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Chloroplast-Based Overexpression of Pharmaceuticals to Develop Cost-Effective Therapeutics

Thesis Info

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External Link

Author

Nurjis, Farwa

Program

PhD

Institute

Quaid-I-Azam University

City

Islamabad

Province

Islamabad.

Country

Pakistan

Thesis Completing Year

2010

Thesis Completion Status

Completed

Subject

Natural Sciences

Language

English

Link

http://prr.hec.gov.pk/jspui/handle/123456789/1605

Added

2021-02-17 19:49:13

Modified

2024-03-24 20:25:49

ARI ID

1676725721450

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Interferon, one of the very powerful components of the immune system, are cytokines in nature, produced and secreted by virtually all eukaryotic cells in response to stimulation by certain viruses, bacteria, antigens and mitogens; they mediate their biological activity by binding to specific receptors on the cell surface and modify cell differentiation and exhibit immunoregulatory activity. Interferon mainly classified as alpha, beta and gamma, based on their common genomic location, structural similarities and competition for binding to the same receptor. The development of recombinant interferon embodied profound hope for the cure of many debilitating and potentially fatal diseases. Various recombinant interferons currently dominate the annual market and are approved to treat many chronic viral diseases as well as a variety of cancers and different types of leukemia. The only type of interferon produced commercially is alpha. The production of interferon alpha from microbial to mammalian expression system, have certain precincts in terms of cost, scalability, safety and authenticity. Modern biotechnology exploits transgenic crops to get large quantities of complex proteins in a cost-effective way. In order to overcome several challenges from biosafety point of view, the chloroplast transformation strategy is one of the best approaches since plastids are strictly maternally inherited in most of the cultivated species. Further, chloroplasts allow hyper expression of recombinant DNA with correct folding of expressed protein due to the presence of chaperonin proteins. Hence transplastomic technology is the best choice, now days, to overexpress pharmaceuticals cost-effectively. In the present study the interferon alpha genes were synthesized by using complex set of oligos. After sequence confirmation of the synthesized genes, the histidine residues along with the thrombin protease site were engineered upstream to the synthetic interferon alpha 5 and 2 genes. The recombinant fragments were then tethered with chloroplast light inducible promoter, rbcL followed by sequential cloning to develop chloroplast transformation vector to target the cassettes into the inverted repeat region of plastome through two events of homologous recombination. Since chloroplast and E. coli transcriptional and translational machineries have similarities to express recombinant genes, therefore the chloroplast specific expression cassettes were first expressed in E. coli and selection was performed on spectinomycin because the vector carries selectable marker gene aadA that encodes aminoglycoside-3`-adenyltransferase and confers resistance to both spectinomycin and streptomycin. The expression of interferon genes was assessed and a reasonable level of recombinant protein was recorded. Encouraged from these experiments with E. coli cells harboring chloroplast transformation vector, sterile tobacco leaves were bombarded using biolistic gun. The putative transgenic plants, obtained as a result of antibiotic selection of chopped bombarded leaves, were subjected to different round of selection and regeneration for homoplasmicity. The spectinomycin-resistant shoots were routinely analyzed for gene presence through PCR and the expression of introduced synthetic genes was recorded using ELISA technique. It was experienced that mature leaves contained high levels interferon compared to young and senescence leaves.
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پلاٹ

علی اکبر ناطق نے بہت ہی منظم انداز میں کہانی کے پلاٹ کو ترتیب دیا ہے۔مسلمانوں  ہندوؤ ں اور انگریزوں کی نفسیاتی کشمکش، کیفیات اس طرح جامع انداز میں منظر عام پر لے کر آئے ہیں گویا محسوس ہوتا ہے کہ ایک ہی وقت میں اس نیان تمام زندگیوں کو جیا ہے اور محسوس کرنے کے بعد اسے ناول کا رنگ دیا ہے۔سکھ ، انگریز ، مسلمان اورجاگیرداروں کی چپقلش کو اس نے وہ عملی جامہ پہنایاہے کہ گرفت بھی قائم رہتی ہے اور اس ہنر مندی نے  پلاٹ کو منظم اور موبپط بھی کیا ہے۔

قرون وسطی میں مصر سے ملتان تک قرامطہ کے سیاسی و مذہبی اثرات

With the decline of strong Muslim Khilafate various sectarian based movements proved a serious danger for the Muslim world. Qramtah movement was most famous among them. During the latter period of Abbassid Khila-fate, Qaramtah appeared very strongly. They had a strong hold in different part of Islamic state. Bahrin was their strong head quarter and then they spread all around in state especially in rural areas. They defeated a large and powerful army of Khalifa with a small army severl time. Qramtah killed a millions of innocent Muslims. They captured Makkah and disgrced “Bait Ullah” and banned Hajj for almost 20 years. They propagated their philosophy and beliefs in all over the Muslim world by force. Qramtah also established a strong government in Multan after the departure of Muhammad bin Qasim. Jalam bin Shaban was a famous Qramtian ruler of Multan In 1004 A.D. When Mehmood Ghaznavi came in Multan at that time Abul Fatih Dawud Qramti was the ruler of Multan Mehmood arrested him and destroyed the power of Qramtah in Multan. Qramtah continued serious unrest in Islamic world for four centuries. This movement effected badly the Muslim world and they have become politically weak against their political rival Christianity.    

Usage Patterns and Impact Fo Smartphone on Social Behaviour of Youth in Pakistan - Challenges and Prospects

Every year, the development in handheld communication technologies is being made at an extraordinary speed and the smartphone has seen a spectacular enhancement throughout the world. Today smartphone has become an essential gadget in our lives. In many ways, the smartphone facilitates our relationships enabling us to contact those who would not otherwise be able to converse face-to-face. In fact, smartphone has been converted into an all-in-one device as it provides access, like SMS, voice and video call, Internet through Wi-Fi, sharing and editing MS word files. It has become an important aspect of youth’s daily life that has moved from a mere ‘technological object’ to a key in ‘social object’. Despite of the fact, this device has projected many challenges and prospects. The overall functions and usage of the smartphone has attracted youth to different purposes and to perform various functions in their daily life. However, the present study is primarily a cross-sectional social survey applying quantitative approach. A questionnaire on five-point Likert scale was developed to collect the data from subjects. Cluster, multistage simple random and purposive sampling techniques were applied to select a sample from four universities. The sample comprised 800 subjects (male and female) studying at BA/BSc and MA/MSc level in selected universities (p.78). Different tests like Regression Analysis, t-test, ANOVA, Chi-Square and Cross-tabulation were applied to analyse the results and interpretation. The reliability of questionnaire was also assessed through Cronbach’s alpha which was 0.79 (p.80). Almost all the subjects (youth) were found using smartphone in different ways and at different times. Their responses showed that they were selective in using social network applications. The male subjects, in majority, used social applications for building social relationship as compared to female subjects. Most of the male subjects liked WhatsApp, Facebook, Round and Viber; whereas, female subjects had been using WhatsApp and Facebook only for social relationships. The overall results proposed that smartphone usage habits had impacted positively on the social behaviour of youth. Most of the subjects were found using smartphone in activities like making friends for social relationship, capturing pictures of friends during social gathering and sharing x them with other friends. They also used emails with smartphone for sharing some social events or other activities with friends. The overall findings documented that smartphone usage had a positive impact on values, beliefs, education, social relationship and social behaviour of youth in Pakistan which was significantly proved. The majority of the subjects agreed that smartphones usage led to increase social relationships, and had a positive impact on their social behaviour. The results of t-test of selected variables also proved that youth (both male and female) was more inclined to the smartphones usage. The results also showed that emerging patterns of social behaviour were associated with the smartphone usage in social connection. Based on findings, it has been suggested that university teachers may establish learning environment for youth like BYOD (By Your Own Demand) or SMILE (Smartphone Interactive Learning Environment). In the nutshell, it is convenient to conclude that the smartphone has become an essential part of the youth’s life for interaction, sharing, liking, etc. The most prominent findings were that youth with higher levels of smartphone usage had a greater tendency to engage in using social networking applications for social relationship.