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Development of Recombinant Proteins for the Control and Diagnosis of Hydropericardium Syndrome

Thesis Info

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Author

Salah-Ud-Din Shah, Muhammad

Supervisor

Javed Anver Qureshi

Program

PhD

Institute

Quaid-I-Azam University

City

Islamabad

Province

Islamabad.

Country

Pakistan

Thesis Completing Year

2014

Thesis Completion Status

Completed

Subject

Natural Sciences

Language

English

Link

http://prr.hec.gov.pk/jspui/bitstream/123456789/2272/1/2793S.pdf

Added

2021-02-17 19:49:13

Modified

2024-03-24 20:25:49

ARI ID

1676725885440

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Hydropericardium syndrome (HPS) is a viral disease of poultry which is caused by Fowl adenovirus (FAdV). This virus belongs to family Adenoviridae and genus Aviadenovirus. In recent years, Hydropericardium syndrome (HPS) has emerged as one of the important diseases occurring in Pakistan and has caused heavy economic loss. Efforts have been made to develop conventional vaccines against this disease. These vaccines were formulated from infected liver homogenate. Unfortunately, formalin-inactivated liver organ vaccines failed to protect the poultry industry in the country. Hence, there is a need to develop a suitable vaccine to combat this disease. Currently, recombinant vaccine candidates are being developed for the prevention and control of some infectious diseases in several laboratories elsewhere. The present work is an effort to develop a recombinant protein, using molecular biology, biotechnological and immunological approaches for effective control and diagnosis of HPS. In the present study, the viral particle was isolated from natural outbreak of Hydropericardium syndrome in broilers, Punjab province of Pakistan using conventional methods. The existence of the virus was initially observed by Scanning Electron Microscopic examination. Icosahedral shaped viral particles of 70 – 80 nm in diameter were observed. Further, the presence of FAdV was confirmed by Polymerase Chain Reaction (PCR) by amplification of 730 bp variable region (L1 and part of P1 loop) of hexon gene. DNA sequence analysis and Phylogenetic analysis of the PCR product revealed that isolate is closely related with Indian fowl adenovirus – 4 isolate. To investigate which gene product encoded by fowl adenovirus plays vital role in immune response against the disease, two genes representing structural proteins of the virus (Penton base & short fiber) and one gene representing non structural protein (100K) were selected to develop recombinant constructs. To achieve this, the Penton base (1587bp), Short fiber (1437bp) and 100K (2397bp) genes were amplified by PCR and cloned in an expression vector (pET28a). The histidine residues along with thrombin protease site were engineered upstream to inserts (viral genes). The presence of recombinant DNA fragments were confirmed by double digestion method, PCR amplification of insert using gene specific primers and DNA sequencing of the inserts. Nucleotide sequences of inserts revealed that two genes (Penton base and Short fiber) of local isolate have >98% homology with the Indian FAdV-4 isolates, while one gene (100K) has 96% homology with the Russian FAdV-10 isolate. The recombinant constructs were expressed in E. coli. The expression of recombinant proteins was assessed by SDS-PAGE. Western blot analysis confirmed the presence of histidine tagged recombinant proteins i.e. short fiber (60Kda), penton base (65Kda) and 100K (95Kda) using anti His tag antibody. The three recombinant proteins were purified by Nickle affinity chromatography. The biological and immunological activity of recombinant proteins were assessed for potential use as antigen in vaccine and diagnostic (ELISA). The purified recombinant proteins were adjuvanted separately with Freund’s complete adjuvant and broilers were immunized. ELISA test was performed and antibody titers were determined against the respective recombinant proteins. The results indicated that protein constructs pSMJ-2 (penton base) and pSMJ-3 (short fiber) are more immunogenic antigens as compared to protein construct pSMJ-1 (100K) and commercial vaccine. Challenge protection test also proved that penton base (pSMJ-2) and short fiber (pSMJ- 3) protein constructs conferred 90% and 80% protection respectively against pathogenic virus challenge. Whereas 100K (pSMJ-1) protein construct and commercial inactivated vaccine provided 50% and 70% protection respectively. The results obtained by ELISA and challenge test in this study indicated that the constructed recombinant proteins are suitable candidates to develop subunit vaccine and diagnostic kit (strip test) thereby can be used for prevention and control of this disease.
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ڈاکٹر وحید مرز ا

آہ! ڈاکٹر وحید مرزا
گزشتہ ماہ ریڈیو سے خبر ملی کہ اس برصغیر کے ایک بہت ہی لائق اور ممتاز اہل علم ڈاکٹر وحید مرزا کی وفات لاہور میں ہوگئی، اس خبر سے بڑا دکھ ہوا، ان سے میری ملاقات زیادہ نہیں رہی، اب سے ۳۵ سال پہلے ۱۹۴۱؁ء میں دہرہ دون میں تو ان سے برابر ملنے کا موقع ملا، اس کے بعد پھر کہیں ان سے نہیں ملا، مگر وہ اس وقت سے برابر دل و دماغ پر چھائے رہے ہیں ۱۹۴۱؁ء میں ایک طویل علالت کے بعد دہرہ دون جاکر تقریباً تین مہینے رہا، سرسید احمد خان ہم عصر اور دوست منشی ذکا اﷲ مرحوم کے نامور فرزند جناب عنایت اﷲ صاحب نے اپنی کوٹھی کے دو کمرے میرے قیام کے لئے دے دیئے تھے، اسی زمانہ میں لکھنؤ سے ڈاکٹر وحید مرزا دہرہ دون آکر اپنے خسر جناب رضاء اﷲ کے یہاں مقیم تھے، جو جناب عنایت اﷲ صاحب کے چھوٹے بھائی تھے، انجینئر کے عہدہ سے ریٹائر ہوکر دہرہ دون میں ایک بنگلہ بنایا تھا، دونوں بزرگوں کا وطن دہلی تھا، مگر عنایت اﷲ صاحب نے دہرہ دون ہی میں ڈالن والاکے سیمی روڈ پر منتقل سکونت اختیار کرلی تھی، ان ہی کے بنگلہ کی پشت پر رضا اﷲ صاحب کا بنگلہ تھا، ڈاکٹر وحید مرزا ان دنوں لکھنؤ یونیورسٹی میں استاذ تھے، وہ دہرہ دون آئے تو عنایت اﷲ صاحب کو سلام کرنے آئے، وہیں ان سے میرا تعارف ہوا، قد لمبا اور جسم دبلا پتلا تھا، دونوں گال پچکے ہوئے تھے، کیونکہ منہ کے تمام دانت نکلوا دیئے تھے، پتلون اور قمیض پہنے ہوئے تھے، بڑے متین اور سنجیدہ نظر آئے، خاموش بیٹھے رہے، عنایت اﷲ صاحب کی باتوں کا جواب بہت ہی مختصر طریقے پر دے کر خاموش ہوجاتے، جب وہ اٹھ کر چلے گئے تو عنایت اﷲ صاحب...

سیرت طیبہ کی تشریعیاتی حیثیت کا تحقیقی و تاریخی مطالعہ A Research review of the legislative Status of the Sirah of Holy Prophet ﷺ

The deeds and actions of the Holy Prophet ﷺ is a practical interpretation of the Holy Qur'an. Allah Almighty has stated his Shari'ah in short but comprehensive words that “take what the Holy Prophet ﷺ has given you and which you forbid”. Therefore, his command is the command of Allah Almighty and not yours. That is why the Holy Qur'an has declared his obedience to be the obedience of Allah Almighty. And from the instructions of the selected scholars of the ummah, it is clear that the legislative status of The Sirah is authenticated. Allah Almighty has given him a special position of Shariah. As for the legislative status of Sira-e-Taiba, both the special and the general legislation are specific or special to him. Special legislation etc i.e. you have legislated for a specific person at certain times like accepting the condition of a person that he will convert to Islam if he prays only two prayers. The other person cannot join. The general legislation in which you have enacted all kinds of legislation for the common ummah is included. The center and axis of Islamic law is your caste. You have complete control over the status and sanctity, likes and dislikes as you like, for whom you can make Shariah whenever you want. It is as if you are a follower of the Shari'ah. He also has full authority in the brief description of the Qur'an, in the adherence to the Absolute, and the explanation of similarities. You are authorized to legislate as a Shari'ah and Shari'ah in all areas of worship, affairs, debates, and crimes. Therefore, in this article, the legislative status of His Sira-e-Taiba has been explained in the light of Qur'an and Hadith, Sahabah and Tabi'een, and the commandments of Imams and jurists.

A Specification-Based Distributed Intrusion Detection Framework for Wireless Sensor Network

Wireless Sensor Networks (WSNs) have a great potential to assist in storing and processing data collected from tiny sensors placed in various environments such as smart homes, vehicles, hospitals, enemy surveillance areas, volcanoes, oceans, etc. The sensors may be implanted to inspect the physical aspects of the external environment such as temperature, moisture, humidity, pressure, motion, magnetic fields, light, sound, gravity, vibration, electrical fields, and others or inspect the physical aspects of the internal environments such as motion of the organism, glucose level, oxygen level and others. The data recorded by these sensors can further be used for several applications as well as services. Here, the data is acquired from sensors through the wireless medium. Recent studies show that WSNs are vulnerable to various kinds of security threats and there is a requirement of a security solution that can safeguard them from lethal attacks. Several security schemes have been proposed in the recent past to counter the attacks launched at different layers of WSNs. Intrusion detection systems (IDS) focus on the detection of malicious activity at the network layer. Most of the proposed IDS based security approaches for WSNs lack completeness with respect to data acquisition, detection policy and the way actions should be taken once the malicious behavior is detected. Further, they lack the proper testing of the proposed schemes with respect to the performance metrics such as energy consumption, throughput, false positive rate, intrusion detection rate, and accuracy etc. Hence, there is a requirement of a purely distributed security scheme that works independently and communicates the anomalous behavior of sensor nodes with the base station (BS). The scheme should be lightweight and is able to perform efficiently with respect to energy efficiency and throughput. Moreover, it should be able to achieve low false positive rate and high detection rate. In this thesis, a novel intrusion detection framework is proposed for securing WSNs from routing attacks. The proposed system works in a distributed environment to detect intrusions by collaborating with the neighboring nodes. It works in two modes: online prevention allows safeguarding from those abnormal nodes that are already declared as malicious, while offline detection finds those nodes that are being compromised by an adversary during the next epoch of time. The proposed framework is a specification-based detection framework that works for a flat WSN scenario. To test the performance of the proposed framework, a simulator is implemented, and results are produced. The results show that a centralized distributed approach cannot properly figure out the actual condition of the network. Therefore, a purely distributed security system is more appropriate for WSNs. The results also show that the specification-based detection scheme achieves higher detection rate and low false positive rate. These results also guide that each node should be treated independently in WSNs, and centralized distributed detection schemes may fail to identify the network behavior whether it is normal or is under any attack. As a second contribution, the low-energy adaptive clustering hierarchy (LEACH) protocol for WSNs is modified by adding the functionality of the proposed intrusion detection framework to secure it from sink-hole, black-hole, and selective forwarding attacks. The modified protocol is called LEACH++. We performed two types of analyses: (1) numerical analysis to check the effect on vi throughput and energy, and (2) simulations in Network Simulator-2 (NS-2) to prove the results found from the numerical analysis. The results are quite promising and favor LEACH++ over LEACH under attack with respect to throughput and energy consumption. The third contribution is to perform a security analysis of the LEACH++ protocol to validate the proposed specification-based detection scheme with respect to accuracy, false positive rate, and detection rate. For this purpose, we simulate LEACH++ by launching various numbers of attacks in different patterns for different configurations. The experiments are carried out against the LEACH++ protocol for black hole and sinkhole attacks in different patterns. The results show that the proposed scheme achieves high accuracy and detection rate for LEACH++ and shows very low false positive rate.