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Home > Mutational Study of Ns5a Gene in Therapy Resistant Patients Infected With Hepatitis C Genotype 3

Mutational Study of Ns5a Gene in Therapy Resistant Patients Infected With Hepatitis C Genotype 3

Thesis Info

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Author

Shaikh, Naila

Program

PhD

Institute

Liaquat University of Medical and Health Sciences

City

Jamshoro

Province

Sindh

Country

Pakistan

Thesis Completing Year

2016

Thesis Completion Status

Completed

Subject

Molecular Biology

Language

English

Link

http://prr.hec.gov.pk/jspui/bitstream/123456789/14122/1/Naila_Shaikh_Molecular_Biology_2016_HSR_LUMHS_Jamshoro_22.08.2016.pdf

Added

2021-02-17 19:49:13

Modified

2024-03-24 20:25:49

ARI ID

1676726740100

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Hepatitis C is wide spread infectious disease, affecting almost 185 million people worldwide. It is more prevalent in developing and third world countries, where people have poor health facilities and related education. In Pakistan, nearly 10 million people are affected with HCV and majority of the patients belong to poor class with limited resources for treatment. The available conventional therapy for HCV is not 100% successful and almost 50% patients fail to achieve sustained virological response. The rate of treatment response depends on genetic components of virus and host. The aim of this study was to analyze the host and viral genetic variations associated with interferon and ribavirin therapy in chronic hepatitis C patients. Genetic changes in ISDR and PKRBD region of NS5A gene of HCV and variations of human immune gene IL28B were studied in relation with response to HCV treatment. Total of 220 HCV patients, were enrolled, 100 were responders and 120 were non responders to conventional therapy. The whole blood samples were collected for viral RNA and human DNA extraction. HCV genotype was successfully determined in 60% non-responder patients The viral genomic segments were directly sequenced while IL28B SNPS were analyzed by RFLP. The results showed multiple mutations in ISDR and PKRBD regions of NS5A gene, including two consistent missense variants, A2252V and F2252I. In 74.2% (52/75) of the patients, more than 30 mutations were found in the regions. RFLP analysis of IL28B SNPS revealed that rs12980275 AA (p<0.0001), rs12979860 CT (p=0.001) and rs8099917 TT (p=0.032) genotypes are associated with response to treatment where as AG/GG genotype of rs12980275, CC/TT of rs12979860 and GT/GG of rs8099917 are associated with non response to treatment. The un-typed HCV genotypes were further explored by sequencing 5’UTR region and various nucleotide changes were observed. The results revealed the significant association of rs12980275 polymorphism with treatment response in HCV patients followed by rs12979860 and rs8099917 and various mutations in NS5A gene showed impact on treatment response. These finding may help to predict the response to treatment with IFN in HCV patients and may reduce the side effects and cost of treatment in predicting non-responder patients.
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یار پرانے چھڈ کے ٹریوں۔۔۔

یار پرانے چھڈ کے ٹریوں نویاں من پسنداں نال
ہتھیں دتیاں ہوئیاں گنڈھاں، بہہ کے کھولیں دنداں نال
ہک پل کول کھلوتیاں میرے، جے کر جگ نے ویکھ لیا
پھیر یقین کسے نہیں کرنا، قسماں تے سوگنداں نال
قدم قدم تے ہے پئی چمدی، منزل پیر مسافر دے
چار دیہاڑے بہہ کے جس نے کڈھے دانش منداں نال
تن من دھن قربان کرایا، دین بچایا نانے دا
شاہ حسینؑ، شہید ہوئے نے، خویشاں تے فرزنداں نال

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Formulation of Controlled Release Hydrogel Microspheres Using Acrylate Derivatives: in Vitro and Invivo Studies

The aim of present study was to develop, characterize, in-vitro and in vivo evaluation of copolymeric pH sensitive microgels/hydrogel microspheres for controlled drug delivery of antihypertensive drugs (nifedipine and diltiazem HCl). Itaconic acid which is a highly hydrophilic monomer having two carboxylic acid groups on side chain and able to form hydrogen bonds with corresponding groups. Due to the double ionization of IA at different pH values, stepwise release behavior was observed for specially adsorbed drugs or other adsorbents by controlling the pH of the medium. Butyl acrylate and 2-ethyl hexyl acrylate (EHA) are hydrophobic monomers which are used to make pH responsive microgels through co-polymerisation with itaconic acid. Nifedipine and diltiazem HCl are calcium channel blockers, are used for the treatment of hypertension and angina pectoris. Due to the poor bioavailability of drugs which undergo first pass hepatic metabolism, pH sensitive copolymeric microgels/ hydrogel microspheres of these antihypertensive drugs were developed. Suspension polymerization technique is used to synthesize pH sensitive copolymeric butyl acrylate-co- itaconic acid p (BA- co-IA) and 2-ethyl hexyl acrylate -co- itaconic acid p (EHA- co-IA) hydrogel microspheres or microgels by using different ratios of monomers with the addition of 5% ethylene glycol dimethacrylate (EGDMA) as crosslinker and 1% benzoyl peroxide (BPO) as an initiator. P (BA- co-IA) hydrogel microspheres or microgels were loaded with nifedipine and p (EHA- co-IA) hydrogel microspheres or microgels loaded with diltiazem HCl by equilibrium swelling method after preparation of empty microgels. Prepared hydrogel microspheres or microgels were evaluated by fourier transforms infrared spectroscopy (FTIR), scanning electron microscopy (SEM), x-ray diffractometry (XRD), thermal gravimetric analysis (TGA), differential scanning calorimetry (DSC). FTIR analysis of both p (BA- co-IA) and p (EHA-co-IA) microgels confirmed the formation of copolymer. FTIR, DSC and X-RD techniques were used to confirm the chemical stability of nifedipine and diltiazem HCl after encapsulation into prepared formulations. TGA of both formulations p (BA- co-IA) and p (EHA-co-IA) indicates that prepared microgels showed much better thermal stability than pure drug nifedipine and diltiazem HCl respectively. SEM images showed that both formulations were spherical in shape. The size distribution of p (BA- co-IA) and p (EHA- co-IA) hydrogel microspheres was found between 4.145μm to 10μm and 4.145 μm to 20 μm respectively using the malvern nanosizer ZS instrument. The nifedipine was encapsulated into prepared hydrogel microspheres maximum up to 67% and maximum % yield was about 72 %. p (EHA- co-IA) hydrogel microspheres were loaded with diltiazem HCl up to 96 % and their % yield were up to 76 %. In vitro release studies of both formulations confirmed the pH sensitivity of the prepared hydrogel microspheres. By appling korsmeyer-peppas equation to cumulative drug release data of both formulations to calculate the diffusion exponent (n), this follows non-Fickian diffusion. These pH sensitive hydrogel microspheres loaded with antihypertensive drugs (nifedipine and diltiazem HCl) are validated in rabbit plasma by HPLC analysis and then in vivo studies of these formulations were performed in rabbits.