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Item Prevalence of untypable hcv variants in different districts of punjab, pakistan and a case report of its treatment strategy(UMT, Lhr, 2017) Ayesha ZafarIn Pakistan, around 11 million people are HCV infected with 6 circulating genotypes and subtypes in Pakistan and the viral burden is still on rise. HCV genotype determination is necessary to help the health care facilitators for recommending the appropriate antiviral therapy. This large scale study was carried out to investigate the epidemiological distribution of genotypes in different districts of Punjab, Pakistan and focuses on prominently increasing one (diagnostically untypable) and its possible antiviral therapy. We have also characterized an untypable HCV variant by sequence and phylogenetic analysis that showed the greatest homology of untypable HCV variant with 3a genotype from Pakistan (Unpublished data). A case study has been followed up with untypable HCV genotype for 24 weeks. Of the 8353 patients enrolled in study, the genotype 3 and subtype 3a (n= 6650, 79.6%) was most significantly prevalent throughout the study followed by undetermined genotype i.e untypable (n= 1377, 16%) by the diagnostic method of Ohno et al. The presence of untypable variants suggested that there are some novel or quasi species prevailing among HCV patients in local community. Case of patient with untypable genotype had been treated effectively with interferon alpha plus ribavirin therapy for 24 weeks and achieved SVR rate of 70-80 %. From the study, it can be concluded that untypable variants can be successfully treated with standard interferon therapy unless the mystery of untyped genotype has been solved with novel and effective diagnostic methodsItem Recovery of seminal material from different types of cloth after washing with commercially available detergents in markets of pakistan(UMT, Lhr, 2017) Asad SaeedSemen is the most significant evidence that can help to investigate a rape crime. All types of rape crimes (sexual assault, sodomy, rape and murder) includes many types of evidence items such as victim and suspect clothing, bedding at crime scene, on floor and walls, on body of victim and suspect and sometime on different objects like condoms and copper-T. From all these, clothing and bedding are most important as analyst can get complete single source DNA profile of assailant by identifying individual stains. Unfortunately, in a country like Pakistan, where there is no sense to deal with evidence items due to low literacy ate, the chances of recovering sperms from clothing is nearly zero. In this study, a comparison of sperm retention after washing among different fabrics was done. For this purpose, commercially available detergents and commonly used female and male fabrics were tested and compared results for sperm retention after washing with detergents and distilled water with different washing periods. This study was designed to assess the effect of washing with different time intervals using five detergents available in the market on various fabrics stained with seminal material. Fabrics used in this study were cotton, wool, polyester and mixture of all these with different proportions. Different results were obtained from different detergents used on different fabrics to remove semen stains. Data obtained was analyzed in different aspects with one variable in each aspect.Item A comparison of validity of genetic analysis and chemical components analysisfor the identification of papaversomniferum L(UMT, Lhr, 2017) AbidNaseerCultivation of Opium Poppy (PapaverSomniferum .L) is controlledthroughout the world due its key role in the manufacturing of illicit drug Heroin. As per law, the positive identification by a Forensic Laboratory is the first and basic requirement to proceed against the criminals. The poppy plant is identified by the chemical analysisof its organic extract. In a second opinion the chemical analysis may not be the only criterion for the exact identification of a species as it has some limitations. This research work was aimed to develop some method independent of chemical analysis to identify the poppy plant. Use of anatomical, morphological and molecular analysis for the identification of an intact plant at any growth stage may support the results of chemical analysis.The DNA based identification of opium poppy can be performed using a total of eight genetic markers. Out of these eight markers six are specific to some poppy plants while the other two are common to many land plants however the sequence of amplicons from these two can discriminate opium poppy from the other poppies. The experimental work performed here can be adopted for forensic analysis as per demand of the law. The results in this research concluded that DNA based identification of the controlled plants is a more authentic way for the identification as it contains less chances of error and can be adopted to identify the plant at any growth stage and from any part of the plant.Item Pattern of hcv genotypes prevalence in different districts of punjab, pakistan(UMT, Lhr, 2017-12-27) Muhammad ImranHepatitis C is generally defined as a disease in which the inflammation of liver takes place. This situation is caused by RNA virus of the family Flaviviridae. It's a worldwide problem and in Pakistan its ratio is 4% in general population. The problem of Hepatitis is also considered as one of the major problems by WHO. During 1980s the hepatitis were known to be a A or B but soon after the discovery of Hepatitis C takes place in 1989 at CDC and the experiments was done on the sera of chimpanzee. After that several patients were diagnosed for that virus which is NANB in nature.Till date there are no vaccines discovered for Hepatitis C hence the disease progression in some areas and some groups of people are high. On the other hand the available drugs for this disease are either costly or not that much efficient, generally a combination of interferon and ribavirin are given to the patients suffering from that virus. Recently a newly a newly discovered drug DAA show a high efficiency in order to cure the disease and to improve SVR. That is also proved by a case study from our patients. Hence hepatitis is blood borne disease so there are some groups who have risk to get infected that include barbers, dentists, sex workers, drug users, piercing of nose ear etc. Some evidences of transfusion through house hold contact also be witnessed. If not diagnosed and treated on time this viral infection can be lethal and may cause liver cirrhosis that can cause to death of person. At acute or minor phase if diagnosed this can be easily curable with no side effects. Some individuals show no physical signs of Hepatitis infection while some show dizziness, loss in appetite, loss in weight and change in mood. Hepatitis C is a virus that keeps on mutating and due to its nature there are several types and subtypes of HCV. There are some methods that can predict the type of HCV, but the most exact and widely used method is typing through nested PGR. This technique involves the extraction and amplification of RNA from serum of infected individual and then amplifying it with type specific primers which is then observed under U.V lite. The specific types give specific position band which is then compared with key to predict results of specific type of HCV. The prevalence of different genotypes is different in different parts of the world. In Pakistan the most prevalent genotype is 3a followed by la and that is also proved by our study, in our study we include 8864 patients out of which 7161(80.77%) were 3a. That pattern of dominancy over other subtypes in Pakistan is also supported by several studies.Item Development of genetic transformation system in okra (Abelmoschus esculentus (L). Moench)(UMT, Lhr, 2017) Dur-E-AemanThis study appraises the development of genetic transformation system in okra (Abelmoschus esculentus L. Moench). Okra plays an important role in human diet and is an excellent source of carbohydrates, proteins, fats and minerals. Okra is affiliated with Malvaceae ancestors and is highly susceptible towards insects and viruses. Reported begomoviruses of okra reported throughout the world are Bhendi yellow vein mosaic virus, Okra yellow vein mosaic virus, Okra yellow crinkle virus, Okra yellow mosaic Mexico virus and Cotton leaf curl Alabad virus. Conditions were optimized by establishing a simple regeneration system of okra. 193 embryos were isolated and 69% total regeneration frequency was calculated. Best regeneration frequency was calculated when embryos were grown on MS medium containing 1g GA/L and B5 vitamin. 342 embryos were isolated for development of transgenic plants by using embryo as explant. The transformation efficiency was 2.92 %. Presence of transgene was confirmed by Green fluorescent protein (GFP) expression and PCR analysis. Expression of GFP were effortlessly and rapidly distinguished when examined under a fluorescence microscope at 40X- 100X resolving power. Microscopic analysis was done by using fluorescence stereo microscope to check GFP expression. A tissue sample of okra plant leaf was placed on glass slide. Expression of Green fluorescent protein genes was quickly distinguished at 40-100X resolution power. PCR analysis confirmed the presence of RNAi constructs in transgenic plants of okra when amplified PCR product of size 417 bp was visualized on 1 % agarose gel stained with ethidium bromide.Item In silico designingof a prospective drug against human herpes virus group(UMT, Lhr, 2017) RABIYA NISARThe infection associated with Human Herpes Virus Group leads to long term effects in terms of various diseases, specifically neurological ones. The Herpes infection is an emerging threat and no clinically approved antiviral treatment is discovered yet to cure the disease. Computational approaches for drug development are proven to be effective and time efficient as these are not based on costly and hectic laborious works. In drug discovery, the molecular dynamics simulations help in the study of the motions of biological macromolecules such as proteins and nucleic acids. Computational mechanisms of biological targets and their related small-molecule ligands are useful in drug discovery; these include identifying the binding sites, virtual screening of numerous compounds libraries and estimation of ligand binding energies. The binding process of the tegument proteins with the inhibitorsilluminates the process of inhibition with efficiency and specificity of that inhibitor. It was aim of the present study to identify potential inhibitors of the virus replication and the tegument proteins were targeted in this study using 2053 plant derived compounds. The successfully docked compounds with high binding affinity and reactivity are reported against the proteins. These are all effective drug like compounds. Due to higher affinities and reactivity of these compounds against the tegument proteins, these compounds can be further analysed using in vitro and in vivo approach to elucidate their inhibitory mechanism against HHV.Item Optimization of kojic acid production from aspergillus flavus and aspergillus oryzae in submerge fermentation(UMT, Lhr, 2017) SAMIYA YAQUBKojic acid is an organic acid obtained from various species of Aspergillus through fermentation. Its demand is very high in cosmetic industries as it is a good alternate of carcinogenic Hydroquinone. Beside its cosmetic use and importance, it has grabbed a vital position in Pharmaceuticals, Food and Agriculture industries. Current study was designed for production and purification of Kojic acid crystals from local Aspergillus species, Aspergillus flavus and Aspergillus oryzae. The effect of pH, temperature, static and non-static (shaker) condition on Kojic acid yield in submerge fermentation was examined. Significant yield of Kojic acid crystal was obtained by A. flavus as compared to A. oryzae. Optimized conditions were pH 4.5 for (A. flavus), pH 3.5 for (A. oryzae), temperature 30 0C, 20 days incubation period. We have found that high yield of Kojic acid crystals are produced under static condition (16 gram per litter in A. flavus and 11 gram per litter in A.oryzae) as compared to non- static (shaker) conditions (6 gram per litter in A. flavus and 5 gram per litter in A. oryzae). Quantitative estimation of Kojic acid was done by Bentley’s colorimetric method and conformation through TLC and HPLC. We were successful to get high yield of Kojic acid under optimized conditions.Item Optimization of kojic acid production from aspergillus flavus and aspargillus oryzae in solid state fermentation(UMT, Lhr, 2017) Afia ZahoorKojic Acid is a fungal secondary metabolite that can be obtained naturally by the fermentation of fungal species. Aspergillus is considered to be the best specie for the production of kojic acid. Kojic Acid has numerous uses in health, cosmetics and pharmaceutical industries. In order to get the highest yield of kojic aid we have used local strains of Aspergillus that was Aspergillus flavus and Aspergillus oryzae. Both strains were giving yield of Kojic Acid in solid state fermentation. Glucose and sucrose were used as carbon source, ammonium nitrate used as a nitrogen source at optimized pH and temperature. Given temperature was 25oC, 30oC, 35oC and 40oC, while given pH was 3.5, 4.5, 5.5, 6.5, 7.5, and 8.5. Highest yield of Kojic Acid was obtained by Aspergillus flavus and Aspergillus oryzae at 30oC. Aspergillus flavus shown best result at pH 4.5 and 3.5 was the optimized pH for Aspergillus oryzae. Here we have get the highest yield of Kojic Acid by using local species of Aspergillus by optimizing the conditions.Item Incidence and resistance pattern in bacteria associated with urinary track infections in pakistan(UMT, Lhr, 2017) MAHVISH KABIRBackground: Urinary track infection is on of the most common type of infection in Pakistan. Poor knowledge about personal hygiene, lack of awareness about diet and unavailability of clean drinking water are major reasons of this infection. Varieties of infectious agents are responsible for UTI incidence but in present study E. coli and K. pneumonia were main causative agent of infection in majority of cases. This high prevalence of E. coli and Klebsiella in UTI patients is because of its resistance towards different antibiotics. These strains contains β-lactamases enzyme which provides resistance to β-lactam antibiotics. Prevalence and incidence of these bacteria is different in different region of the world. In present study bacterial resistance and susceptibility has been determined in UTI produced by E. coli and Klebsiella species against different antibiotics. Method: Total 250 samples were obtained from patients suffering from urinary track infection from Genomic Research Lab and Diagnostic Center Rawalpindi, Pakistan. The samples were tested for presence of different microbial infectious agents. Samples had been tested for the presence of different pathogenic bacteria by using various kinds of biochemical test such as Gram staining, Lactose fermentation test, Motility test, Catalase test and Colony formation on MacConkey agar, Indole test, Citrate utilization test. The susceptibility pattern and resistance pattern had also been evaluated by using fourteen different antibiotics. Mullar Higton agar was used for the determination of susceptibility and resistance pattern. The susceptibility and resistance pattern had also been determined for each type of bacterial strain. The effect of each antibiotic had been tested on various samples and results were compiled to determine which antibiotic was most effective against UTI. Result: Fourteen antibiotics were used for susceptibility while ten antibiotics were selected at a cut off value of 100 samples for E. coli and 20 samples for Klebsiella, then these samples were tested and their results were compared against each type of antibiotic on E. coli and K. species. The result showed that the effect of one antibiotic against various infection agents was not same its susceptibility varies with the type of infectious microbe. Over all, the most susceptible antibiotic was Amikacin with 93.5% susceptibility while the most resistant was shown against Ciprofloxacin with 76.8% resistance. The results in case of E. coli showed that the most susceptible antibiotic was Imipenam with 97.5% susceptibility and most resistance was against Ciprofloxacin 81.7% resistance. Where as against K. species most susceptible antibiotic was Amikacin with 94.9% susceptibility and most resistant was against Tobramycin with 76.1% resistance.. Conclusion: It is concluded from the present study that the effect of each antibiotic is different with respect to type of infectious agent; therefore, it is recommended that the doctor should determine the prime type of infectious agent and its susceptibility response before prescription of any antibiotic. It will help to cure the disease in most effective way. This study confirms that the susceptibility pattern varies from person to person. More over comparison with pervious studies had shown an increase in resistance against various antibiotics. It is suggested that these kinds of studies should be conducted on the large scale to determine the susceptibility of antibiotics against each kind of infection at national level.Item Cloning, expression and characterization of α-amylase gene from pyrobaculum calidifontis(UMT, Lhr, 2017) Ayesha SadiqaThe present study describes the cloning, expression and characterization of alpha amylase from Pyrobaculum calidifontis. α-amylase is an extracellular enzyme and belongs to GH13 family. It is a starch degrading enzyme which hydrolyzes the α-1, 4-glycosidic linkages. In recent biotechnology applications the enzyme is being used in fermentation, food, paper and textile industry. Cloning of alpha amylase (Pcal_1039) gene was done in expression vector pET-21a(+). Pcal_1039 gene was expressed in E. coli BL21 and analysis of protein was done on SDS-PAGE. Recombinant protein was refolded by using in vivo and in vitro methods. The size of the gene was 1.37 kb and molecular mass of protein was 53.5 kDa. After expression the activity of α-amylase was measured by DNS method at OD540. Before refolding the inclusion bodies showed a minor activity while highest activity of 3.3 U/ml and specific activity of 23.6 U/mg/ml was measured by taking expression at 17°C (in vivo).Item Cloning, expression and characterization of trehalose synthase from pyrobaculum calidifontis(UMT, Lhr, 2017) Rabbia MuzaffarTrehalose is a multifunctional disaccharide sugar which is produced in different organisms like plants, animals, fungi and other microbes. It is known for its ability to preserve the important proteins and biomolecules of organisms inside the body under stress conditions i.e. temperature fluctuations and desiccation. Due to this potent nature, it can be used in pharmaceutical, food and cosmetic industries as stabilizer. It is difficult to attain this commercially valuable product from those organisms in high amount. Therefore, enzymes can be used for in vitro synthesis of trehalose at large scale. The most efficient enzyme for the production of trehalose is the Trehalose synthase, a glycosyltransferase. It involves a one-step pathway that convert maltose into trehalose. The enzyme of trehalose synthase was successfully cloned and expressed from Pyrobaculum calidifontis. The ORF of the gene was Pcal_1359. The gene was expressed in E. coli expression system through pET-21a (+) vector. The protein was produced as insoluble inclusion bodies. That was refolded by various strategies. The considerable activity was observed by refolding with Urea. The 3D structure was not available, therefore it was made by homology modelling technique. Furthermore, the structure was refined by Molecular dynamics (MD) simulation studies that have indicated that the enzyme can survive at high temperature range from 300K to 400K. The docking studies was also performed by using maltose as substrate and acrabose, cathomycin and ValidamycinA as inhibitors. The active site residues of Pcal_1359 was Asp119, His139, Arg222, and Glu308. The binding affinity was highest with cathomycin that indicated it as a potential inhibitor for the enzyme.Item Microbiological analysis of the guava skin grown in soa assal lahore(UMT, Lhr, 2017) MADIHA RAHMANGuava is well known tropical tree which grows up to the 35 feet tall in height. It is abundantly grown in tropic areas for fruit. It belongs to the Myrtaceae family. Guava tree and its all parts have a old history of medicinal values. In this study micro and macro nutrients of guava skin is evaluated. The nutritional values, aflatoxin analysis, antioxidant activity, anti bacterial anti fungal activity of guava skin was evaluated. The purpose of this study was to find the quality and efficiency of guava skin grown in the Soa Asal. Nutrition values were found out by the recommended methods of AOAC. The antioxidant activity was find out by the DPPH and FRAP method. Gel ager method was used for the antibacterial analysis. Results of nutritional values indicated that the moisture content, total ash content, fiber content, protein content, fat content and carbohydrates contents were 5.25%, 7.32%, 17.5%, 3.2%, 0.65% and 20.85% respectively. Phytochemical analysis results showed that the ethanolic extract have maximum phytochemicals. Tannin, Phenol, carbohydrates and sterol were in high content. Aflatoxin analysis showed that no aflatoxin from four types was present in the guava skin. Antioxidant activity analysis showed that the %age inhibition and the reducing capacity were maximum in the yellow ripened skin. This was concentration dependent. Antibacterial activity was highest in the ethanolic extract Salmonella spp. and it was also concentration dependent. Antimicrobial analysis showed that guava skin has a significant quantity of pathogens. Results of the study indicated that guava skin of this area’s guava is safe for use and it can be use for nutritional and medicinal purposes.