1.
Estimation Of Heavy Metals In The Drinking Water Of Residential/Industrial Areas Of Lahore By Atomic Absorption
by Waheed Ahmad | Dr. Abu Saeed Hashmi | Dr. Sualeha | Miss Shagufta Saeed.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 2010Dissertation note: Heavy metals are chemical elements with a specific gravity that is at least 5 times the specific gravity of water. The elements studied were mercury, lead, arsenic, cadmium and chromium. Heavy metals have no useful biological function in the body but might be highly toxic as they cause precipitation of proteins especially the enzymes. This investigation was therefore carried out to estimate concentration of these metals and their influence on biological system. For this purpose drinking water samples were collected in one litre polyethylene bottles adding 5 mL of concentrated HNO3 as preservative to adjust the PH<2.00 to maintain heavy metal concentrations during analysis. Samples were marked with unique numbers with dates for the study of Acid Extractable metals. Similarly samples were prepared and preserved for micro biological testing.
The metallic ions were estimated by Atomic absorption spectrophotometer of Perking Elmer Model A. Analyst; 2003 at recommended wavelengths for metal ion. Acetylene gas was used as fuel (at 8 psi) and air as an oxidizer.
Statistical analysis was done. The calibration curves were prepared separately for all the metals by running suitable concentrations of the standard solutions. It was evident that concentration of chromium, lead, mercury, arsenic and cadmium were high in several drinking water sources in Lahore. This problem is particularly alarming for ground water sources. Almost all water sources are contaminated with lead. According to WHO maximum acceptable limit 10 ppb ,8 water sources had mean chromium concentration in water samples above maximum acceptable limit of WHO (50 ppb), 94 water samples were contaminated with cadmium according to WHO maximum acceptable limit (10 ppb), 13 water sources had arsenic concentration above maximum acceptable limit according to WHO (50 ppb) where as 7 water samples were having concentration of arsenic less than minimum acceptable limit according to WHO (10 ppb) and only 5 water sources meet the criteria of WHO for concentration of mercury, the acceptable limit of 2 ppb.
Multitube Fermentation Technique/MPN Method as described by Mackie & McCartney was used for microbiological analysis i.e. Colifcrm bacteria. The results of this study revealed that both samples i.e. tap and ground water do not show conformity with the standards for safe portable water recommended by WHO. The most frequently encountered pathogen in this study was Escherichia Coli which was isolated more in ground water than tap water.
It is therefore concluded that by using Atomic Absorption Spectrophotometer concentration of heavy metals in water can be determined and thus on the bases of this work precautionary measures can be taken to prevent the health hazards of these toxic metals. Similarly microbiological analysis of drinking water has provided the evidence that most of the water sources are contaminated with microbes.
Availability: Items available for loan: UVAS Library [Call number: 1170,T] (1).
2.
Dna Fingerprinting Of Pakistani Buffalo Breeds (Nili-Ravi, Kundi) Using Microsatellite And Cytochrome B Gene
by Rashid Saif | Prof.Dr.Masroor Elahi Babar | Mr. Asif | Prof. Dr. Abu Saeed Hashmi.
Material type: ; Format:
print
Publisher: 2010Dissertation note: Customarily, classification of breed was based on phenotypic traits. In some cases, recent genetic studies have found differences in the structure proposed. In buffalo ,one has to bear in mind that morphological changes were not the result of adaptation to the environment, but have a social significance thus may not be indicative of the genetic relationship. In recent years microsatellites have proved to be very useful for the determination of genetic relationship among population. Comparative studies beiween microsatellite and protein markers have highlighted the advantages of the former.
The water buffalo (Bubalus bubalis) holds tremendous potential in livestock sector in many Asian countries, particularly in Pakistan but the genetic data of different buffalo breeds like Nili-Ravi and Kundi is lacking, which need to be established for their genetic identification. Blood samples of unrelated true representative of both breeds (Nili-Ravi and Kundi) were collected from different government livestock farms in Punjab and Sindh respectively. DNA was extracted by inorganic method and amplification of the mitochondrial Cytb gene and microsatellite was done with especially designed primers in Molecular Biology and Genomics Laboratory at Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Lahore. Cytochrome b gene markers have been proved as an efficient and powerful tool for breed characterization and species identification of buffaloes. Several panels of microsatellite markers have also been reported for this purpose. In this study, a panel of nine microsatellite markers has highly Polymorphism Information Content (PlC) were selected, Specific primers were designed for these microsatellite and Cytb gene partial amplification using primer3 software. Then primers were optimized for successful amplification with minimum reagent concentration. PCRs were performed for amplification of these microsatellite and Cytb markers on each sample, Genotyping and sequencing was conducted on all amplicons to find out the different SNP to design haplotypes with the help of bioinformatics software e.g. Blast 2sequence and Chrornas Lite, Further statistical analysis was done by the help of some other software e.g. Popgene version 1.31, Power Stat., Genetic diversity, Allele frequencies, observed and expected homozygosity and heterozygosity, Hardy Weinberg equilibrium, F-Statistics and Gene Flow for all Loci, population's dendogram, Nei's genetic identity and genetic distance! diversity was calculated. The results obtained from this study can contribute to the establishment of routine DNA typing services, beneficial for the buffalo industry as well as in animal forensics for litigation and expedite the police investigation services in Pakistan, which will also be useful for breed characterization and phylogenetic study of aforementioned breeds of buffalo.
Availability: Items available for loan: UVAS Library [Call number: 1183,T] (1).
3.
Detoxification Potential Of Yeast Sludhe Ahainst Ochratoxin In Broiler Chicks
by Huma Mujahid | Dr. Abu Saeed Hashmi | Dr. Aftab | Miss Asma Waris.
Material type: ; Format:
print
Publisher: 2011Dissertation note: Ochratoxin the fungal secondary metabolite is a potent natural contaminant of poultry feed. Mycotoxins present in poultry feeds from the raw material used in their production is the major cause of toxic feed. The intake of very low levels of Ochratoxin-A result in overt ochratoxicosis resulting in impairment of immune system and acquired resistance to infections causing health problems which lead to economic losses in the form of reduced productivity The research study was conducted to study the harmful effects of Ochratoxin on broiler chicks and the adsorptive potential of yeast sludge against Ochratoxin in broiler chicks .
Aspergillus ochraceus was grown on Sabraud's Dextrose Agar and ochratoxin was produced on fermented wheat grains .One fifty day old Chicks of broiler breed were purchased from Big birds hatchery and were raised on commercial broiler diet till 7 days.
Four diets A,B,C and D were formulated A diet serve as control, B diet contained OTA 500ppb, C diet contained OTA 500ppb and 1% Yeast sludge and D diet contained OTA 500ppb and 2% Yeast sludge. These four diets were assigned randomly to the chicks, such that there were three replicates on each ration and each replicate contained 10 chicks. Vaccination against N.D and IBD was performed according to the schedule. During feeding trial weight gain , feed consumed, FCR and mortality rate was determined.
Group B (500ppb OTA) showed a decrease in weight gain and feed consumption as compared to group A (control diet) , C (1% yeast sludge and 500ppb OTA) and D (2% yeast sludge and 500ppb ochratoxin). Group D showed more improvement in weight gain, feed consumption and FCR as compared to group C.
Blood serum and tissue samples were collected from the birds slaughtered at the end of experimental trial. Concentration of serum total protein, albumin and activity of alanine transaminase were determined. Blood Serum levels of total protein and albumin were lower in the group B (500ppb OTA) than group D having 2 % yeast sludge but the group C fed on 1% yeast sludge did not show much improvement in those parameters. Activity of ALT was found to be significantly higher (P<0.05) in group C as compared to all other groups. Whereas blood serum ALT activity of the birds fed on ration B was significantly high (P< 0.05) as compared to blood serum ALT of group A
The Level of Ochratoxin in Liver and Kidneys was also determined and it was found to be highest in Group B (500ppb OTA) and lowest in Group D (500ppb OTA + 2% yeast sludge).
Based upon the observations obtained in this study it can be concluded that ochratoxin-A is a nephrotoxic and hepatotoxic agent. But supplementation of 2% yeast sludge in the broiler diet can effectively detoxify the effects of ochratoxin as compared to supplementation of 1% yeast sludge in the chicks diet.
Availability: Items available for loan: UVAS Library [Call number: 1313,T] (1).
4.
Antibacterial Activity Of Indihenous Hernal Exteacts Ahainst Urease Profucinh Bacreria
by Rubina Yasmeen | Dr. Abu Saeed Hashmi | Dr. Aftab | Miss Shagufra Saeed.
Material type: ; Format:
print
Publisher: 2011Dissertation note: Poultry farming is a profitable business but is facing serious ammonia environment particularly during winter season when ventilation frequency is reduced to maintain the shed's temperature. Urease producing bacteria in droppings are main cause of emitting ammonia in the sheds. The ammonia poultry environment is inducing reduced weight gain, immuno-suppression, enhanced susceptibility to respiratory pathogens, etc. Aqueous and alcoholic extracts of 14 local herbs (Aloe Vera, Azadirachta indica, Allium sativum, Calotropis procera, Cannabis sativa, Carum capticum, Eucalyptus camaldulensis, Lantana camara, Mangifera indica, Mentha piperita, Nigella sativa, Opuntia ficus indica, Piper nigrum and Zingiber officinalis) and four commercial herbal products (Mentofin, Suduri, Safi, Yucca) were evaluated for their in-vitro antibacterial activity against Proteus mirabilis by serial dilution method. It was observed that with reference to rise in pH, Ammonia concentration and urease activity in aqueous and alcoholic extracts of Allium sativum (pH: 8.5560, 8.8480, Ammonia:4.42, 3.52 µg/mL, Urease: 0.009, 0.007 U/mL respectively) had shown best results as compared to control positive (pH: 9.03, Ammonia: 6.7µg/mL, Urease: 0.013 U/mL). Alcoholic extracts of Mangifera indica (8.8820, 5.42µg/mL, 0.010 IU/mL), Mentha piperita (8.8880, 4µg/mL, 0.008 U/mL) Carum capticum (8.9540, 4.84µg/mL, 0.009 U/mL) and aqueous extract of Opuntia ficus indica (8.8100, 5.22µg/mL, 0.010 U/mL) had weak activity against P. mirabilis. Both aqueous and alcoholic extracts of Eucalyptus camaldulensis (pH: 8.91, 8.96, Ammonia: 5.16, 5.06 µg/mL, Urease: 0.01, 0.01 U/mL) has weak inhibitory effect. All commercial products had shown strong antibacterial activity (pH: 4.8-6.8, Ammonia: 0µg/mL, Urease: 0 U/mL). Results of remaining herbal extracts were not significantly different (p<0.05) from positive control. It was concluded that all herbal products had strong antibacterial activity against P. mirabilis. Mentofin had shown best results with optimum inhibitory concentration (1/1000 mL). Alcoholic extracts of few herbs had shown weak bactericidal activity. These herbs might give better results in-vivo.
Availability: Items available for loan: UVAS Library [Call number: 1314,T] (1).
5.
Isolation And Characterization Of Collagen Type Ii From Poultry Trachea
by Sidra Ashraf | Dr. Abu Saeed Hashmi | Dr. Sualeha Riffat | Zahid Mushtaq.
Material type: ; Format:
print
Publisher: 2011Dissertation note: This project was designed to use poultry waste to isolate and characterize collagen type II
from its trachea. Collagen type II is being used along with condroitin sulfate and
glucosamine for the treatment of osteoarthritis and is also available as a neutraceutical
product in the market. For project purpose, trachea of slaughtered broiler birds were
collected from the market and after removing adhering tissue and debris, it was then
washed thoroughly first with distilled water and then with deionized water. Tracheal
cartilage was then cut into small pieces and defattened with chloroform: methanol (2: 1
v/v) solution. After this, the cut pieces were properly cleaned with deionized water. 0.5%
Pepsin solution in 0.5 M acetic acid was prepared. Cartilage was then hydrolyzed by the
already prepared 0.5 % pepsin (in 0.5 M acetic acid) at 4 ° C for 48 hours. The extract
was then separated from the tracheal pieces and the viscous solution obtained was
centrifuged at 12000 rpm for 1 hr at 4 "c. Now the collagen was expected to be in the
supernatant which was salted out by adding NaCI to a final concentration of 2.5M and
kept for almost 12-16 hrs. This collagen was again centrifuged at 12000 rpm for 1 hr at
4 C. The obtained collagen pallet was redissolved in 0.5 M acetic acid and then it was
dialyzed against 0.1 M acetic acid followed by dialysis with distilled water. The sample
after dialysis was put in petri dishes and kept in freezer for overnight to let it be prepared for
lyophilization. The frozen collagen sample was then lyophilized. After lyophilization, the sample
gave an appearance of a white mesh. This sample was reconstituted in PBS with pH 8 to run it on
SDS-PAGE. The procedure of SDS-PAGE in non reducing conditions was adopted for the
characterization of collagen type II in the sample. The description of results of SDS-PAGE is given
below:
Lane M contains protein markers of different molecular weight. Lane 1, 2 and 3 contains
samples at different steps of the whole procedure showing clear bands of collagen type II.
Lane 4 contains lyophilized sample of collagen type II showing the thickest band (alpha
chain of collagen type II). In this research, poultry waste has been used for making health improving
product. As in our country poultry is used in bulk quantity so if its waste might be used in any
medicinal product then it might not only be useful but also economical for such a developing country
as ours. Another thing is that as this collagen Type II has been extracted from poultry trachea, it
shows that tracheal cartilage is a rich source of such collagen type. Collagen Type II is used in the
cure of arthritis especially rheumatoid arthritis so through this research, it has been made clear that
poultry waste can be utilized in a positive way in medicinal industry and also that collagen Type II
acts as an effective neutraceutical.
Availability: Items available for loan: UVAS Library [Call number: 1330,T] (1).
6.
Preparation Of Turnip Peroxidases And Its Application To Remove The Phenolic Content Of Sannerty Effluent
by Muhammad Usman Amin | Dr. Abu Saeed Hashmi | Miss. Faiza Masood | Mr. Tanveer.
Material type: ; Format:
print
; Literary form:
drama
Publisher: 2011Dissertation note: Peroxidases are heme-containing oxidizing enzymes, which are wide spread in nature. They have the ability to catalyze the oxidation of many organic and inorganic electron donor substrates through a reaction with hydrogen peroxide or organic hydrogen peroxides. In this study peroxidase were purified from turnip using ammonium sulphate precipitation, poly ethylene glycol precipitation and zinc sulphate precipitations in order to find some simple and less expensive procedure for partial purification of peroxidases. Ammonium sulphate and PEG (6000) in the presence and absence of NaCl were used to make aqueous two phase system. Aqueous two-phase system (ATPS) without NaCl purified enzyme most efficiently. (NH4)2SO4 layer was subjected to dialysis and for further purification on sephadex gel which gave maximum enzyme activity of 1544u/mg protein. SD-PAGE analysis was done to determine enzyme purity. Purified enzyme was charged into the tannery waste water along with H2O2 to remove toxic phenolic content up to 98.24%.
Availability: Items available for loan: UVAS Library [Call number: 1356,T] (1).
7.
Production And Characterization Of Hemicellulaase Activities From Aspergillus Flavus
by Hamna Qayyum | Ms.Faiza Masood | Dr. Abu saeed hashmi | Mr. Tanveer.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 2012Dissertation note: The study was conducted with objective of optimized xylanase using local raw materials from indigenous isolate of Apergillus jlavus.
The fungus was grown on different carbon sources including wheat bran, rice polishing for the production of xylanase enzyme. All four fungi produced xylanase activity in the medium containing wheat bran and rice polishing (1%) at pH 7.0 for 4 days. Maximum activity (14.3U/mL) was depicted by A. jlavus3 in medium with wheat bran. On the basis of better xylanase production A. jlavus3 was selected for further production and characterization of enzyme studies. The highest xylanase activity was achieved in cultivation with wheat bran (lS.8U/mL).
A slightly higher quantity of xylanase was produced by the strain in wheat bran-supplemented medium (18.5 U/mL) followed by rice polishing (17.9 U/mL) when 3% carbon sources were used. The effect of supplementation of different source of nitrogen on xylanase activity by A. flavus was studied with 3 % carbon source. Of all the nitrogen sources investigated, yeast extract (organic source) was the most promising and the corresponding xylanase production was 19.9 UlmL (wheat bran) and 18.3 U/mL (rice polishing). Com step liquior was used to enhance the activity of xylanase which was approx. 10 % higher than that of control medium lacking com step liquior. The highest level of xylanase activity as well as extracellular protein using wheat bran was reported .Maximum xylanase production occurred at pH 5.5 and activities of enzyme were obtained at temperature 30°C for A. jlavus.
The enzyme was purified by gel filtration, ion exchange chromatography. The enzyme activity was characterized on different temperatures and pH ranges.
Availability: Items available for loan: UVAS Library [Call number: 1385,T] (1).
8.
Alayisis Of Sodium Channel Subunit Beta-1 ( Scnib ) Mutations Involved In Generalized Epilepsy With Febrile Seizures
by Salma siddique | Dr.Muhammad Wasim | Dr. Abu saeed hashmi | Dr. Atif Hanif.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 2011Dissertation note: Epilepsy is, one of the most common disorders of the brain, a common neurological
condition defined by recurrent and unprovoked seizures. Epilepsy affects 50 million
people worldwide, including one in every 200 children. Febrile Seizures (FS) are not
thought of as a true epileptic disease but rather as a special syndrome characterized by its provoking factor (high grade fever) and a typical range of 6 months to 6 years. The
patients with generalized epilepsy with febrile seizure plus (GEFS+) show febrile
seizures initially, lasting beyond 6 years of age, and afebrile seizures occur with multiple
types, mainly with generalized seizures but sometimes with focal seizures. Studies have
shown that genetic factors play an important role in the pathogenesis of GEFS+ and other types of epilepsy. Mutations in a number of genes have been identified that leads to the various types of epilepsy. Sodium channel subunit beta-l (SCN1B) was the first gene identified to be associated with febrile seizures. However, very little work has been done on SCNl B gene in epilepsy patients, especially in Pakistan. The present study was
conducted to understand the comprehensive role of SCN1B gene in GEFS+ patients.
Blood samples of unrelated true representative of generalized epilepsy with febrile
seizures plus were collected from psychiatry and preventive pediatrics departments of
various hospitals of Lahore. DNA was extracted and amplified with specially designed
primers and sequencing of the peR products was also done. Analysis of the sequences
and SNPs/mutations was done with the help of appropriate bioinformatics softwares. In
the present study, polymorphism analysis of human SCNIB gene isolated from healthy
and diseased Pakistani individuals (suffering from neurological disorder, GEFs+) have
been investigated for genetic association. Novel mutation IVS2-1 G> T in splice acceptor
site of exon 3 have also been identified from Pakistani GEFS+ patients. This mutation
was absent in the healthy (control) sample. This is first report of gene characterization
and polymorphism of Human SCNI B gene from Pakistan. Likewise, in the last 15 years,
several mutations in the genes have been identified which were associated with GEFs+.
In addition to detecting new mutations and identifying new genes, further studies are
required to elucidate the particular role of furtive mutations, genetic milieu, environment,
or random events on the individual's phenotype. This study has opened a new avenue in
medical sciences in Pakistan, which will help the scientists to work on genetic disease
following the methodologies used in this study. The outcome of this study can further be
used to confirm the hypotheses through animal modeling and proteomics. The mutation
found in this study may add the information in gene databanks, which ultimately help the
scientist to develop the gene therapies for genetic diseases.
Availability: Items available for loan: UVAS Library [Call number: 1388,T] (1).
9.
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).
10.
Effect Of Date Palm Pollen On The Plasma And Intra-Testicular Testosterone Levels Of Male Albino Rats
by Yasir Arfat | Dr. Muhammad Tayyab | Dr. Abu Saeed Hashmi | Dr. Ali Raza.
Material type: ; Format:
print
Publisher: 2012Dissertation note: Considerable evidence exists for the efficacy and safety of short courses of low
dose testosterone therapy for treating infertility and delayed puberty. This treatment is
associated with high levels of patient satisfaction. There is not yet sufficient evidence for
the routine use of other therapies. Experimentally, date extract had been shown to
increase sperm count and increase stimulating concentration of testosterone count in
guinea pigs and to enhance spermatogenesis, follicle stimulating hormone (FSH) and
luteinizing hormone (LH) in rats. Intratesticular testosterone (ITT) is thought to play a
key role in the control of spermatogenesis but is rarely measured.
The present study is therefore designed to examine the effect of date palm pollen
(DPP) (Phoenix dactylifera) on the plasma and intra-testicular testosterone levels using
male albino rat as an experimental animal with the hope that the result of this study may
pave the way for treating male infertility and delayed puberty.
Adult male albino rats were divided into two groups (control and experimental).
Experimental group were given date palm pollen (DPP) suspension in a single oral dose
of 120 mg/kg of body weight for 35 days. Where as the control were given equal amount
of distilled water. Blood samples of control and experimental groups were taken for
measurement of serum testosterone levels at day 0, 12, 24 and finally at day 36.Aanimals
were sacrificed. Testes were removed for gross and biological studies. Intra-testicular
testosterone levels were measured at the end of experimental studies.
There were no statistically significant differences in the variable of control group.
Experimental group who received DPP suspension for 35 days showed statistically significant increase in body weight, weight of paired testes, serum and intra- testicular testosterone levels as compared to control group.
Availability: Items available for loan: UVAS Library [Call number: 1411,T] (1).
11.
Paternal Lineage Analysis In Sahiwal, Cholistani And Dajal Breeds Of Cattle Through Sry And Zfy Genes Analysis.
by Anwar Saeed | Prof.Dr.Masroor Elahi Babar | Dr. Abu Saeed Hashmi | Dr. Asif Nadeem.
Material type: ; Format:
print
; Literary form:
drama
Publisher: 2012Dissertation note: Livestock sector plays a vital role in the economy of Pakistan. Main contribution of milk comes from buffaloes and cattle. Cattle are the major elements of livestock in the country and possess great importance for economy in the form of milk and meat production. Cholistani, Sahiwal and Dajal are the major cattle breeds of Pakistan.
Conventional classification of breeds was based on phenotypic traits. In some cases, recent genetic studies have found differences in the structure proposed. In cattle ,one has to bear in mind that morphological changes were not the result of adaptation to the environment, but have a social significance thus may not be indicative of the genetic relationship. In recent years Y chromosomal genes have proved to be very useful for the determination of genetic relationship among population. Comparative studies have highlighted the advantages of the SRY and ZFY genes of Y chromosome. These genes have been considered as competent and powerful tool for the purpose of breed characterization and species identification of cattle.
Blood samples from true representative animals of each of the three cattle breeds (Cholistani, Sahiwal and Dajal) were collected from different Government livestock farms and their respective home tracts in Punjab. DNA was extracted by inorganic method and amplification of the SRY and ZFY (exon 5) genes of Y chromosome was done with especially designed primers using Primer3 software in Molecular Biology and Genomics Laboratory at Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Lahore. Specific primers are designed for these genes amplification. Then primers were optimized for successful amplification with minimum reagent concentration. PCR was 58
performed for amplification of SRY and ZFY (exon 5) genes on each sample. Sequencing was conducted on amplicons to find out the different single nucleotide polymorphism (SNP) to make haplotypes with the help of bioinformatics software like Blast 2sequence and Neighbor Joining phylogenetic tree was constructed by using MEGA version 5. The results obtained from this study now can contribute to the establishment of routine DNA typing service to the advantages of the cattle in livestock industry.
Availability: Items available for loan: UVAS Library [Call number: 1459,T] (1).
12.
Production, Purification And Characterization Of Xylanase Enzyme From Mutant Aspergillus Flavus Strain
by Rukhsana Tahir | Miss. Faiza Masood | Dr. Abu Saeed Hashmi | Dr. Aftab.
Material type: ; Format:
print
Publisher: 2012Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1506,T] (1).
13.
Bioconversion Of Wheatbran To Glucose By Gluoamylase From Aspergillus Fumigatus
by Hassan Ali | Dr. Muhammad Tayyab | Dr. Abu Saeed Hashmi | Dr. Asif Nadeem.
Material type: ; Format:
print
; Literary form:
drama
Publisher: 2012Dissertation note: Background:
Glucose is produced by hydrolysis of starch. Many crops like maize, rice and wheat can be used as the source of starch. Wheat bran is an agricultural waste byproduct which can be converted to glucose using glucoamylase. Wheat bran is very cheap source for carbohydrates. It is mainly composed of carbohydrates; hemicelluloses, cellulose and starch. Glucoamylase is an enzyme that yields glucose from the nonreducing chain of amylose and amylopectin by hydrolyzing ? -1,3, ?-1, 4 and ?-1,6 linkages of starch. Glucoamylases are produced by plants, animals and microorganism. Microbes, including bacteria, yeast and fungi are major source for the production of glucoamylases. Aspergillus fumigatus is found in soil and in decaying organic matter and it has an essential role in carbon and nitrogen recycling.
Hypothesis: A. fumgiatus might be a good source for the production of glucoamylase through submerged fermentation conditions.
Parameters/Methodlogy: Aspergillus fumigatus was identified macro and microscopically. Enzyme production was measured by DNS method. The effects of different sources of carbon, phosphorous and nitrogen on glucoamylase production were also examined. In order to get the optimum production of glucoamylase, the effect of temperature, pH and incubation period was analysed separately.
Methodology: Initially the A. fumigatus was isolated and conditions were optimized for the growth and production of glucoamylase. Production of enzyme was examined by DNS method. The effects of various carbon, nitrogen and phosphorous sources were examined on the production of glucoamylase. From the present study it was concluded that maximum production of glucoamylase can be obtained from A. fumigatus using wheat bran as the substrate at pH of 4.8, temperature of 40oC with an incubation time of three days.The use of wheat bran as substrate wheat bran for the production of glucoamylase will reduce the cost for the production of glucoamylase.
Availability: Items available for loan: UVAS Library [Call number: 1509,T] (1).
14.
Bioconversion Of Agriculture Waste To Lysine With Brevibacterium Flavum (Wild) And Its Biological Evaluation In Broiler Chicks
by Amber Nawab | Dr. Abu Saeed Hashmi | Dr. Masroor | Ms. Asma Waris.
Material type: ; Format:
print
; Literary form:
drama
Publisher: 2012Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1511,T] (1).
15.
Association Of Myogenic Factor 5 (Myf5) Gene Polymophism With Meat Quantity And Quality Traits In Sahiwal
by Faiza Maqbool | Prof. Dr. Masroor Ellahi Babar | Dr. Abu Saeed Hashmi | Dr. Atif Hanif.
Material type: ; Format:
print
; Literary form:
drama
Publisher: 2012Dissertation note: Pakistan has a large population of farm animals, which are consuming for many purposes. Livestock is the major source of money for any country. Livestock is the major machines and factories which convert roughage (grasses and shrubs etc.) into quality food i.e. Meat and milk.
The Myf5 gene is a member of basic helix-loop-helix family of transcription factors which is involved in the regulation of myogenesis. Its main role in muscle growth, development, proliferation, muscle fibers formation and muscle functioning makes it a candidate gene for molecular marker of meat production in livestock.
Myf5 gene in cattle has been mapped to chromosome 5 and has a length of 3236 bp (Gen Bank accession no. NC-007303). It consists of three exons and two introns. Exons have the lengths of 659, 76 and 1245bp. Role of Myf5 gene in muscle development and growth makes it a candidate gene for meat production in farm animals. In this study association of myogenic factor 5 (Myf5) gene polymorphism with meat quality and quantity traits in Sahiwal cattle was checked out.
In this study blood samples were collected from Sahiwal cattle breeds and DNA was extracted from leukocytes. DNA amplification was done by PCR. Then sequencing of amplified gene was done by Genetic Sequencer.
Allele frequencies and genotype frequencies were statistically analyzed by using SNPator software. The relationship between SNP marker genotypes of myogenic factor 5 (Myf5) gene with meat quality and quantity traits was evaluated by using SNPator software. This study will be a helpful tool for marker assisted selection of beef cattle.
Availability: Items available for loan: UVAS Library [Call number: 1561,T] (1).
16.
Bioconversion Of Industrial Wastes To 6-Aminopencillanic Acid With Escherichia Coli.
by Hasan Javed | Ms. Shagufta Saeed | Dr. Abu Saeed Hashmi | Dr. Muhammad.
Material type: ; Format:
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Publisher: 2013Dissertation note: 6-aminopenicillanic acid is ?-lactam nucleus produced by penicillin acylaseupon hydrolysis of penicillin. 6-APA is main component of semi-synthetic penicillins. Penicillin acylase is most valuable enzyme and is produced by many microbes such as Escherichia coli. Different media and method were used for the isolation, identification an characterization of E. coli. Total 30 strains of E. coli were isolated from fecal matter of equine species and tested for the penicillin acylase activity. About 13 isolates gave the enzyme activity.
For the production of cell mass, different low cost media was used to cut down the price of production. Corn steep liquor, molasses, milk whey and wheat bran was tested for the growth of E. coli. These industrial wastes can minimize the production cost of 6-APA which has a high demand for the production of semi-synthetic penicillins. Corn steep liquor showed better growth of E. coli and can be used as the cheap source of carbon and nitrogen.Phenylacetic acid was also used in the growth medium and it was used as the inducer for enzyme. Without phenylacetic acid in medium, enzyme production decreases.
Corn steep liquor is the best sources for production of cells which is 0.520 mg mL-1 Molasses also better for fermentation and highest value is 0.336 mg mL-1. Milk whey media needs further studies for the better production of cells with using different concentrations.it gave best production 0.112 mg mL-1 Wheat bran is not proper source for cell production and does no showed E. Coli growth. All the strains showed growth in corn steep liquor, milk whey and molasses but not in wheat bran. Among all the strains horse sample (Ho-9) showed better cell production in all the media used.
Availability: Items available for loan: UVAS Library [Call number: 1571,T] (1).
17.
Bioconversion Of Whey To Beta-Galactosidase By Aspergillus Niger
by Muhammad Tayyab Younas | Ms. Huma Mujahid | Dr. Abu Saeed Hashmi | Ms. Sehrish.
Material type: ; Format:
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; Literary form:
not fiction
Publisher: 2013Dissertation note: Beta-galactosidase (lactase) has catalytic property to hydrolyze lactose into glucose and galactose. It is extensively used for the synthesis of milk made products through fermentation. Food rich in lactose have variety of application in industrial and environmental processes.
In present study production, purification andcharacterization of ?-galactosidase synthesized by Aspergillus niger has been considered as a great challenge. Beta-glactosidase is an important enzyme involved in conversion of lactose into glucose and galactose and produced on industrial scale for its large applications in the field of health, and food. The production of beta-galactosidase was carried out from fungal culture of Aspergillus niger using whey as a substrate. Optimization of different physical parameters such as temperature, pH, addition of corn steep liquor and production, purification and characterization of beta galactosidase enzyme from Aspergillus niger were studied. Optimum concentration of whey (4mL) were found 13.42 IU/mL and activity of beta galactosidase was found maximum at 72 h of incubation period and further incubation period decline the activity.Optimum pH (13.50 IU/mL)and temperature (17.67 IU/mL) were found 5.5 and 40°C respectively. Addition of corn steep liquor was enhanced the activity of beta galactosidase. Maximum activity was found with 0.6% of corn steep liquor which was 19.4IU/mLas compare to the other nitrogen sources. Finally, addition of ammonium sulphate ?-galactosidase was purified. ?-galactosidase was characterized considering ortho-Nitrophenyl-?-galactoside (ONPG) and whey as a substrate The purified beta-galactosidase was confirmed by SDS PAGE analysis which has molecular weight of 74kDa. The study could also establish that whey could effectively be utilized for ?- galactosidase production thus alleviating water pollution problems caused due to its disposal into the water streams.
Availability: Items available for loan: UVAS Library [Call number: 1387,T] (1).
18.
Identification Of Pesticide Residues In Different Vegetable Collected From Market Of Lahore, Pakistan.
by Anam Munawar | Dr. Muhammad Imran | Dr. Abu Saeed Hashmi | Dr. Muhammad.
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Publisher: 2013Dissertation note: Pesticides are the chemicals which are used to kill or repel the unwanted objects such as pests. Different types of pesticides are present which undergo a different mechanism and kill the pests. Four different types are being used in Pakistan such as organophosphate, organochlorine, pyrehtroid and carbamates. Use of organophosphate and organochlorine become less due the presence of residues. Use of pesticides is increased for a number of purposes such as to increase the rate of production, to decrease the damage of crops and to increase the saving time of different vegetables.
Vegetables are the main source of income of Pakistan, and vegetables are common in our use. Vegetables contain different nutritional elements of our diets. That's why vegetables play an important role in the nutritious diet of a person. The spray of different chemicals on vegetables not only decreases the nutritional elements but also increase the risk of different diseases.
As pesticides leave their residues in vegetables, different techniques can be used to detect the residues and their maximum residue limit, at which limit these pesticides are harmful for humans. Pesticides can also act on unintended individual such as human beings and cause different acute and chronic diseases.
Different vegetables were selected for analyses that are common in use and available in every season. Pesticides which were selected are that which are common in Pakistan and from different pesticide classes.
In present study vegetables of different areas of Lahore were collected and analyzed through HPTLC and GC/MS. HPTLC was used to analyze and calculate the concentration and GC/MS was used for the confirmation of results, and it was concluded that which vegetable contain the high concentration of pesticides. It was studied that which vegetable absorb large amount of pesticides. Potato, tomato, egg plant, okra and cucumber of different markets of Lahore contain high concentration of pesticides as compared to the other vegetables.
Availability: Items available for loan: UVAS Library [Call number: 1510,T] (1).
19.
Suitability Of In-House Developed Pt-Pcr Fro The Detection And Serotyping Of Dengue Virus In Pakistan
by Kashif Iqbal Sahibzada | Dr. Abu Saeed Hashmi | Dr. Aftab | Ms. Asma Waris.
Material type: ; Format:
print
Publisher: 2013Dissertation note: Dengue Virus (DENV) belongs to the genus Flavivirus of family Flaviviridae having four serological different serotypes such as DENV1, DENV2, DENV3 and DENV4 (Bai et al., 2008) Being a Flaviviridae member, the dengue virus is transmitted to human by genus Aedes, mainly Aedes agypti. Over the years dengue fever has become a significant infectious disease in different parts of the world that leads and increases the growth of mosquitoes. It has become epidemic in more than 100 countries on the globe with more than 2.5 billion people at the risk of infection. Pakistan has witnessed some severe outbreaks of dengue viral infection which results to major morbidity and mortality since mid of 90s. There is a need to overcome this infectious and in many cases fatal disease. Imprecise fatality morbidity and statistics underrate the magnitude of dengue as a regional health problem. Medical and public health services have been incapable to diminish this infection since there is no current vaccine available to prevent infectious disease, no effective medical treatments that avert the development of severe symptoms and no sustainable control measures against the vector that guarantee protection of affected communities.
Management of dengue patients and principally dengue hemorrhagic fever (DHF)/Dengue shock syndrome (DSS) cases are the alarming challenges now a day and in the upcoming episodes in this country. To deal with this challenge a sensitive and specific technique is required for its early diagnosis along with the knowledge of dengue serotype to increase the specificity of diagnosis and treatment. This study was designed to check the usefulness of nucleic acid based molecular determination of dengue virus along with nucleic acid sequencing/ analysis of different Dengue serotypes through phylogenetic studies.
Total 50 Blood samples were collected from the dengue suspected patients in 2011 outbreak of dengue. Samples were analyzed by PCR based detection and were compared with IgG, IgM detections to check the usefulness of PCR based nucleic acid detection. In second phase of study nucleic acid sequencing was done
The study has recommended PCR as a suitable and sensitive method for the rapid detection of dengue virus as it was found more sensitive than other utilized techniques including antibodies detection however it was not found useful to differentiate between primary and secondary infection for which a combination of IgG, IgM is more helpful choice. Nucleic acid analysis helped to define the common serotypes/genotypes of dengue virus circulating in Pakistan. In addition the present study has correlated our studied serotypes to other serotypes circulating in the globe which showed 98% homology with Srilankan strain and find out sequence similarities of our serotypes to the other serotypes distributed worldwide through phylogenetic analysis.
Availability: Items available for loan: UVAS Library [Call number: 1551,T] (1).
20.
Biochemical Identification Of Various Causes Of Anemia In Females From District Pakpattan
by Hafiz. Muhammad Toqeer | Dr. Muhammad Tayyab | Dr. Abu Saeed Hashmi | Mr. Muhammad.
Material type: ; Format:
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Publisher: 2013Dissertation note: Anemia is estimated to be affecting almost 600 millions people all over the globe and is regarded as deficiency in Hemoglobin concentration. The decreased amount of hemoglobin in blood could not been able to fulfill the oxygen demand of tissues in body. Keeping in view the above situation, a study was planned to investigate the various types of anemia in dist. Pakpattan.
One hundred blood samples were collected from females randomly selected from various parts of district Pakpattan. The samples were divided into two groups on the basis of age. Group A contains the patients with age between 14 to 26 years where as Group B consist of patients with age 27 to 40 years. Samples were processed in-order to estimate Complete Blood Count, serum iron level, serum ferritin levels, vitamin B12 assay and HPLC based estimation of various variants of hemoglobin.
The results demonstrated that 62% of the total female population of dist. Pakpattan was found to be anemic. Among Group A, 66.66% were anemic due to iron deficiency and 33.33% were due to chronic disease. Group B contained 59.09% anemic, out of these patients, 57.69% were anemic due to iron deficiency, 38.46% due to chronic disease and 2.27% due to deficiency of Vitamin B12. Iron deficiency was found to be the major cause of anemia that is followed by anemia due to chronic disease and Vitamin B12 deficiency. The intensity of anemia was 5% higher in young age females (Group A) as compared to the elder age females (Group B).
This work provided the information about the prevalence of various types of anemia in the population of dist. Pakpattan. The data will be helpful for developing strategy for the control of anemia in future. Further study with a large number of samples, is required throughout the country for the establishment of a data base that will be a good step to control various types of anemia.
Availability: Items available for loan: UVAS Library [Call number: 1611,T] (1).
21.
Decolorization And Degradation Of Azo Dyes In Textile Effluent By Candida Tropicalis
by Urooj Chaudhry | Dr. Abu Saeed Hashmi | Dr. Asif Nadeem | Ms. Asma Waris.
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drama
Publisher: 2013Dissertation note: Azo dyes are synthetic organic compounds widely used in the textile, paper, cosmetics, pharmaceutical and food industries. It consist of one or more azo bonds (-N=N-) associated with one or more aromatic systems. Studies indicate that these dyes are toxic, harmful to the environment and form carcinogenic and/or mutagenic aromatic amines. These are not readily biodegradable in textile effluent treatment.
To decolorize and degrade the textile industry dye effluents by treatment with microorganism Candida tropicalis (yeast) to an extent to make it least harmful to the water habitat and also to make fit for irrigation purposes. The influencing parameters that affect the percentage of decolorization rates are optimized in still culture fermentation. Spectrophotometric analysis method was used to estimate decolorization of textile effluent at its?max 390 nm. The optimal values of parameters such as effluent to water ratio, fermentation time and pH and carbon to nitrogen ratio are found to be 1:5, 72 hours, 6.0and 1:1.72 respectively. The concentration of ionic saltof CaCl2 was also optimized for maximum decolorizationand optimized concentration was 0.15% for Candida tropicalisrespectively. The decolorization of effluent was carried out on large scale in a flask of 2.5 L by applying the predetermined optimum levels. In this case the maximum percent of decolorization of the effluent was found to 80.34% with Candida tropicalis.
Availability: Items available for loan: UVAS Library [Call number: 1629,T] (1).
22.
Molecular Study Of Apolipoprotein E Gene In Hypercholesterolemic Families
by Nasir Ali | Mr. Akhtar Ali | Dr. Abu Saeed Hashmi | Dr. Asif Nadeem.
Material type: ; Format:
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; Literary form:
not fiction
Publisher: 2013Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1630,T] (1).
23.
Lysine Production On Pilot Scale By Brevibacterium Flavum And Its Characterization, Purification And Crystallization
by Muhammad Faisal | Dr. Abu Saeed Hashmi | DR. Aftab | Mrs. Shagufta Saeed.
Material type: ; Format:
print
Publisher: 2013Dissertation note: Food and feed protein demands have increased due to raise in population. Therefore continuous efforts have been progressed to enhance the production rate by conventional and non conventional methods. Fermentation technology have participated decisive role for a long time period and presently the amino acids formed by fermentation set apart principal biotechnology products significantly. By consuming low-cost carbon supply mutants originate potential to the inexpensive built-up for amino acids. L-lysine demand is steadily rising in the sector of feed stuffs, soft drinks, food ingredients, pharmacy and biological fluids, etc. In order to meet the market demand and accomplish growing and assorted L-lysine requirements, microbial metabolic engineering and recombinant DNA technology is the only hope and possibility for advancing the strains. Purification and isolation of material produced is a very significant element extremely influences fermentation practice usefulness and manufacturing expenses. It demands enhancement in the recycling procedure of amino acids, mainly L-lysine.
The present study was designed to produce lysine on pilot scale by using Brevibacterium flavum. A variety of agricultural byproducts like wheat bran, sugar cane molasses and rice polishing were utilized as substrate for lysine production through fermentation by using Brevibacterium flavum. Primarily optimum conditions were determined through fermentation for lysine production on micro scale. Subsequently these conditions were employed for biosynthesis of lysine on pilot scale. Qualitative assay of lysine was performed by TLC and quantitative assay by spectrophotometrically. It was found that amongst all the substrates 4% molasses was produced maximum lysine at 300C. Different inorganic and organic material like 0.4% CaCO3, O.4% MgSO4.7H2O, 0.1% NaCl, 0.8% KH2PO4, 2.5 % (NH4)2SO4, 0.5 % urea, 0.04 mg % biotin and 0.6 % corn steep liquor were found to be optimal for maximum lysine yield. After pilot scale production of lysine in fermentor, different techniques of downstream were applied. The biomass liquor thus produced was purified and crystallized through different techniques to transform in to L-lysine crystals. The information thus attained was subjected to statistical analysis by using one way ANOVA on optimization of different parameters for L-lysine production and comparison of mean values was done by Least Significant Difference (LSD).
Based on the above observations it was concluded that molasses is the most suitable substrate among other agriculture wastes for maximum lysine production with Brevibacterium flavum.
Availability: Items available for loan: UVAS Library [Call number: 1631,T] (1).
24.
Isolation, Purification And Characterization Of Xylanase From Aspergillus Flavus (Wild Stin) Using Agriculture Waste as Substrate
by Hadia Rehman | Ms. Asma Waris | Dr. Abu Saeed Hashmi | Dr. Muhammad.
Material type: ; Format:
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; Literary form:
drama
Publisher: 2013Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1634,T] (1).
25.
Identification Of Polymorphisms In 6Th & 7Th Exons Of "Parkin Gene" And Their Relationship With Parkinson'S Disease.
by Sadaf Niaz | Prof. Dr. Masroor Ellahi Babar | Dr. Abu Saeed Hashmi | Dr. Aif Nadeem.
Material type: ; Format:
print
Publisher: 2013Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1638,T] (1).
26.
Identification Of Pesticides Residues In Defferent Samples Of Milk
by Neelam Shahzadi | Dr. Muhammad Imran | Dr. Abu Saeed Hashmi | Dr. Muhammad.
Material type: ; Format:
print
; Literary form:
drama
Publisher: 2013Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1646,T] (1).
27.
Biosafety Studies Of Transgenic Sugarcane Developed By Camb
by Rizwan Abid | Miss Asma Waris | Dr Abu saeed hashmi | Miss Maryam.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 2013Dissertation note: GM crops confer multiple number of benefits yet it is required to evaluate these crops from every aspect in terms of toxicity, allergenicity or if they cause any immune response. Through brisk improvement in biotech field, a number of transgenic crops have come into prominence and permitted by regulatory authorities for farming and commercialization globally. The potential risk assessment associated with transgenes effect on non-target organisms is of great concern.
The present work was carried out to study the effect of Herbicidal resistant EPSPS protein on animals. For this purpose 40 rabbits were selected i.e., Albino red eye (Newzealand breed). Rabbits are mammals and herbivores and have 95% sequence homology and similar cellular and enzymatic functions like human. Several physical, molecular, histological and biochemical analysis had confirmed the safety of EPSPS protein on non target animals.
The first goal was risk assessment of EPSPS (glyphosate tolerant gene) on rabbits. A total number of forty (40) rabbits of approximately 5-7 weeks old were selected at the start of experiment. These rabbits were placed in 4 groups with comparable body weights, i.e. A, B, C, and D respectively having 10 animals in each group. The 4 groups of animals consisted of purely control diet group (A), non transgenic diet group (B), the 33% transgenic sugarcane diet group (C) and the 40% transgenic sugarcane diet group (D). The groups were fed their particular diets for 90 days. Weight data of each group was recorded after intervals of seven days which showed no difference between these four groups. The weight and growth of all the rabbits increased with the passage of time. Molecular analyses i.e. SDS-PAGE and PCR was also confirmed the absence of EPSPS in blood and urine samples. Furthermore, histological studies gave no evident difference in cellular architecture of transgenic and non transgenic fed rabbits. Finally biochemical tests i.e., Blood urine nitrogen, Alanine transferase, Aspartate transferase, Creatinine, BUN and Cholesterol were observed. Physiological changes of organs were not confirmed in experimental groups when compared to control.
Present studies will help in successful deployment and commercial release of genetically modified sugarcane in Pakistan. Data will also be helpful in evaluating more biosafety concerns about transgenic plants and their potential impact on animals.
Availability: Items available for loan: UVAS Library [Call number: 1701,T] (1).
28.
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).
29.
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).
30.
Extraction, Purificaton And Characterization Of Proteolytic Enzyme From Fig (Ficus Carica)/ Karachi
by Haseeb Akram Sindhu | Dr. Abu Saeed Hashmi | Dr. Aftab | Ms. Faiza Masood.
Material type: ; Format:
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; Literary form:
not fiction
Publisher: 2013Dissertation note: Today, the enzymes are generally used in various industrial applications and require for more stable, highly active and specific enzymes are growing rapidly. Global market for industrial enzymes is reported to be €1 billion in 1995 (Godfrey and West, 1996) whereas, it was increased to $2.3 billion in 2007 and was expected to increase to over $2.7 billion by 2012.
In this piece of research work, purification and characterization of papain (a proteolytic enzyme) from Kachri (Cucumis trigonus) and Ficus (Ficus carica) were carried out. Extraction of papain was done using 0.1M alkaline phosphate buffer of pH 8.00, 70% ethanol and dist.water. Purification of papain was carried out by Ammonium Sulphate precipitation and dialysis followed by Gel filtration by Sephadex G-50. Then characterization of papain such as protein estimation, determination of proteolytic activity (international Unit) of enzyme and SDS-PAGE analysis were performed to determined molecular weight. Finally, the yield and proteolytic activity of papain was measured and compared with the commercial product available in the market.
Crude preparation of enzyme has a wide specificity due to the presence of various proteinase and peptidase isozymes. The performance of the enzyme depends on the plant source, the climatic conditions for growth, and the methods used in its extraction and purification, for example, if the fruit is healthy, then enzyme found is more active.
Papain is used in many industries such as breweries, pharmaceuticals, food, leather, cosmatics, detergents, meat and fish processing for a variety of processes. Therefore, the end use segments are many in signifying that papain has high export demand (Ezekiel and Florence, 2012).
Outcomes
In case, Kachri and Ficus contain high concentration of proteolytic enzyme. These enzymes being present in natural fruit were free from any toxic effect. Hence can be used in food and pharmaceutical industries.
Statistical analysis
Student's t-Test was used for comparing the means of two samples Kachri (Cucumis trigonus) and Ficus (Ficus carica).
Availability: Items available for loan: UVAS Library [Call number: 1722,T] (1).
31.
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).
32.
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).
33.
Genetic Effect Of B-1, 4 Galactosyltransferase-I Gene Polymorphism On Milk Quality In Nili Ravi Buffalo
by Aamir Sohail | Dr. Asif Nadeem | Dr. Abu Saeed Hashmi | Mr. Akhtar Ali.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 2013Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1820,T] (1).
34.
Effect Of Orally Adminisrered B-Gulcan From Different Sourves On Lipid Profile Of Hypercholestrolemic Rata
by Fatima razzaq | Miss.Faiza masood | Dr. Abu saeed hashmi | Dr.Aftab ahmad.
Material type: ; Format:
print
; Literary form:
drama
Publisher: 2014Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 1874,T] (1).
35.
Biologigal Biochemical And Histopathological Responses Of Rats Fed With Detoxified Jatropha Curcas Seed Meal
by Sunnia Sharif | Ms. Faiza masood | Dr. Abu saeed hashmi | Dr. Asif nadeem.
Material type: ; Format:
print
Publisher: 2014Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 2063,T] (1).
36.
Bioconversion Of Agricultural Wastes To Lysine And Its Biological Evaluation In Broiler Chicks
by Shagufta Irshad | Dr. Abu Saeed Hashmi | Dr. Ali Raza | Dr. Masroor Ellahi Babar.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 2014Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 2100,T] (1).
37.
Biochemical Effects Of Ginger And Turmeric Extracts In Diabetic And Dyslipidemic Model Of Rats
by Naveed Hussain | Dr. Abu Saeed Hashmi | Dr. M. Wasin | Mrs. Shagufta Saeed.
Material type: ; Format:
print
Publisher: 2014Dissertation note: Abstract Availability: Items available for loan: UVAS Library [Call number: 2101,T] (1).
38.
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).
39.
Production, Purification And Characterization Of Laccase From White Rot Fungus
by Afrah Shafique (2012-VA-577) | Ms. Faiza Masood | Dr. Abu Saeed Hashmi | Dr. Tanveer Hussain.
Material type: ; Literary form:
not fiction
Publisher: 2014Dissertation note: Laccase (oxidoreductase, EC 1.10.3.2) are blue copper dependent oxidases and the mainligninolytic enzyme produced by white rot fungus. Laccase catalyze the oxidation of large snumbers of phenolic compounds (Kunamneni et al. 2007; Poonkuzhali et al. 2011). These enzymes have a molecular weight 60-90 kDa and consist of 15–30% carbohydrate. Laccases are the earliest and maximum investigated enzymatic systems. Laccase was initially found by Yoshilda in 1883 in the sap of Japanese laquer tree named as Rhusvernicifera. After a while in 1896, Bertrand and Laborde determined that laccase is a fungal enzyme.(Shraddha et al. 2007; Giardina et al. 2010).
Laccases are present extensively in nature, originating from plants, bacteria and fungi (Poonkuzhali and Palvannan 2011). In fungi, laccases are widely distributed in ascomycetes, deuteromycetes and basidiomycetes. The laccase producing fungus include Trametes versicolor, Pleurotus ostreatus, Polyporus, Trametespubescens, Cerrenaunicolour,PhanerochaetechrysosporiumandFunaliatrogiietc (Dwivediet al. 2011). Laccases occur morein fungi, than in the higher plants. Laccases are also present in few bacteria such as S.lavendulae, S.cyaneus, and M.mediterranea(Viswanath et al. 2008; Arias et al. 2003). In vegetables laccases have been recognized in turnips, apples, pears, cabbages, potatoes, beets, asparagus and various other vegetables (Jhadav et al. 2007).
Enzymes are produced by every living organism, however enzyme produced by microbes have various benefits over the enzyme originated from plants and animal origins.Laccases by nature
are important because of its huge diversity of catalytic activities, economical in production and comparatively more stable than other enzymes.The field of biotechnology proposes expanding possibilities for the production of several enzymes from microorganisms. New methods and techniques have been advanced by using enzyme as biocatalysts to produce big added value products like growing food requirements,good quality chemicals and medicines. Moreover enzymes are also utilized for environmental actions and for diagnostic and analytical motives. (Buchholz et al. 2005). Microbial enzymes are used as cost effective and environmentally sensitive substitutes for chemical processing in several industries and bioremediation. Therefore the commercial demand for microbial enzymes is increasing (Radhika et al. 2013).
Fungal laccases have boundless biotechnological functions across the globe like the decolouration and detoxification of industrial effluent, bleaching of pulp, phenolicselimination from wines, in preparation of biosensors in detergents blockindye transfer- functions (Yaver et al. 2001).It is also used in the formation of anticancer drugs, and included in few cosmetics to lessen their toxicity (Couto and Herrera 2006).In recent years, laccase have been skillfully practiced to the field of nanobiotechnology due to its capacity to mobilize electron transfer reactions without further addition of cofactor(Shraddha et al. 2007).
Laccase is ample in several white- rot fungi that are involved in lignin metabolism (Bourbonnais et al. 1997, Leontievskyet al. 1997). Fungal laccases have immense redox potential (up to +800 mV) than bacteria or plant laccases. The action of these laccases seems to be appropriate in nature and also has significant applications in the field biotechnology. These laccases are associated with the deterioration of lignin and also in the elimination of conceivably lethal phenols appear during the breakdown of lignin (Thurston et al. 1994).
The white rot fungus is corporeal in preference to morphological and composes of those fungi that are adequate of degrading lignin, which is a heterogenous polyphenolic compound in huge amount within the lignocellulose wastes(Eaton and Hale. 1993).Theircapability to deteriorate cellulose, hemicellulose, these are the polysaccharides forming the essential part of lingo cellulose is the basic metabolic processbetween the fungi and happen under the span of environmental conditions.The degeneration of lignindoesn’tprovide net energy so it is degraded during the secondary metabolism in order to gain polysaccharides present in lignin and carbohydrate complexes, supplying energy to which the organisms don’t have access(Jeffrics. 1990).The white rot fungi varyingly secrete one or more three extracellular enzyme namely manganese peroxidase, lignin peroxidase and laccase that are fundamental for degradation of lignin, ant they are generally mentioned as lignin modifying enzymes LMEs (Pickard et al. 1999).
Laccase is the subjects of demanding research in the recent years, because of their several properties like extensive substrate relevance, doesn’t required the inclusion of cofactors because they use oxygen as cofactor which is frequently present in the environment (Eugenio et al. 2009). Maximum number of laccases produced by various organisms is excreted as extracellular enzymes and this makes the purification process quite accessible. Laccase commonly display appreciable extent of stability. Due to these properties laccases are ideally applicable in diverse biological processes such as the treatment of industrial effluent, biopulping and biobleaching (Eggert et al. 2006).
The huge potential of laccase requires advancement in its production and, with huge activities and low cost (Herrera et al. 2007). The use of lignocellulosic agricultural waste as substrates is a tradition for the production of enzyme like laccase because it is ligninolytic in nature (Niladeviet al.2011). It is highly crucial to optimize the fermentation parameters for the adequate production of laccase (Revankaret al. 2007). .
The advantages of agro-industrial leftovers for cultivation media is of immense concern as agriculture waste cut down the expenditure of enzyme production and enhance the understanding on energy protection and recycling (Mansuret al. 2003).These agriculture wastes are comparatively economical and also contain ample nutrients such as lignin, cellulose andhemicellulose. These nutrients serve as inducer to energize the production of enzyme (Vassil et al. 2000).Due to these properties these agricultural waste can be used as substrate for the production of ligninolytic enzymes during the process of fermentation.
Laccase can be produced at varying rates by using a wide range of organisms grown on different substrates and by using several methods of fermentation, such as solid state, semisolid state, and submerged (Rodriguez et al. 1999; Boran et al. 2011). However, for effective laccase production, it is very important to use efficient laccase-producing organisms, suitable fermentation methods, and cheap and widespread sources. Accordingly, one of the most suitable approaches for the production of this enzyme is to use the most efficient agricultural wastes for increasing the production of the ligninolytic enzymes (Elisashviliet al. 2008).
Pakistan is an agricultural country and each year manufactures tons of agricultural by products. These agricultural wastes are accessible in markets at a very reasonable price and can be utilized as substrates in fermentation technique (Minussi et al. 2007). Agricultural waste products like rice husk, wheat bran, corn cob, millet husk and cereal huskhave been utilized by various scientists for laccase production (Osma et al. 2011; jhadav et al. 2009).The chemical properties of these agricultural wastes make them important and economical fermentation medium for biotechnological purposes(Giardina et al. 2010).The cellulose and hemicellulose constituents of lignocellulose wastes are widely used by several organisms but lignin, which is the maximum contrary material to microbial degradation, is transformed conveniently by only few organism of thw white rot fungus (Dwivedi et al. 2011).Lignin serves as a barrier that protects cellulose and hemicellulose from enzymatic attack, however, white rot fungi can attack this barrier in order to obtain energy from cellulose. These fungi produce different extracellular ligninolytic enzymes such as laccase, manganese peroxidase, and lignin peroxidase (Couto et al. 2006).
Fermentation is a biological approach that is used for the transformation of complicated substrates into basic composites by different microorganisms like bacteria and fungi. In the procedure of this metabolic breakdown the microorganisms also release various added compounds like carbon dioxide and alcohol asidefrom the conventional products of fermentation. These added compounds are known as secondary metabolites (Pandey et al. 1999). These Secondary metabolites span from enzymes, antibiotics, peptides and growth factors (Balakrishnan and Pandey. 1996; Machado et al. 2004; Robinson et al. 2001). They are also known as bioactive compounds becausethey carry biological activity(Demain et al. 1999).
Submerged fermentation is a type of fermentation in which components are present in a liquid media like broths and syrup. The co-active composites are poured into the fermentation broth. In this media the substrates are employed quiet immediately, due to this reason the nutrients in the media are either fortified or regained continuously. This type of fermentation approach is optimum for microorganisms such as bacteria, fungi because they depend upon on immense moisture content. The increased benefit of this approach is that the purification of the desired products or enzymes is quiet effortless. Submerged fermentation is especially used in the abstraction of secondary metabolites that are utilized in liquid form (Subramaniyam et al. 2012).
Furthermore 75% of the commercial enzymes are made by using submerged fermentation, it also supports the usage of genetically modified organisms to a large expanse then solid state fermentation. Submerged fermentation is also used on large extent because it doesn’t require equipment concerning solid state. On the contrary solid state fermentation is a mechanism operated in absence of free flowing water by utilizing solid support in form of natural substance ( Poonkuzhali et al . 2011). .
The major purpose of conducting this research is to design optimized fermentation process which produces effective amount of enzyme by using agricultural wastes. The use of agricultural wastes as substrates is economical and increase awareness on energy conservation .The enzyme can be used further for bioremediation because it not substrate specific and can act on broad range of substrates.
Availability: Items available for loan: UVAS Library [Call number: 2213,T] (1).
40.
DNA Based Characterization Of Arginase Gene From Geobacillus Sp. SBS-4s
by Raabia Bibi (2012-VA-537) | Dr. Muhammad Tayyab | Dr. Abu Saeed Hashmi | Dr. Muhammad Imran.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Geobacillus is a group gram-positive, rod-shaped, aerobic, endospore-forming and obligate thermophilic bacteria, isolated from the diverse habitats, hot springs, thermal environments, terrestrial soils, deep sea sediments (Zeigler, 2014), petroleum and soil of desserts (Claus and Berkeley 1986). It grows at a wide range of temperature from 45 to 75°C and pH ranging from 6.2 to 7.8 (Nazina et al. 2001). These bacteria survives at higher temperature where most of other living species fail to survive (Claus and Berkeley 1986). Geobacillus have achieved a significant population with a worldwide distribution, probably in large part due to adaptive features of their spores (Zeigler, 2014). These can be found singly or in short chains and motile by means of peritrichous flagella and is capable of secreting a wide variety of extracellular and intracellular enzymes i.e amylase, lipase, carboxypeptidase, cellulase, xylanase, protease and galactosidase (Fogarth et al. 1974; Obeidat et al. 2012).
Geobacillus sp. SBS-4S was isolated from hot spring located in Gilgit, Northern areas of Pakistan. It was found to be an aerobic, gram-positive and rod-shaped bacteria having ability to hydrolyze a variety of sugars, carboxylic acids and hydrocarbons at elevated temperatures from 45 to 75°C. SBS-4S was found to be involved in the production of various intra and extra cellular enzymes (Tayyab et al. 2011).
Arginase is the enzyme responsible for the degradation of arginine resulting in the production of urea and ornithine (Kaur et al. 2009). It is accomplished by the cleaving of the guanidinium group from arginine which yields urea (Turras et al. 2008). Arginase present in many mammals (Homo sapiens), Bacilli (cyanobacteria), protozoa (Entamoeba histolytica), yeast (Saccharomyces cerevisiae), fungi (Neurospora crassa) and plants (Lathyrus sativus) etc (Kaur et al. 2009). The crystal structure of arginases have been determined by X ray crystallographic studies. This is a manganese dependent enzyme. The enzyme shows its activity through the metal ion. Metal ion is actively responsible for the incorporation of water molecules essential for the activity of the enzyme. A second proposed mechanism, based on electron paramagnetic resonance (EPR) studies postulates direct coordination of the substrate to manganese and disruption of the aqua bridge. Arginases are homo-oligomers, with a typical subunit mass of 32 to 36 kDa (Bewley et al. 1999).
There are two types of arginases, arginase-I and arginase-II, located in the cytoplasm and mitochondria, respectively. The principal ureagenic enzyme activity arginase-I is most abundant in normal mammalian liver and acts in coordination with the other enzymes of the urea cycle to sequester and eliminate excess nitrogen from the body. The second form arginase-II can be found in many organs, with the highest levels found in kidney and prostate where as lower levels in macrophages and lactating mammary glands (Iyer et al. 2002).
Important role of arginase in controlling the cellular levels of arginine and ornithine, which are required for various critical metabolic processes, including protein synthesis and the production of creatine, polyamines, proline and nitric oxide (NO). Type II arginase is found in a variety of different tissues and have a key role in the regulation of urea cycle arginine metabolism by regulating levels of arginine in the cell (Bewley et al. 1999). The enzyme arginase plays key role in the pathogenesis of pulmonary disorders such as asthma through dysregulation of L-arginine metabolism and modulation of nitric oxide (NO) homeostasis and it also play role in the development of chronic airway remodeling through formation of ornithine with downstream production of polyamines and L-proline, which are involved in processes of cellular proliferation and collagen deposition (Benson et al. 2011). Arginase involved in tissue repair processes by the synthesis of L-ornithine, which is the precursor of polyamines and proline that are involved in cell proliferation and collagen synthesis (Maarsingh et al. 2009).
Genetically engineered arginase as fusion protein with prolonged half-life and increased efficacy are used to treat different tumor lines that inhibit cell proliferation and impaired cellular migration in vitro and in vivo (Li et al. 2013). This is a arginine-degrading and ornithine producing enzyme and is used to treat arginine-dependent cancers (Yu et al. 2013). Chemically modified arginase-II has been employed for the treatment of taper liver tumor and L5178Y murine leukemia (Kaur et al. 2009). The enzyme was cloned and expressed in E. coli and subsequently conjugated to polyethylene glycol to increase the circulating half-life and decrease the immunogenicity of the recombinant mycoplasma enzyme. The human hepatocellular carcinoma, melanoma cell lines and tissue samples do not express argininosuccinate synthetase (ASS), making them auxotrophic for arginine and thus reasonable candidates for arginine deprivation (Yang et al. 2010).
Arginase is induced in murine myeloid cells mainly by T-helper 2 cells cytokines and inflammatory agents and participates in a variety of inflammatory diseases by down-regulation of nitric oxide synthesis, induction of fibrosis and tissue regeneration. In humans, arginase I is constitutively expressed in polymorphonuclear neutrophils and is liberated during inflammation. Myeloid cell arginase-mediated L-arginine depletion profoundly suppresses T cell immune responses and this is a fundamental mechanism of inflammation-associated immunosuppression. Pharmacological interference with L-arginine metabolism is a novel promising strategy in the treatment of cancer, autoimmunity or unwanted immune deviation (Munder, 2009).
Arginase has very important role in nitrogen fixation and fruit ripening (Yu et al. 2013). Putrescine (1,4-butanediamine) is the product obtained from arginine with the highest market value and it is used as an intermediate in a large number of industries, including the pharmaceutical industry, agrochemical industry and textile industry (Turras et al. 2008).
Arginine is a semi-essential amino acid and is the precursor for the formation of nitric oxide (NO) by nitric oxide synthases (Getz and Reardon, 2006). One of the major functions of arginine within the body is as an intermediate in the urea cycle. In the cytosol of hepatocytes, arginase-I removes the guanidine group from arginine to produce urea and ornithine. Urea is then transported from the hepatocyte into the bloodstream and ornithine is used to regenerate arginine within the hepatocyte. Arginine deficiency causes several disorder like, hyper cholesterolemia, hypertension, diabetes mellitus, kidney failure, hyper homo-cysteinemia, smoking, and aging (Alvares et al. 2012). Arginine is used to modulate the cellular immune response during infection. The generation of nitric oxide from arginine is responsible for efficient immune response (Das et al. 2010).
Arginine is synthesised in humans and other mammals from citrulline in two steps through the urea cycle enzymes, argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL). ASS catalyses the conversion of citrulline and aspartic acid to argininosuccinate, which is then converted to arginine and fumaric acid by ASL (Yang et al. 2010).
Ararinase play important role in conversion of arginine to 1,4–butanediamine (a building block for nylon-4,6), through two main transformations: the hydrolysis of arginine to ornithine and urea; and the decarboxylation of ornithine to 1,4–butanediamine and carbon dioxide. Both steps can be catalyzed chemically or enzymatically (Turras et al. 2008).
The present study deals with the characterization of arginase gene.
Availability: Items available for loan: UVAS Library [Call number: 2244-T] (1).
41.
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).
42.
Polymorphism Of The Slc11a1 Gene Associated With Resistance To Bovine Tuberculosis.
by Qamar Raza Qadri (2009-VA-569) | Dr. Asif Nadeem | Dr. Tahir Yaqoob | Dr. Abu Saeed Hashmi.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Bovine Tuberculosis (bTB), caused by Mycobacterium Tuberculosis is a health threat to
livestock. Information on genetic resistance or susceptibility because of polymorphisms of
candidate genes could be used in making selection decisions. Solute carrier family 11 (protoncoupled
divalent metal ion transporters), member 1 gene (SLC11A1), is a known candidate gene
which is associated with natural resistance to infection by Mycobaterium spp in buffalo.
Polymorphism in this gene can be studied for breeding disease resistance animals. Blood samples
were collected from Nili Ravi buffalo breed of Pakistan. 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 polymorphisms were identified in
exonic region of gene. This might be due to less sample size. Genetics play important role in
fighting against pathogens. Identifying the genes involved can lead to marker-assisted selection
strategies. Availability: Items available for loan: UVAS Library [Call number: 2332-T] (1).
43.
Mutational Analysis Of Hepatitis C Virus Ns4b Gene Encoding Protein
by Faiza Nisar Bukhari (2013-VA-12) | Dr. Muhammad Imran | Dr. M.Yasir Zahoor | Dr. Abu Saeed Hashmi.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Hepatitis C virus (HCV) infection has an estimated global prevalence of 2%–3% with approximately 270 million infected people worldwide of which 10 million belongs to Pakistan. HCV is a positive-sense single-stranded RNA virus from the family of Flaviviridea. Its genome encodes 10 proteins including a 261 amino acids protein NS4B. NS4B is a non-structural protein that performs hyperphosphorylation of NS5A, transactivates interleukin 8 promotor, suppresses HCV translation and modulates the ER stress response. Mutation in NS4B encoding gene may be targeted to develop an effective vaccine against HCV before virus invasion in host immune system.
Serum was collected from 20 patients who are infected with HCV. RNA was isolated from these samples to reverse-transcribe it into cDNA. This cDNA were PCR-amplified and amplicons were detected by UV light after their separation through agrose gel electrophoresis followed by seguencing of NS4B encoding gene.
Variations in HCV NS4B gene was analyzed by using Mega 6 software and sequence 2 blast analysis NCBI tool. Protein modeling was performed with phyre 2, DIANNA tool and I-Tasser online server and quality of model and antigenicity was checked by Vaxajen V 2.0 and Epijen softwares. Conservative analysis and epitope mapping were performed by using Immune Epitope Database (IEDB).
A systematic approach was employed for the prediction of potential epitopes in NS4B protein. Vaxijen V 2.0 was used to determine overall antigenicity of NS4B using cut off value of 0.4. Values above this threshold level show probable antigens in the protein. The topology of NS4B was determined by TMHMM server with the help of membrane topology data. Regions outside membrane and transmembrane were eradicated for epitopes prediction. T cell epitopes propred was used with proteosome cleavage site filter of 4% threshold. From the following data analysis 12 to 15 possible T-cell epitopes can be predicted; only these T cell epitopes were included in conservation analysis.
The present study provides information about mutational changes in HCV protein NS4B and thus preliminary data for the development of vaccine against HCV. This can also lead to future prospects for diagnosis, treatment and prognosis of HCV.
Hepatitis C virus (HCV) infection has an estimated global prevalence of 2%–3% with approximately 270 million infected people worldwide of which 10 million belongs to Pakistan. HCV is a positive-sense single-stranded RNA virus from the family of Flaviviridea. Its genome encodes 10 proteins including a 261 amino acids protein NS4B. NS4B is a non-structural protein that performs hyperphosphorylation of NS5A, transactivates interleukin 8 promotor, suppresses HCV translation and modulates the ER stress response. Mutation in NS4B encoding gene may be targeted to develop an effective vaccine against HCV before virus invasion in host immune system.
Serum was collected from 20 patients who are infected with HCV. RNA was isolated from these samples to reverse-transcribe it into cDNA. This cDNA were PCR-amplified and amplicons were detected by UV light after their separation through agrose gel electrophoresis followed by seguencing of NS4B encoding gene.
Variations in HCV NS4B gene was analyzed by using Mega 6 software and sequence 2 blast analysis NCBI tool. Protein modeling was performed with phyre 2, DIANNA tool and I-Tasser online server and quality of model and antigenicity was checked by Vaxajen V 2.0 and Epijen softwares. Conservative analysis and epitope mapping were performed by using Immune Epitope Database (IEDB).
A systematic approach was employed for the prediction of potential epitopes in NS4B protein. Vaxijen V 2.0 was used to determine overall antigenicity of NS4B using cut off value of 0.4. Values above this threshold level show probable antigens in the protein. The topology of NS4B was determined by TMHMM server with the help of membrane topology data. Regions outside membrane and transmembrane were eradicated for epitopes prediction. T cell epitopes propred was used with proteosome cleavage site filter of 4% threshold. From the following data analysis 12 to 15 possible T-cell epitopes can be predicted; only these T cell epitopes were included in conservation analysis.
The present study provides information about mutational changes in HCV protein NS4B and thus preliminary data for the development of vaccine against HCV. This can also lead to future prospects for diagnosis, treatment and prognosis of HCV.
Availability: Items available for loan: UVAS Library [Call number: 2339-T] (1).
44.
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).
45.
Mutation Anlysis Of DTNBP1 Gene In Pakistani Patients With Schizophrenia Disorder
by Hafiza Sidrah Yasin (2013-VA-11) | Dr. Asif Nadeem | Dr. Maryam Javed | Dr. Abu Saeed Hashmi.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Schizophrenia (SCZ) disorder is a mental complex, heterogeneous and chronic neurodegenerative disorder with a cumulative prevalence of 1%. SCZ is an idiopathic disorder of the cortex and hippocampus. Environmental as well as genetic factors contribute to its complex pathogenesis. A functional repeat polymorphism in the Dystrobrevin Binding Protein 1 (DTNBP1) gene promoter conveys susceptibility for SCZ disorder. The DTNBP1 has been implicated in rare autosomal dominant forms of SCZ disorder because of mutations associated with severe disease progression and a typical physical signs and symptoms, indicative of neurodegeneration. Mutation in DTNBP1 gene has association with change in dysbindin protein which leads to change in abnormal neurotransmitter trafficking which leads to decrease in neuronal size, brain atrophy and reduced glutamate release in schizophrenia disorder. A systematic approach was applied to proceed the present study in order to identify the single nucleotides polymorphisms in schizophrenic patients. Blood samples (n=40) were collected from schizophrenia disorder patients. DNA was extracted by organic method. Primers were designed using Primer3 software. The 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 was done by CHROMAS software. Sequence was aligned by Blast tool of NCBI. Difference between allele and genotype frequency of studied gene was evaluated and analyzed by using “SNPator”. The present study provides information about the susceptibility and genetic basis of the individual towards this disease and identified polymorphisms provides the opportunity to diagnose the disease earlier on the basis of particular SNPs in Pakistani patients. Availability: Items available for loan: UVAS Library [Call number: 2382-T] (1).
46.
Mutational Analysis Of Hcv Gene Encoding E1 Glycoprotein
by Muhammad Saad Junjua (2013-VA-893) | Dr. Muhammad Imran | Dr. Wasim Shehzad | Dr. Abu Saeed Hashmi.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Hepatitis C virus (HCV) is a positive single stranded RNA virus that belongs to Flaviviridae family and causes liver related issues like hepatocellular carcinoma, cirrhosis and chronic liver disease. HCV is affecting people worldwide; more than 170 million peoples have been affected so far and the number is increasing day by day. Its prevalence in Pakistan is about 3% to 6%. There is lot of variation in its genome and it is classified into 6 major genotypes and these genotypes are further classified into many subtypes. Size of HCV is about 9500 bps which only encodes single polyprotein. This 3000 to 3300 amino acids polyprotein is processed by cellular and viral proteases to generate 10 polypeptides consisting of 4 structural (Core, E1, E2 and P7) and 6 non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A, NS5B). The most immunogenic gene from all the genes is E1. It involves in the interaction with the host cell and easily escapes from the immune system of host due to the presence of hypervariable regions in E1 gene.
To isolate the E1 gene, RNA extraction was done using the kit method. RNA was converted to cDNA which was then amplified in two rounds of PCR using nested primers from HCV core region. After confirming the presence of HCV RNA in serum samples, PCR amplification of HCV E1 gene was carried out using gene specific nested primers. Amplified E1 gene products were Sanger sequenced and aligned with standard sequence to find out genetic variations. E1 gene sequences were converted to protein sequences for which secondary protein structures were made and analyzed. No noticeable change was seen in these secondary protein structures. The protein sequences were also analyzed for the presence of B-cell and T-cell epitopes; two T-cell epitopes (QAFTFRPRR, FLVGQAFTF) were found which may inform the development of a proper vaccine against HCV. Availability: Items available for loan: UVAS Library [Call number: 2399-T] (1).
47.
Physical, Chemical and Biological Treatment of Rice Husk to Improve Its Nutrative Value
by Rahat Naseer (2003-VA-196) | Dr. Abu Saeed Hashmi | Dr. Muhammad Tayyab | Prof. Dr. Habib ur Rehman.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: Thesis submitted without CD. Availability: Items available for loan: UVAS Library [Call number: 2450-T] (1).
48.
Bio-Conversion of Molasses to Phytase Through Solid State Fermentation With Aspergillus Niger
by Faseeha Nasim (2012-VA-633) | Dr. Abu Saeed Hashmi | Ms. Faiza Masood | Prof. Dr. Saima.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: CD Corrupt. Availability: Items available for loan: UVAS Library [Call number: 2484-T] (1).
49.
Nucleotide Sequence Variation In Heat Shock Protein 70-1 Gene Of Capra Aegagrus Blythi
by Fehmeeda Fatima (2014-VA-775) | Dr. Asif Nadeem | Dr. Maryam Javed | Dr. Abu Saeed Hashmi.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: Heat shock protein 70 (HSP 70) plays a vital role in survival of an organism by providing
cytoprotection against various kinds of stresses. Among all the HSPs present in the cell, the
ubiquitous HSP 70 proteins are the most abundant and temperature sensitive. Considering the
importance of HSP70-1 gene in conferring thermotolerance, present study has been designed to
characterize this gene in Sindh ibex which is a wild goat species of Pakistan. The
characterization of HSP70 gene might be helpful for deriving phylogenetic relationship among
different species and identifying new functions among the related species. Blood/meat samples
(n=25) were collected from Kirthar national park, Sindh. Standard DNA extraction method was
used for DNA extraction. PCR primers were designed by Primer3 software and amplification of
gene was done by Polymerase Chain Reaction. PCR product was sequenced bi-directionally by
Big DyeTM Terminator on ABI 3130XL Genetic Analyzer. Multiple sequence alignment was
performed for polymorphism identification. Genetic diversity was calculated by using DnaSP
v.5.0. Phylogenetic analysis using the MEGA v.6.0 software package was performed and
neighbor joining and UPGM trees were constructed. The results indicated that Sindh ibex
HSP70.1 gene was highly similar to of domestic goat, sheep, cattle, buffalo, camel and horse
which indicates their origin from a common ancestor. The results of this data might be helpful in
designing effective conservation strategies for Sindh ibex. Availability: Items available for loan: UVAS Library [Call number: 2524-T] (1).
50.
Biochemical And Homology Analysis Of Jak2 Gene In Canines And Hominidae
by Marya Saadullah Khan (2014-VA-324) | Ms. Huma Mujahid | Dr. Abu Saeed Hashmi | Dr. Muhammad Yasir Zahoor.
Material type: ; Literary form:
not fiction
Publisher: 2016Dissertation note: Cancers are considered to be the most lethal of all diseases known out of which myeloproliferative neoplasms comprise of a very little percentage.The frequency of these disorders is known in human beings and a lot of work has been done on humans. But there is a lot of scope for research on this area in canines. As dogs were found to have strong homology with human beings, we compared canine cJAK2 exon 13 sequence with the humanhJAK2 exon 13 and found 96 % homology. Mutations in JAK2 gene are well known to cause three types of disorders i.e. polycythemia vera caused by a well-known point mutation in exon 14 causing substitution of valine for phenylalanine in JH2 domain of the protein.Essential thrombocythemia and idiopathic myelofibrosis may also be caused by this mutation but similar clinical conditions arise without the presence of this mutation. Studies have revealed that other point mutations such as deletion, addition or substitution are also responsible for these disorders.
JAK2 is an intracellular protein which performs phosphorylation of STAT molecules upon their activation. Although the whole protein in its good state is important for its function but the two domains JH1 and JH2 are vital. JH1 domain acts as a tyrosine kinase enzyme and its activity is controlled by JH2 domain also known as pseudo tyrosine kinase domain. Any mutation in these domains leads to protein conformation defect and thus prevents its performance. Besides V617F mutation, other mutations are being discovered in this part of gene. Researchers have found mutations in exon 12, 13 and 15 that have been found to be involved in development of myeloproliferative neoplasms in different cases of patients.
Blood picture do not reveal any direct clue except for increased erythrocytes alone or along with other cells like increased platelets. Therefore blood indices are not reliable parameter to indicate the type of mutation involved in these disorders. Also LDH and EPO levels are not correlated with the disorder. Although EPO test must be done to exclude the possibility of secondary PV and erythropoiesis.
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