 40% culture and 60% adjuvant. Final vaccine contained 1ml of inactivated bovine rotavirus harvested culture, 1.8ml of Montanide ISA 70, 0.2ml of PBS and 0.05% of Thiomersal sodium. Efficacy of the vaccine was checked in rabbits.
For vaccine efficacy testing twenty one month old rabbits were procured. Rabbits were reared in individual isolator units in the shed facility of Quality Operations Laboratory, UVAS, Lahore. The collected rabbits were divided into two groups, vaccinated and unvaccinated rabbit groups. Each group had 10 rabbits. One ml of rotavirus vaccine was administered intramuscularly in vaccinated rabbits group. In unvaccinated rabbits group 1ml of normal saline was injected intramuscularly. The second dose of vaccine was administered at 24 days post-vaccination of first dose. The rabbits from both groups were bled at 0, 14, 28 and 42 days post-vaccination. The antibody response of rabbits to rotavirus vaccine was determined through using Antibody detection kit. The rabbits were challenged on day 42 post-vaccination using live field strain of rotavirus having TCID50 1 × 108.5. The rabbits were observed daily up to 14 days post-vaccination for appearance of diarrheic signs. The stool samples of ELISA positive were further confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR) at least 14 days post-vaccination.
The field trials were conducted at Livestock Production Research Institute, (LPRI) Bahadurnagar, Okara. The field study was done to evaluate the prepared rotavirus vaccine for prevention of neonatal calf diarrhea. For this trial, 100 dams were selected. The dams were divided into two groups and each group consisted of 25 pregnant cows and 25 pregnant buffalos. A total of 50 dams (25 cattle and 25 buffalo) were vaccinated intramuscularly with 3ml of prepared inactivated rotavirus vaccine. The 50 remaining dams (25 cattle and 25 buffalo) were kept unvaccinated.
SUMMARY
119
The blood samples were collected for serum separation after 0, 14, 28 and 42 days post vaccination in dams. The antibody titers were measured using antibody detection ELSIA kit.
After calving, newborn calves were fed with the colostrum obtained from the vaccinated dams daily for 5 consecutive days. Similarly, the calves from unvaccinated dams were fed on colostrum from their unvaccinated dams.
The 5 calves from vaccinated and 5 from the unvaccinated dams were isolated in individual isolators. These calves were challenged orally with 1ml of live field strain of rotavirus having 1 × 108.5 TCID50 and the animals were observed for diarrheic signs for 7 days.
All of the collected data was subjected to statistical analysis of (one way) ANOVA and t-test using SPSS. The <0.05 p-value determined the significance of the results through this study.
The data collected through this study allowed for the creation of valuable inferences. According to the current results of this study, the prevalence of bovine rotavirus was shown to be 6% in Punjab. This 6% included 40% and 20% from the districts of Lahore and Faisalabad respectively. Keeping these results in mind, it is to be noted that the recorded prevalence percentage from this study is higher than the prevalence of 2% in Lahore according to a previous study done in the country. It is to be noted that while the 6% prevalence of rotavirus in Punjab detected through ELISA is lower than the prevalence of 16.83% which was detected by ELISA in diarrheic calves from pervious researches, the 12% prevalence detected by ELISA in this research is higher than the prevalence of 7.25% detected by ELISA in diarrheic calves from past data.
In the present study of this thesis it was observed that the use of killed vaccine for bovines produced more efficient immune response in calves. It also enhanced the clostral rotavirus antibody titers as compared to previous studies where the use of the same strain of modified-live virus in a commercial vaccine administered IM with or without adjuvant did not significantly
SUMMARY
120
elevate colostrum antibody titers. The results collected from the present research showed that the average antibody titers in the 25 cattle dams at 0, 14, 28 and 42 days post vaccination were 0%, 57%, 68% and 78% respectively. In a similar manner the average antibody titers in the 25 buffalo dams at 0, 14, 28 and 42 days post vaccination were 0%, 55%, 70% and 82% respectively. These results indicated
the protective maternal antibody level against the rotavirus which will be transferred passively to calves. The results indicate that vaccinated dams were able to provide passive immunity to both buffalo and cattle calves in order to provide protection against the deadly virus. Availability: Items available for loan: UVAS Library [Call number: 2570-T] (1).
56.
Salook o Tasawuf Ka Amli Dastoor
by Prof. Dr. Tahir ul Qadri.
Edition: 9th ed. Material type: ; Literary form:
not fiction
Publisher: Lahore: Minhaj ul Quran Publications; 2005Availability: Items available for loan: UVAS Library [Call number: 297.4 Tahir 21752 9th 2005 Sufism] (1).
57.
Safa e Qalab o Batan
by Prof. Dr. Tahir ul Qadri.
Edition: 1stMaterial type: ; Literary form:
not fiction
Publisher: Lahore: Minhaj ul Quran; 2006Availability: Items available for loan: UVAS Library [Call number: 297.72 Qadri 21901 1st 2006 Islam] (1).
58.
Alqul Al-waseeq fi Manaqib Al-Sadeeq (R.A.)
by Prof. Dr. Tahir ul Qadri.
Edition: 1stMaterial type: ; Literary form:
not fiction
Publisher: Lahore: Minhaj ul Quran; 2005Availability: Items available for loan: UVAS Library [Call number: 297.72 Qadri 21834 1st 2005 Islam] (1).
59.
Jahad Bilmal
by Prof. Dr. Tahir ul Qadri.
Edition: 1stMaterial type: ; Literary form:
not fiction
Publisher: Lahore: Minhaj ul Quran; 1990Availability: Items available for loan: UVAS Library [Call number: 297.72 Qadri 21749 1st 1990 Islam] (1).
60.
Molecular Epidemiology Of Mycobacterium At The Animal Human Interface And Its Co-Morbidity With Diabetes Mellitus
by Zarfishan Tahir (2011-VA-624) | Prof. Dr. Mansur-ud-Din Ahmad | Dr. Abdul Majeed Akhtar | Dr. Muhammad Hassan Mushtaq | Prof. Dr. Tahir Yaqub.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: Tuberculosis (TB) is a common and fatal infectious disease which has afflicted mankind for several millennia. At the moment, TB is positioned at number five when it comes to the most common causes of fatality worldwide. TB is curable if it is properly diagnosed and treated. In 2015, it was estimated that 1.5 million deaths (an equivalent of 4,000 deaths per day) and 9 million new TB cases have been reported. Diabetes Mellitus is also widely distributed and estimated to affect 366 million people by 2030. The co-morbidity of DM and TB is re-emerging because of the progressive epidemiology of both diseases especially in the developing countries. Endemicity of TB and DM is growing in developing countries because of low socio-economic status and poor living conditions.
In this study, a total of 500 tuberculosis positive patients were selected under TB DOTS program from five tertiary care hospitals of Lahore. Sputum samples were collected from all the enrolled patients and smear microscopy was performed for TB confirmation. Blood samples were collected from the same patients for screening of diabetes mellitus. Sputum samples were also processed for culture and drug sensitivity on LJ medium. Molecular identification by PCR technique was carried out on all positive cultured strains and results were compared with reference strain H37RV. For DNA sequencing, PCR products were sent to Singapore where sequencing was performed by Sanger method.
Data was compiled and variables including gender, age, drug resistance and treatment history and correlation among different variables was analyzed using chi-square test and Fischer’s exact test method at P-value of ≤0.05. SPSS (Statistical Package for Social Sciences, Version 20.0) was used for statistical analysis. The count data was statistically analyzed using
SUMMARY
124
descriptive statistical tools. On screening for fasting blood sugar level, 74 (14.8%) patients were recorded as diabetics as well i.e. blood sugar level ≥ 126 mg/dl. Out of these 74 patients, 22 patients had previous history of diabetes whereas remaining 52 patients were newly diagnosed at the time of screening. The maximum distribution of TB-DM patients was found in age group > 57 years. Mean age of the group without DM was 39 years and with DM was 48 years. Coexistence of DM in TB patients was higher in males (62.2%) as compared to female study subjects. However, the gender difference is statistically non-significant (p value 0.243).
The distribution of education level revealed that out of the total participants, maximum number of patients (n=220) were illiterate and similar trend was observed in diabetic patients with 54 (73%) individuals belonging to the illiterate group of the subjects. There is statistically significant difference between existence of DM and literacy level in tuberculosis patients. Among social and behavioral risk factors in tuberculosis patients, majority of the patients were unemployed (24%) in TB-DM group. Significant correlation p value ≤ 0.05 was found between coexistence of TB-DM and tobacco use. TB cases with diabetes were known to have history of smoking with 73% (n=54) while non-smokers were 27% (n=20).
On sputum smear microscopy frequency of 3+ results showing high bacterial load, was profoundly higher i.e. 67.6% in diabetic tuberculosis patients as compared to non-diabetics which was 4.9% only. Total culture yield was 363 out of 500 sputum samples. There were 193 samples that were sensitive to all drugs, 9.4% were MDR strains (resistant to Isoniazid and Rifampicin). MDR-TB is significantly higher in TB-DM patients i.e. 13.5% as compared to 8.7% in TB only patients.
In our study, DNA sequence data for drug resistance was studied by the sequence of rpoB gene of the wild type MTB strain. Sequencing results showed mutations at various spots of rpoB gene.
SUMMARY
125
Most common mutational sites identified were at codon 531, 526 and 516 with frequency of 70%, 15% and 7.5%, respectively. Moreover, mutation sites at 512 and 574 codon had also been reported. In this study, predominantly two phylogenetic variants were identified. Majority of the isolated strains were Central Asia Strain (CAS) with a prevalence of 88.2% and rest were Beijing strain. However, attempts to find zoonosis could not be established. A total of 900 raw milk samples were also screened for M. bovis and no positive sample could be detected.
The present study emphasizes the importance of screening for DM in TB patients, which had not been done in routine. This practice may prove to be helpful in reducing the disease burden of TB patients as well as DM patients. Thus it is recommended that the screening for DM should be implemented in TB/DOTS clinics.
Emergence of Multi drug resistant Mycobacterium tuberculosis is also a serious challenge for clinicians. A very large financial implication in terms of treatment, duration of chemotherapy and spread of MDR TB strains is being faced. Treating MDR TB is more complicated than treating drug sensitive TB. Patients with MDR TB require longer, much more costly treatment and experience higher mortality rates. Such a long time to initiate the treatment is not affordable, thus there is a dire need for some rapid technique like molecular based diagnostics for MDR detection, which can provide quick results and making it possible to start treatment at earlier to minimize transmission, morbidity and mortality. Availability: Items available for loan: UVAS Library [Call number: 2710-T] (1).
61.
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).
62.
PREVALENCE OF MAJOR BACTERIAL AND VIRAL POULTRY DISEASES IN LAHORE DIVISION OF PAKISTAN
by Wasiq mehmood (2009-VA-420) | Prof. Dr. Tahir Yaqub | Dr. Aamir Ghafoor | Dr. Yasin Tipu.
Material type: ; Literary form:
not fiction
Publisher: 2017Dissertation note: Poultry is a huge industry as an emerging agribusiness in Pakistan that contributes a lot in GDP of country but infectious diseases contribute as a major obstacle in profitable production of poultry. There is need to study the most current scenario of major diseases together with all the parameters involved. This can help in making effective preventive measures to minimize the losses. In this study we investigated 1008 cases of poultry diseases received at GP Lab, Lahore. Sick and dead birds were received from different locations of Lahore division during November, 2015 to November, 2016. Whole year was divided in to five seasons as winter (15-Nov to 15-Feb), spring (16-Feb to 14-April), hot summer (15-April to June), hot humid summer (July to 15-Sept) and autumn (16-Sept to 14-Nov). Disease was diagnosed on the basis of flock’s history, postmortem findings, isolation and identification of pathogens using various techniques of bacteriology, virology and molecular biology along with different serological techniques. The result of this study revealed that both bacterial and viral health risks are prevailing in Lahore division of Punjab however bacterial problems are greater in number in comparison of viral infections. Prevalence of E. coli infection (Colibacillosis) was greater than any other disease which could be due to poor disinfection and cleaning of control sheds along with poor management of flocks. Avian influenza and ND shared more than 90% of viral problems throughout the year. Mean prevalence of IBD was found to be 1.46% in recent year whereas CAV and adenoviral infection remained up to negligible extent. Very few cases of CRD and necrotic enteritis were reported. Prevalence of diseases has a strong correlation with seasons with incidence highest in winter and hot summer which could be due to challenging management of flocks because of severe climate conditions. Incidence in spring was found out to be 19.46. Hot humid summer and autumn were found to be least harmful seasons with prevalence of 11.23%
Summary
66
and 7.11% respectively. In order to cope up with health issues, up to date studies on prevailing poultry diseases needs to be done in upcoming years as well. Availability: Items available for loan: UVAS Library [Call number: 2822-T] (1).
63.
Study Of Tyrosine Hydroxylase (Th) Gene Sequence Variations In Association With Δ9-Tetrahydrocannabinol (Thc) Dependence
by Ali Raza (2015-VA-446) | Dr. Maryam Javed | Dr. Asif Nadeem | Prof. Dr. Tahir Yaqub.
Material type: ; Literary form:
not fiction
Publisher: 2017Dissertation note: Anti-Social Personality Disorder (ASPD) is ability of an individual to adopt social norms. These ASPDs are driving force in majority of criminal activities. Dopamine being important neurotranmiter of nervous system controls major behavioral traits. Low level of dopamine can be causative factor for an individual to start drug abuse to restore it because majority of drug are proven to enhance dopamine production. In this study genetic exploration of genes coding for dopamine producing enzymes. Tyrosine Hydroxylase (TH) gene was selected and its two regions (Intron 1 and exon 3) were amplified and analyzed through Sanger’s sequencing method followed by statistical analysis.
Total five SNP were recorded at locus TH1, TH2, TH3, TH4, and TH5. One insertion, three transversion and only one mutation was transition. No exonic mutation was recorded hence no change in protein structure was found. Mutations at TH1 and TH4 were found to be highly associated with addiction. Mutant “B” allele were also present but still wild “A” was most common allele in our population. TH1, TH2, TH4 have positive correlation with addiction, TH3 is correlated with Nicotine and TH5 shown protective role against nicotine. There are few genetic changes in our population that can be associated with drug addiction statistically but still there prevalence in our gene pool is very low. We can conclude on the basis of these findings that drug addiction in our population is more likely a social issue rather than genetical.
Limitations of our research was sample size. There are further possibilities for this project to investigate on mRNA level. Advanced neurobiological techniques can also be applied on subjects to analyze dopamine level in different brain regions. For genetic studies epistatic role of few other genes can also be considered for validation. Availability: Items available for loan: UVAS Library [Call number: 2858-T] (1).
64.
Genetic Variation In The Promoter Region Of Pro Inflammatory Cytokine Tnf-Α Among Hiv Infected People In Lahore
by Shahid Nawaz (2015-VA-437) | Prof. Dr. Tahir Yaqub | Dr. Arfan Ahmed | Dr. Asif Nadeem.
Material type: Publisher: 2017Dissertation note: Despite of the fact that HIV is one of the most studied virus of the present time, so far there is no legitimate cure for the treatment of HIV AIDS, and in most cases AIDS is often fatal. Immune response has always been vital to cope with any disease. In context of immune response TNF-α is mainly released by the macrophages as a pro inflammatory cytokine. Studies have shown that HIV infected persons have higher concentration of TNF-α released. A particular single nucleotide polymorphism (SNP) is observed at -308 position of the promoter region of TNF- α gene due to which TNF is categorized into TNF1 and TNF2 allele. TNF2 allele is associated with higher concentration of TNF- α which in turn is associated with HIV infection. In order to know the particular association in the population of Lahore we designed this study. The hypothesis was that SNP at -308 position of the TNF- α may be associated with HIV infection. Methodology was designed as follows: 15 HIV positive samples and 15 HIV negative samples were taken and categorized into group A and B respectively. Whole Blood samples were taken from the patients and subjected to DNA extraction using commercially available kit (Favorgen blood DNA mini extraction kit). Specific primers were used to amplify the particular region (-308) of TNF- α through PCR. A small amount of PCR products were subjected to restriction enzyme Ncol. Sequencing was done and the results were analyzed using specific bioinformatics tools such as NCBI. -308 region of the TNF-alpha was analyzed for the SNPs (TNF1 and TNF2). Collected data was analyzed through SPSS for association studies. Outcome could be as follows: The study is designed to identify SNPs at -308 position of the promoter region of TNF-α gene. It will enable us to understand the possible association between TNF- α and HIV AIDS. It will be the first ever study regarding TNF- α and AIDS in Pakistan. Availability: Items available for loan: UVAS Library [Call number: 2833-T] (1).
65.
Comparative Efficacy Of Pre And Post Exposure Prophylaxis Using Indigenous Rabies Vaccine By Im Route
by Kaneez Fatima (2010-VA-202) | Prof. Dr. Tahir Yaqub | Dr. Aamir Ghafoor Bajwa | Dr. Maryam Javed.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 2017Dissertation note: Rabies is a viral zoonosis that is known to be present in more than 150 countries, including Pakistan. It is a very serious health problem especially in countries with limited resources and poor awareness. It has significant economic impact and almost 100 % mortality if not properly managed.Dogs are responsible for up to 99% of all rabies transmissions to humans. Rabies is vaccine preventable viral disease. The vaccine is very expensive and a significant factor in patient’s compliance in Pakistan especially in rural areas where the main problem exist i.e. more stray dogs and increased probability of being bitten. Availability of a cheap indigenously produced effective vaccine can be very helpful in reducing the cost and overall improvement of the rabies problem in Pakistan.
We randomly selected a total of 50 patients visiting IPH. Among them 10 of pre-exposure prophylaxis and 40 for post-exposure prophylaxis. Twenty patients of the post-exposure group were children and twenty were adults. The NIH-anti rabies vaccine was administered intramuscularly to the persons visiting the IPH for pre and post exposure prophylaxis using Essen regimen. For pre exposure patients three doses on day 0,7,28 was given and for post exposure patients five doses on day 0, 3, 7, 14, and 28 was administered. The 3ml blood was collected on day 0, 28, 60 and 90 following vaccination. Serum was examined by ELISA Kit (Bio Rad Platelia rabies II Kit) for protective antibody titer.
Pakistan is importing anti-rabies vaccine which is much costly, and sometimes unavailability is a serious concern for patients. In the present study we concluded that indigenous rabies vaccine was very effective and protective levels of rabies antibody titers was detected following vaccination in all patients of the study. By widespread utilization of this vaccine we can reduce demand of imported vaccine, thus lessen the economic burden.
Availability: Items available for loan: UVAS Library [Call number: 2875-T] (1).
66.
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).
67.
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).
68.
Bacterial Profiling And Development Of Molecular Diagnostic Assays For Detection Of Bacterial Pathogens Associated With Bovine Mastitis
by Aqeela Ashraf (2012-VA-388) | Dr. Muhammad Imran | Prof. Dr. Tahir Yaqub | Dr. Muhammad Tayyab.
Material type: ; Literary form:
not fiction
Publisher: 2017Dissertation note: The livestock sector plays a critical role in strengthening the economy of Pakistan. Control of livestock diseases is the primary objective of government livestock departments. Bovine mastitis is among the most significant diseases of livestock as reported by various field surveys in Pakistan. Despite considerable knowledge about mastitis and its etiology, this disease is still prevalent in many dairy herds; it remains most difficult to eradicate or control. It is an inflammation of mammary gland resulting in decreased milk production, veterinary care costs and culling losses.
In animal health improvement, there is a paradigm shift from treatment of clinical illness to disease prevention. Recognition of disease is the foundation of disease control and prevention. California mastitis test and somatic cell counting are the most commonly used methods for diagnosis of bovine mastitis. These methods are unable to identify the causative agent. Detection of pathogen is critically important for better control of mastitis. Microbial culturing and biochemical tests are traditionally used methods for pathogen identification. But, these methods are very time consuming and can only detect viable bacteria from the sample and can lead to false negative results. The progress in molecular methods based on PCR has improved the veterinary diagnostics.
For the identification of bovine mastitis pathogens, an economical, rapid and sensitive molecular diagnostic assay was developed using multiplex PCR, detecting the pathogenic species-specific DNA. The target species areS. aureus,E. coli, S. uberis, S. agalactiae, S. dysagalactia, S. haemolyticus, S. epidermidis, S. chromogenes andM. bovis. Multiplex PCR assay was developed for the detection of these significantly important bacterial pathogen
causing bovine mastitis. Species specific primers were designed which have the ability to specifically amplify the particular gene in the target species. For this purpose various gene regions were selected for different bacterial species which included 16S rRNA, cpn60, phoA and rdr. Initially monoplex PCRs were optimized individually for each target species. For optimizing multiplex PCR assay, various combinations of individual PCRs with varying concentrations of primers and template DNA were used. The final protocol included all the nine sets of primer pairs, every set targeting a unique mastitic pathogen. Multiplex PCR assay was checked for its specificity and analytic sensitivity was calculated. Mastitic milk samples were collected aseptically from various farms. Initial screening was based on Surf field mastitis test and California mastitis test. Milk samples were cultured on nutrient agar, blood sheep agar and MacConkey’s agar. The bacterial isolates were identified and further sub-cultured in nutrient broth. All the isolates were identified on the basis of 16S rRNA sequencing analysis.
The developed multiplex PCR assay was used to detect the target bacterial pathogens from the collected milk samples. Limit of detection of developed assay was up to 50 pg for DNA isolated from pure cultures and 104 CFU/ml for spiked milk samples. The results obtained by 16S rRNA sequencing, bacterial culture based identification and multiplex PCR assay were compared. Sensitivity and specificity were calculated using latent class analysis, specificity was up to 88% and sensitivity was up to 98% for targeted mastitic pathogens. The developed multiplex PCR detected nine bacterial species in a single reaction. Multiplex PCR assay has also detected the bacterial pathogens in a few culture-negative mastitis milk samples. This is the first multiplex PCR assay which can efficiently detect nine important mastitic bacterial pathogens in a single reaction. The development of multiplex PCR assay is useful in early diagnosis and prevention & control of bovine mastitis.
Mycoplasma is often ignored as a major mastitis-causing pathogen due to lack of rapid and accurate diagnostic tools. In this study a LAMP assay was developed for the identification of M. bovis from clinical mastitic milk samples. LAMP primers were designed from three gene regions including uvrC, 16S rRNA and gyrB. Bacterial reference strains and mastitic milk samples positive for M. bovis were collected from Quality Milk Production Services, Cornell University, Ithaca, NY. Bacterial strains were further cultured on Hayflick medium containing 15% horse serum and incubated for up to 7 d at 37°C with CO2 enrichment. DNA was isolated from mastitic milk samples and bacterial culture using Qiagen DNeasy Blood and Tissue Kit (Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions with few modifications. PCR and LAMP assay was performed for all the samples obtained. Analytic sensitivity was calculated and the limit of detection was up to 50pg/reaction for LAMP assay which is higher as compared to PCR.
Sensitivity and specificity was calculated for each of the three tests. Cohen’s kappa values obtained were 0.940 for uvrC, 0.970 for gyrB and 0.807 for 16S rRNA. All three tests showed a high level of agreement between test results and the true mastitis status, indicated by the receiver operating characteristic (ROC) curve. A robust, sensitive and specific LAMP assay has been developed for the detection of M. bovis from mastitic milk. These novel molecular assays could be helpful for correct and timely identification of bovine mastitic pathogens, which is crucial for the control and treatment of the disease.Molecular diagnostic assayshave been developed in the current study based on multiplex PCR assay and loop-mediated isothermal amplification assay.
Availability: Items available for loan: UVAS Library [Call number: 2930-T] (1).
