Institut Penyelidikan Perubatan Molekul - Tesis

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Phage Display Of Naïve Human T-Cell Receptors In Single Chain Format Against Isocitrate Lyase (Icl) And Tumor Necrosis Factor Alpha (Tnfα)
(2014-04)
Ch’Ng Chiew Wen, Angela
T-cell receptors (TCRs) bind with peptide-MHC or lipid-CD1 complexes while antibodies directly bind to antigen. The binding region of T-cell receptors (TCRs) and antibodies exhibit similar three-dimensional (3D) structures. Both composed of two chains and each chain contains two folded domains of one variable and one constant which associate together to create the antigen binding site between them. Since both antibodies and TCR have similar structures, there exists a potential for TCRs to serve as a binding scaffold to bind full protein antigens, much like antibodies. Consequently, the entire experimental study was designed with the aim of investigating whether TCRs can indeed bind to full antigen proteins just like how antibodies bind to antigen proteins. The recognition site of the TCR comprises of the alpha and beta variable regions. Consequently, both alpha and beta variable regions are interconnected through a glycine-serine linker, resulting in the formation of a single-chain TCR. Additionally, a naïve scTCR library was meticulously crafted using phage display technology with a library size of 1010. Then, the scTCR library was used to isolate scTCR binders against two designated antigens recombinant isocitrate lyase (rICL) and recombinant tumor necrosis factor alpha (rTNFα). A total of three monoclonal scTCR binders to rICL and two to rTNFα respectively were isolated. All of the human antibody constant (CH1 or CK) scTCR fusion proteins expressed best at 30 °C except a-rICL-2G10 scTCR.
Mechanism Of Polyalthia Longifolia (Sonn.) Thwaites Pol Yphenols Action In Hela Cells In Relation To Microrna Regulation
(2017-05)
Vijayarathna Soundararajan
Polyalthia longifolia (Sonn.) Thwaites is a lofty, evergreen tree that extensively known for its medicinal beneficial in treating various ailments. Though P. longifolia ethnomedicinal benefits had been recognised, studies concerning its anticancer and apoptotic cell death activity in relation to microRNAs (miRNA) and their mechanism had never been studied in detail.
In Vitro And In Vivo Studies On The Complementary Effects Of Standardized Ethanolic Orthosiphon Stamineus Extract On Pancreatic Cancer Cells
(2018-03)
Salem Yehya, Ashwaq Hamid
Pancreatic cancer is globally known as the fourth most lethal cancer in the world. Despite increasing understanding in tumor biology; the efficiency of treatment in pancreatic cancer remains challenging as patients demonstrate resistance to standard treatments. Although the first line agent, gemcitabine has produced clinical response to treat pancreatic cancer, the prognosis remains dismal.
Transcriptome Study Of Salmonella Enterica Subspecies Enterica Serovar Typhi Biofilm
(2018-08)
Jason, Chin Khee Chian
Typhoid fever is an acute systemic infection of humans caused by Salmonella enterica subspecies enterica serovar Typhi (S Typhi). During infection the bacteria forms biofilm in the gallbladder to protect itself from the harsh environment of the host. Biofilm formation has been associated with increased resistance towards antibiotics and the host immune system, and promotes bacterial persistence. However, the mechanism of biofilm formation is unknown. High-throughput sequencing using the Illumina HiSeq 2500 platform was performed to study the transcriptome of S. Typhi planktonic, intermediate and mature biofilm cells to identify the genes involved in biofilm formation and to postulate the mechanism of action. Planktonic S.
Intensification Of Core Streptavidin Production Through High-Cell-Density Cultivation Of Recombinant Escherichia Coli And A Temperature-Based Refolding Method
(2019-05)
Chua, Leong Huat
This study describes the development of an intensified process that enables high level production of recombinant core streptavidin (core SAV), a non-glycosylated tetrameric protein utilised in a wide range of applications. Due to widespread utility and commercial importance of SAV, recombinant SAV production has been studied extensively. However, in most studies, recombinant SAV was expressed in a soluble form, resulting in in vivo sequestration of biotin. Biotin sequestration is detrimental to cell growth and results in low volumetric yield. To improve volumetric yield, in this study, core SAV was expressed as inclusion bodies (IBs) followed by induction at highcell- density. A pH-stat fed-batch feeding strategy was employed to cultivate host cells to high-cell-density, the effect of induction at different cell densities (OD 20, 60 and 100) on volumetric and specific yield were then studied. Highest volumetric yield of core SAV (1.55 g L"1) was obtained from induction at OD 100.
Development Of A Chronological Life Span Assay For Ageing Studies In Saccharotnyces Cerevisiae
(2019-06)
Ong, Tee Gee
The Saccharomyces cerevisiae. chronological life span (CLS) is the length of time that yeast cells survive in a non-dividing state. In the laboratory, CLS is typically measured by its optical density at the post-diauxic phase that begins approximately two days after initial inoculation. In this study, the development of a CLS assay on a 96-well microplate with different yeast strains and growth parameters (media and time) were carried out. After the evaluation of four different strains, yeast strain BY25929 was found to be the most suitable in tenns of growth for the CLS assay. A total of 24 ageing-related proteins were selected for assessment in the CLS assay.
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.
Isolation And Characterization Of Shark Vnar Antibody Against Malaria Pfhrp2 Protein From Semi-Synthetic Library
(2019-08)
Cheong, Wei Shien
Ujian diagnostik pantas (RDT) malaria adalah penguji imun berasaskan antibodi yang menpercepatkan diagnosis malaria. Walau bagaimanapun, reagen antibodi yang digunakan secara meluas untuk RDT malaria tertakluk kepada degradasi pada suhu sekitar yang tinggi yang boleh memendekkan jangka hayat RDT malaria.
Salt-Induced Phase Separation Of Chaps And Potential Application For Hydrophobic Molecule Extraction
(2019-08)
Lian, Ai Ai
CHAPS is a zwitterionic detergent commonly utilized in protein biochemistry based applications. Despite widespread utility, understanding of CHAPS phase behaviour remained poor. In this study, the effect of salts on CHAPS phase behaviour was first investigated. Consolute solution temperature of CHAPS-(NH4)2SO4 systems were determined, followed by phase diagram construction. Potential of CHAPS-(NH4- )2SO4 systems for hydrophobic molecule extraction via phase separation was then evaluated using Coomassie Brilliant Blue (CBB) and recombinant cytochrome P450 (CYP) 2C19. Sulphate and phosphate salts were successful in inducing phase separation of CHAPS. CHAPS and (NH4)2SO4 systems exhibited an upper consolute solution temperature (UCST). CBB was found to partition extensively to micelle-rich phase (MRP) of the 4.5% CHAPS and 8% (NH4)2SO4 system with an extraction yield of 83±0.32%. Extraction yield of recombinant CYP 2C19 in MRP of the 13.5% CHAPS and 10.8% (NH4)2SO4 system was 46±7.58%. The outcomes of this study propound the scope for application of CHAPS as a component of phase separation systems.
Synergistic Activity Of Ceftriaxone And Polyalthia Longifolia Leaves Extract Against Methicillin Resistant Staphylococcus Aureus (Mrsa)
(2021-09)
Ranjutha A/P Valiappan
Methicillin‐resistant Staphylococcus aureus (MRSA) infection has become one of the most significant pathogenic bacterial infection health issues in developing countries. Furthermore, the most important mecA gene that is encodes a novel penicillin-binding protein PBP2a contributes to the methicillin resistance in MRSA strains. Therefore, new alternative strategies are needed to address this issue by developing a new antimicrobial agent, modifying the existing antibiotic activity with a combination of plant extracts as resistance modifying agents, or using the plant extract combined with existing antibiotics against resistant bacteria. Consequently, the current study was conducted to evaluate the antibiotic modifying activity and synergistic effects of methanol extract Polyalthia longifolia (Sonn.) Thwaites (Annonaceae family) against MRSA in combination with ceftriaxone. The antibiotic modifying activity and synergistic effects of P. longifolia leaf methanol extract (PLLME) were evaluated in this study by disc diffusion method, broth microdilution method, checkerboard microdilution method, and detection of the mecA gene suppression by multiplex Polymerase chain reaction (PCR).
Chemopreventive Effects And Proteomic Analysis Of Myo Inositol Treatment On Human Prostate Cancer Cell Line (Du-145)
(2022-02)
Islam, Mohammad Jahidul
Prostate cancer remains the second most frequent diagnosed cancer in men and is the third leading cause of cancer related death, despite many available treatment options. The inefficiency of existing therapies and their adverse effects have led scientists to seek new treatments for this disease. The identification and development of new anti-proliferative agents for prostate cancer is a major innovation in treating this deadly disease. Although the synthetic compound Myo-inositol is widely used in cancer research, the actual anti-cancer function of these chemicals is still not well understood. This study was therefore intended to unveil the chemopreventive effects of Myo-inositol on prostate cancer cells (DU-145). The growth inhibitory effect and the IC50 value were demonstrated by Myo-inositol at 0.06 mg/ml (**p<0.05). It is suspected that cell deaths are related to apoptosis induction and cell-cycle arrest. Treatment with Myo-inositol has been further identified by induction of early and late apoptosis (***p<0.01). Apoptosis can also be detected using DNA fragmentation and Hoechst 33342 fluorescent dye stain analysis. Myo-inositol caused an alteration to the cell cycle regulation on DU-145 cell line at G0/G1 and S phase, respectively (***p<0.01). Protein identification results via 2D electrophoresis and LC-MS/MS analysis showed 19 and 23 proteins identified in control and Myo-inositol treated samples, respectively.
Isolation, Characterization, And Production Of Recombinant Monoclonal Antibodies Against Rnie For Development Of A Strongyloides Antigen Detection Assay
(2022-05)
Balachandra, Dinesh
Strongyloides stercoralis is a soil-transmitted helminth that causes strongyloidiasis. It is estimated to infect more than 600 million people worldwide. Asymptomatic chronic infections in immunocompromised people can lead to fatal hyperinfection. Serodiagnosis by detecting specific IgG antibodies can be challenging due to potential cross-reactivity with infections by other parasites. An antigen detection assay, a direct detection method, can help the diagnosis and is useful for post-treatment follow-up. This study used phage display technology to produce recombinant monoclonal antibodies (rMAb) against NIE recombinant protein (rNIE) and develop a Strongyloides antigen detection test. rNIE is an established protein for the diagnosis of strongyloidiasis. rNIE was expressed, purified, and then used to select rMAb candidates via biopanning of an immune helminth phage display library. It isolated of 104 ELISA-positive clones and sequence analysis showed that 30 clones had full-length light and heavy chains. Four unique gene families were identified, i.e., IgHV3-LV6 (86.66%), IgHV1-LV3 (3.33%), IgHV5-KV3 (3.33%), and IgHV3-LV3 (6.66%). Randomly, one representative clone from each gene family was selected for further studies, i.e., (a) rMAb5 representing IgHV1-LV3, (b) rMAb6 representing IgHV3-LV6, (c) rMAb14 representing IgHV5-KV3, and (d) rMAb23 representing IgHV3-LV3. The rMAb gene sequences from the phage display vector were subcloned into the pET51b+ expression vector and transformed into Escherichia coli Shuffle T7 Express host cell.
Evaluation Of Toxicity, Pharmacokinetic And Clastogenicity Profile Of Ethyl 2-[4[(Piperidin-1-yl) Phenyl]-1h-benzimidazole-5-carboxylate (Bzd9l1), Novel Sirtuin Inhibitor
(2022-05)
Lee, Yeuan Ting
The mammalian sirtuins (SIRTs) have been linked to various diseases including cancer, diabetes, neurodegenerative and cardiovascular diseases. Thus, SIRTs are attractive targets for the development of pharmaceuticals. The lack of potential SIRT inhibitors in cancer clinical trials highlights the need to develop a potent SIRT modulator as an anti-cancer therapeutic. Studies in the lab have highlighted the capability of a lead sirtuin inhibitor (BZD9L1) to reduce colorectal tumour growth in vitro and in vivo by modulating different cancer pathways in colorectal cancer with different mutation profiles. Also, synergistic effects of BZD9L1 in in vitro assays and tumour xenograft study when used in adjunct with 5- Fluorouracil (5-FU) further support the promising therapeutic effects of BZD9L1 as anticancer regime. Nevertheless, the toxicology profile of this compound remains unknown. To our knowledge, no prior work has reported the regulation of SIRTs in the liver and kidney by a small molecule inhibitor in a drug toxicity study. Therefore, this project aims to determine the toxicology, molecular regulation of toxicity, pharmacokinetics and clastogenicity profiles of BZD9L1 in in vitro or in vivo models.
Exploring The Immunomodulation Profiles Of The Thp-1 Human Macrophage-derived Cell Line Mediated By Shigella Flexneri
(2022-06)
Shabani, Nor Raihan Mohammad
Shigellosis is the primary cause of severe diarrhea, especially among children less than five years old. The occurrence of multi-drug resistant strains of Shigella species has contributed to the ineffectiveness of the existing treatment. Vaccination has become an essential requirement for preventing shigellosis. The invention of an epitope-based vaccine has directed the development of a new vaccine design model. Macrophages are specialized APCs that process antigens and then present the processed antigens to T-cells through HLA molecules. The immunomodulatory profiles of the macrophages infected by the clinical strains of S. flexneri 2a have remained undefined. THP-1-derived macrophages, as the model of human macrophages, were infected independently with mild and virulent strains of S. flexneri 2a at standard growth conditions. The gene expression level of inflammatory mediators was determined using qPCR, while NO production was measured using a commercial NO assay kit. The significant up-regulation of TNFα, IL-1β, IL-6, IL-12, iNOS, and NO indicates the pro-inflammatory reaction to S. flexneri 2a infection. The virulent strain also markedly increased the expression levels of the anti-inflammatory cytokine mRNAs. HLA class II gene expression was also investigated to extend the immunopeptidomics study and found the significant up-regulation of HLA class II by the Shigella-infected macrophages. LC-MS/MS-based immunopeptidomics strategy was employed to identify the peptides that can be used as potential candidates for the epitope-based Shigella vaccine development
Role Of Polysaccharides From Pleurotus Sajor-caju In The Immunomodulation Of Thp-1 Human Macrophage Cells
(2022-06)
Mokhtar, Munirah
Medicinal mushrooms have a diverse range of biological effects, which are excellent sources for pharmaceutical and nutraceutical use. They possess various valuable medicinal properties, including antitumor, hypocholesterolemic, anti-atherogenic, and anti-oxidative properties. Pleurotus sajor-caju is one of the Pleurotus spp. belonging to phylum Basidiomycota, well-known as oyster shaped mushroom (basidiocarps) and generally called oyster mushroom (OM). The present study was aimed to determine the ability of the water-soluble polysaccharides extract from Pleurotus sajor-caju extracts (PSC) in inducing the proliferation, immunostimulating and anti-inflammatory effects on THP-1 macrophage cells. First, the total carbohydrate and β-glucan contents were determined using the individual assay test. Next, the cell proliferation response was evaluated using the MTS assay. To understand the immunostimulating effect of PSC extract at the gene level, the expressions of selected cytokine genes in THP-1 cells were evaluated using real-time PCR. Nitric Oxide (NO) production and inducible nitric oxide synthase (iNOS) expression level were measured in the LPS-stimulated macrophages. The results obtained showed that 95% of total carbohydrate and 89% of β-glucan contents were detected in 50 mg PSC extracts, respectively. PSC extract was found to induce a marked increase in proliferative response on macrophage THP-1 cell in a dose-dependent manner, ranging from 10 μg/ml up to 80 μg/ml. At transcription level, PSC stimulated the expression of tumor necrosis factor-alpha (TNF-α), interleukin 1β (IL-1β), and interleukin 6 (IL-6) significantly (p<0.05) at various dosages of PSC extract.
Genomic Expression Profile Of Human Papillomavirus 16 And 18 – Associated Pre-cancerous Lesions And Cancer Of The Cervix
(2022-06)
Balasubramaniam, Shandra Devi
Cervical cancer is one of the most common cancers in women and is caused by high-risk human papillomavirus (HPV) infection. The integration of HPV into host cervical epithelial cells causes genetic alterations and consequent changes in gene expression affecting downstream molecular pathways, leading to the development of cervical cancer. This study aimed to profile the genomic signatures involved in the pathogenesis of HPV 16 and 18 associated pre-cancerous lesions (cervical intraepithelial neoplasia, CIN) and squamous cell carcinoma (SCC) of the cervix, in comparison to the normal cervix. The differential mRNA expression profiles were determined, to evaluate the expression of up-regulated and down-regulated host genes and to evaluate the perturbed molecular pathways involved in cervical carcinogenesis. In this study, 29 formalin-fixed paraffin-embedded (FFPE) tissues including low-grade CIN (LGCIN), high-grade CIN (HGCIN), SCC, and normal cervix were screened for HPV 16 and HPV 18 using immunohistochemistry and qRT-PCR method. Of these, 9 HPV-positive (3 LGCIN, 3 HGCIN, 3 SCC) and 3 normal cervix (HPV-negative) tissue samples were microdissected to obtain regions of interest in the squamous epithelium before RNA extraction. Transcriptomic profiling was performed using the Affymetrix, GeneChip Human Transcriptome Array 2.0 (HTA 2.0) and the nCounter® PanCancer Pathway Array, Nanostring to identify differentially expressed genes (DEGs) and significantly associated pathways in each stage of cervical cancer development.
Mechanism Of Action Of A Benzimidazole Sirtuin Inhibitor, Bzd9l1, In Colorectal Cancer
(2022-08)
Tan, Yi Jer
Sirtuins (SIRTs) are NAD+-dependent deacetylases that is implicated in various epigenetic diseases including cardiovascular and neurodegenerative diseases, diabetes, aging and cancer. The emergence of SIRTs as therapeutic targets and limitations of existing SIRT inhibitors led to the discovery of a novel SIRT inhibitor: BZD9L1. As testing of the effects of BZD9L1 on the enzymatic activities of SIRTs have been hampered by the availability of commercial kits, BZD9L1 interactions on SIRTs were studied using molecular modelling and docking studies. Molecular docking studies revealed that BZD9L1 may bind to SIRT1-3, 6 and 7 with similar confirmation but with different affinities, thereby expanding the therapeutic potential of BZD9L1 in metabolic diseases. In addition, in silico approaches were deployed to further elucidate BZD9L1-modulated mechanisms based on existing experimental data. In silico analysis of BZD9L1-regulated targets showed that the proliferation and apoptosis of HCT 116 cells may be due to p53-dependent signalling pathways. BZD9L1 was found to be effective against different cancer cell lines especially colorectal cancer (CRC), where its first-line chemotherapy regimen 5-fluorouracil (5-FU) often result in treatment failure due to drug insensitivity and severe side effects. As current efforts to overcome these boundaries involved sensitizing tumours through adjuvant treatments, this project also aims to provide novel insights into the potential development of BZD9L1 as an adjuvant to 5-FU in CRC therapy using in vitro and in vivo models. The combination of BZD9L1 and 5-FU was found to be more effective against HCT 116 CRC cell line in reducing cell viability and survival compared to sole treatment via synergistic effect.
Assessment Of Antitumor Activity Of Standardized Polyalthia Longifolia Leaf Extract In Hela Cell Tumor Xenograft Mouse Model
(2022-08)
Braganza, Cilwyn Shalitha
P. longifolia is a traditional medicinal plant with a rich source of biologically active phytochemicals. Investigations on methanolic leaf extract were reported to exert potent in vitro anticancer effects on HeLa human cervical cancer cells. Current treatment strategies for cervical cancer are facing challenges such as increased drug toxicity, chemoresistance, and limited treatment options, thus making it imperative for the discovery of safe and effective green anticancer agents. Though P. longifolia is a promising anticancer plant, the in vitro findings by themselves are insufficient to translate to human use and therefore require in vivo preclinical testing. Currently, there are no reports on further investigation of P. longifolia leaf extracts on in vivo animal tumor models, which limits the clinical utility of this plant. Hence this study was conducted to evaluate the in vivo antitumor effects of P. longifolia methanolic leaf extract on HeLa cells xenografted tumors developed in athymic nude mice.
Thiazolidinediones Pretreated Bone Marrow-Derived Mesenchymal Stem Cells As An Anti-Cancer Approach Against Mcf-7 Breast Cancer Cell Line
(2022-09)
Lim, Shern Kwok
Breast cancer is the most prevalent type of cancer among women globally. Although treatments are currently available, most of these therapies are associated with adverse effects. For that reason, new treatment strategies utilising approved drugs may prove to be beneficial. Pioglitazone (PIO) and rosiglitazone (ROSI) are thiazolidinediones (TZDs) drugs that are clinically used to treat type 2 diabetes mellitus. Although PIO and ROSI have been linked to several adverse effects, there have been reports about their anti-cancer properties. For that reason, this study was conducted to improve the overall efficacy of TZDs therapy by utilising PIO and ROSI in combination to treat MCF-7 breast cancer cells. Additionally, the study also investigated the combination therapy of PIO and ROSI as a pretreatment strategy for bone marrow-derived mesenchymal stem cells (BM-MSCs) to indirectly treat MCF-7 cells via cell-cell interaction. The results revealed that PIO and ROSI when used in combination, induced higher inhibition of cell viability with lower levels of cellular lipid accumulation in MCF-7 cells at 48 and 96 hours of co-culture. Furthermore, the viability of TZDs pretreated BM-MSCs remained uninhibited for up to 96 hours and no signs of cell stress or death was observed at Day 7.
Identification Of Antimalarial Compounds And Their Mode Of Actions
(2022-10)
Mahmud, Fauze
Malaria is one of the most significant infectious diseases in the tropics, claiming around half-million lives annually, mainly due to Plasmodium falciparum (P. falciparum) infections. To date, P. falciparum has developed full or partial resistance towards all three classes of antimalarials, including against the artemisinin. Hence, it may jeopardize the efficacy of the current antimalarial treatment regimen, the Artemisinin Combination Therapy (ACT), in the near future. Therefore, there is an utmost need to identify a new antimalarial compound with a novel mode of action (MoA). This study mainly focused on identifying antimalarial compounds from soil microorganisms isolated from Malaysia and Japan. The crude extracts of these strains showed potent antimalarial activity in a preliminary assay against Pf 3D7 (wild type). The crude extracts of Malaysian strains also inhibited the activity of human GSK-3β (75 % similar to P. Falciparum GSK-3 (Pf GSK-3)).

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