Institut Penyelidikan Perubatan Molekul - Tesis

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Generation And Characterization Of Scfv Antibody Fragment Targeting Japanese Encephalitis Virus Nsl From A Semi-Synthetic Phage Display Library
(2019-08)
Chong, Hui Ying
Japanese encephalitis (JE) which is caused by Japanese Encephalitis Virus (JEV) is a flavivirus disease and remains to be the major form of viral encephalitis in Asia. JEV infection causes meningitis, encephalitis and in some cases, permanent damage to the brain or even death. As JEV causes severe pathophysiological conditions, we set to generate antiJEV NS 1 antibodies for future immunodiagnostic development.
Development Of Dengue Immunodiagnostic Assay By Targeting Denv Type 2 Ns1 Using Shark Single Domain Vnar Antibody
(2024-07)
Kok, Boon Hui
Denv infection diagnosis requires early and rapid detection which is accurate and specific to provide effective treatment for the patients. However, problems such as limitations of rapid diagnostic test, antibody instability, cross-reaction among denv serotypes and other flaviviruses are causing the dengue diagnosis to be challenging. Alternatively, vnar sdab derived from shark ignar has proven some promising characteristics such as improved stability, thermostability and ability to bind on cavities or clefts hidden on targeted surface. These could possibly overcome the limitations encountered while using conventional antibodies. In this study, a potential binder namely anti-ns1 vnar-z8 was isolated from semi-synthetic shark vnar library and successfully expressed as soluble protein using e. Coli expression system. This soluble recombinant anti-ns1 vnar-z8 shown improved specificity (1.70 ± 0.11) towards denv type 2 ns1 antigen as compared to commercial anti-ns1 mab (1.17 ± 0.07). Besides, both antibodies were sensitive within the detection range of 0.03 μg/ml to 60 μg/ml. The good thermostability characteristic of anti-ns1 vnar-z8 was also proven in this study with retainment of binding affinity towards ns1 antigen after thermal treatment at various temperature (25°c to 60°c) for up to 1 week. On the other hand, the early lfa prototype was successfully developed using aunps conjugated anti-ns1 vnar-z8 protein. Throughout the prototype stability characterization,
Characterization And In Vitro Study Of Protoporphyrin Ix (Haem) Aptamer In Reversing Drug-Resistant Malaria
(2024-07)
Abdulwahab, Aliyu
The global health challenge of malaria, compounded by drug resistance, necessitates innovative approaches for effective treatment. Aptamer technology is a promising tool towards combatting drug-resistant malaria especially chloroquine (cq) resistance. Preliminary study on protoporphyrin ix (haem) dna-aptamers (oka_24 and oka_26 ) demonstrated an anti-malarial property but lack the ability to internalise into the parasite-infected red blood cell (rbc). This research investigates the potential of cholesterol-tri ethylene glycol (col-teg) modified haem aptamers in addressing drug-resistant malaria. The research employs a multifaceted approach, including in silico techniques for predicting aptamer structures and molecular docking studies to assess binding behaviour. Additionally, reverse-phase high-performance liquid-chromatography (rp-hplc) was utilized to evaluate serum stability, while uv-absorption spectral titration and square wave voltammetry (swv) provided insights into the specificity and affinity of modified aptamers for haem. Cellular internalization assays, conducted using fluorescence-microscopy and flow cytometry, determine the efficiency of col-teg-modified aptamers in entering red blood cells. The study also examines the antimalarial activity of modified aptamers against cq-sensitive (3d7-strain) and cq-resistant (w2-strain) plasmodium falciparum. Docking analysis reveals that the transformation of oka_26 to col-teg-oka_26 does not alter binding behaviour,
Expression Of Recombinant Denv Ns2b/Ns3 Protease: In Silico Screening And In Vitro Validation Of Novel Antiviral Compounds For Dengue Virus Inhibition
(2024-05)
Harun, Norshidah
Globally, the relentless rise in dengue cases worldwide has been further intensified and inadvertently neglected in the backdrop of the persistent covid-19 pandemic. Despite the escalating global impact of dengue virus (denv) infection, there remains a conspicuous absence of its effective treatment. Hence, the exploration and advancement of dengue antiviral treatments remain a primary focus of research. An appealing target for the anti-denv drugs is the virally encoded trypsin-like serine protease (ns3pro) and its related cofactor (ns2b). The denv ns2b/ns3 protease complex is responsible for cleaving the viral polyprotein into distinct functional viral proteins, making it crucial for virus replication. In the past 15 years, the production of an active denv ns2b/ns3 pro has mostly relied on constructs that link the cterminus of the hydrophilic cofactor domain of ns2b to the n-terminus of ns3 pro using a flexible glycine linker. Despite thorough analysis, no potential inhibitory drugs have been discovered thus far. Furthermore, the impact of the synthetic linker introduced between the protease and its cofactor remains uncertain. Recently, an alternative strategy for producing a catalytically active denv ns2b/ns3 pro complex that is not covalently connected has been reported by professor paul young from the university of queensland, australia, and his team. By employing the non-covalent plasmid construct, this study successfully expressed and purified a high-solubility protein of recombinant denv ns2b/ns3 pro under optimal conditions, with betaine supplementation.
Synthesis Of Novel Carbon Dots From Polyalthia Longifolia Leaf For In Vitro Cytotoxicity Against Hela Cells
(2024-09)
Shuraish, Paveethra
Carbon dots (CDs), known for their outstanding fluorescence properties, have found extensive applications in medicine and pharmacy owing to their biocompatibility and nano-sized attributes. However, the potential cytotoxic effects of CDs derived from Polyalthia longifolia rich with various pharmacological activity remain underexplored. To address this gap, the current study synthesized, characterized, and investigated the toxicity against RAW264.7 cells, as well as photoinduced cytotoxicity against HeLa cells of the CDs derived from P. longifolia. Various methods, including transmission electron microscopy (TEM), X-ray diffraction (XRD), ultraviolet-visible (UV-vis) spectroscopy, zeta potential, and Fouriertransform infrared spectroscopy (FTIR), were employed to characterize the CDs. Additionally, the toxicity of the synthesized CDs was evaluated against RAW 264.7 cells, and the photo-induced cytotoxicity was assessed against HeLa cells under white light. Moreover, the impact of photo activation on ROS generation in HeLa cells also investigated. The synthesized spherical black spheres exhibited an average size of 6.29 ± 0.31 nm (TEM), consisting of both crystalline and amorphous phases of carbon (XRD), and a negative zeta potential of -20.7 ± 2.01 mV. The synthesized CDs emitted blue fluorescence when exposed to UV light at 365 nm and exhibited a prominent UVvisible absorption peak in the UV region. FTIR analysis revealed the existence of diverse functional groups on the CDs. The toxicity evaluation of synthesized CDs indicated low toxicity, promoting RAW264.7 cell viability within the 500–8000 μg range (P<0.05).

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