Development Of Denaturing High Performance Liquid Chromatography (Dhplc) In Detection Of Common Polymorphisms In The Multi-Drug Resistance (MDR1) Gene Among Malay Patients With Leukemia [QH324.9.L5 B138 2006 f rb].

Although the use of chemotherapy has evolved remarkably during the past 30 years for treatment in leukemia patients, it remains a difficult disease to treat where the main problem is drug resistance. This is due to over expression of multi-drug resistance gene (MDR1) and it’s over expression has been described in many tumors resistant to cytotoxic drugs including leukemias. Walaupun keberkesanan penggunaan kemoterapi dalam rawatan terhadap pesakit leukimia adalah jelas terbukti, namun leukimia tetap merupakan penyakit yang sukar dirawat terutamanya dalam masalah kerintangan pesakit terhadap ubat-ubatan (dadah)

Analysis of time-dependent transcriptomic and phenotypic changes associated with repair and regeneration in the airway epithelium

The airway epithelium demonstates the ability to quickly repair following physical injury. The morphologic features of this dynamic repair process have been well characterised at the anatomic and cellular level using a number of animal model systems and these studies have provided a solid foundation upon which our understanding of normal repair is build. With the advent of molecular and bioinformatic tools and resources the opportunity exists to extend the value of these models in defining the molecular pathways and interactions that underlay the normal repair process. This thesis represents a realisation of this opportunity. A large animal model was developed in which selected areas of airway epithelium were subjected to bronchial brush biopsy as part of routine bronchoscopic examination prcedures in anaethetised sheep. The process resulted in a physical perturbation of the normal pseudostratified structure of the sheep airwway epithelium at specific locations. By careful experimental design it was possible, within the same animals. to identify and sample from sites undergoing repair at different intervals subsequent in injury. To supplement the histological evaluation of the repair process and align findings with extablished small animal models of airway epithelial repair proliferative cell labelling strategies were implemented in order to study the location and extent of cellular proliferation occurring duringthe repair process. Molecular approaches towards defining the transcriptional response to physical injury comprised application of microarray technology using a commercially sources array platform. Such approach demanted preliminary effort directed towards optimising RNA integrity and yield from airway samples. Following preliminary studies directed towards optimising the model conditions patterns of airway epithelial repair following bronchial brush biopsy were studies in eight sheep at degined time points (6 hours, 1,3, & 7 days) post-injury. Bronchial brush biopsy resulted in the acute removal of the epithelial cell layer and components of the underlying structures. repair processes were rapidly implemented through initial epithelial dedifferentiation, proliferatino and migration at the wound margins and subsequent time-depentend changes in the proportion of subepithelial structures, including smooth muscle and blood vessels, as the epithelial surface moved towards repair. Transcriptional analysis revewaled that over 13,000 probes showed evidence of differential expession at some point during the repair process (p<0.05), whilst of these, 1491 probes had in excess of a two-fold change in expression. array results were validated against conventional semi-quantitative RT-PCR for selected genes. Differentially expressed genes with previously characterised roles in epithelial migration, prolifereation and differentiation were identified during the repair process. The relative emphasis of gene products with particular functional roles varied during the course of repair. Indeed gene ontology (GO) terms identified included those associated with the inflammatory response, cellular migration, extracellular matrix activities, differentiation, proliferation, cellular development, cell cycle activities, cellular adhesion, apoptosis and mitosis. In addition the Kyoto Encyclopedia of Genes and Gneomes (KEGG) databases were queried and such process indicated the involvement of cell communication, 053 and complement and coagulation cascade pathways throughout the repair process, initial (6h) Toll-like receptor and cytokine-cytoine receptor interaction pathways, and the progressive involement of cell cycle, focal adhesion and extracellulaar matrix (ECM)-receptor, and cytokine interaction pathways as the epithelium repaired. The model of airway epithelial injury developed in this thesis generated features broadly consistent with those previosly described in relation to various small animal model systems. Importantly, and in addition, this thesis defines the molecular features associated with repair in this model system and provides a useful resource with which to assess the comparative fetures of the airway transcriptional response to physical injury, It is through such comparison, using analogous methodology, that the fundamental pathways and interactions that underlay normal repair and regeneration can be identified and therafter extended towards inderstanding the basis for variation associated with natural and experimental diseas

Rapid Screening for C3435t Polymorphism in Exon 26 of the Multi-Drug Resistance (MDR1) Gene in Malay Patients with Acute Leukemia

The over expression of P-glycoprotein has been found to be associated with therapy resistance in hematological malignancies including acute leukemias. Recently, a single nucleotide polymorphism (SNP) in exon 26 (C3435T) in the MDR1 gene has shown to have functional consequences with altered expression of P-gp. Denaturing HPLC has been established as a rapid method for screening SNP in the form of heteroduplex of the DNA samples under partially denaturing condition

A comparative study of non-viral gene delivery techniques to human adipose-derived mesenchymal stem cell

Mesenchymal stem cells (MSCs) hold tremendous potential for therapeutic use in stem cell-based gene therapy. Ex vivo genetic modification of MSCs with beneficial genes of interest is a prerequisite for successful use of stem cell-based therapeutic applications. However, genetic manipulation of MSCs is challenging because they are resistant to commonly used methods to introduce exogenous DNA or RNA. Herein we compared the effectiveness of several techniques (classic calcium phosphate precipitation, cationic polymer, and standard electroporation) with that of microporation technology to introduce the plasmid encoding for angiopoietin-1 (ANGPT-1) and enhanced green fluorescent protein (eGFP) into human adipose-derived MSCs (hAD-MSCs). The microporation technique had a higher transfection efficiency, with up to 50% of the viable hAD-MSCs being transfected, compared to the other transfection techniques, for which less than 1% of cells were positive for eGFP expression following transfection. The capability of cells to proliferate and differentiate into three major lineages (chondrocytes, adipocytes, and osteocytes) was found to be independent of the technique used for transfection. These results show that the microporation technique is superior to the others in terms of its ability to transfect hAD-MSCs without affecting their proliferation and differentiation capabilities. Therefore, this study provides a foundation for the selection of techniques when using ex vivo gene manipulation for cell-based gene therapy with MSCs as the vehicle for gene delivery

Kinetics Extraction Modelling and Antiproliferative Activity of Clinacanthus nutans

Clinacanthus nutans is widely grown in tropical Asia and locally known “belalai gajah” or Sabah snake grass. It has been used as a natural product to treat skin rashes, snake bites, lesion caused by herpes, diabetes, fever, and cancer. Therefore, the objectives of this research are to determine the maximum yield and time of exhaustive flavonoids extraction using Peleg’s model and to evaluate potential of antiproliferative activity on human lung cancer cell (A549). The extraction process was carried out on fresh and dried leaves at 28 to 30°C with liquid-to-solid ratio of 10 mL/g for 72 hrs. The extracts were collected intermittently analysed using mathematical Peleg’s model and RP-HPLC. The highest amount of flavonoids was used to evaluate the inhibitory concentration (IC50) via 2D cell culture of A549. Based on the results obtained, the predicted maximum extract density was observed at 29.20 ± 14.54 hrs of extraction (texhaustive). However, the exhaustive time of extraction to acquire maximum flavonoids content exhibited approximately 10 hrs earlier. Therefore, 18 hrs of extraction time was chosen to acquire high content of flavonoids. The best antiproliferative effect (IC50) on A549 cell line was observed at 138.82 ± 0.60 µg/mL. In conclusion, the flavonoids content in Clinacanthus nutans water extract possesses potential antiproliferative properties against A549, suggesting an alternative approach for cancer treatment

In vitro evaluation of three-dimensional printed thermoplastic polyurethane and polylactic acid scaffold for tracheal tissue engineering

Introduction: Three dimensional (3D) printed scaffold using polymer composite is a potential technique for the replacement and regeneration of damaged tissue. The scaffolds must meet several requirements to be physiologically functional to provide an ideal environment for cells and act as a vital physical substrate for cell attachment, proliferation, differentiation, and integration into the host tissue. These include degradable, nontoxicity, and a favourable surface for cell-matrix adhesion and proliferation. The purpose of this study was to evaluate the cell proliferation and attachment of the 3D-printed TPU/PLA scaffolds in a cell culture experiment. Methods: 3D printing was performed through the fused deposition modelling (FDM) technique using our custom-made thermoplastic polyurethane (TPU) and polylactic acid (PLA) filaments. The degradation rate was determined, and the pH of the extracts was evaluated towards the proliferation of human normal bronchial epithelial cells (BEAS2B). Direct cell attachment of the cells on the scaffolds was also assessed. Results: The viability of cell culture is shown by the proportion of viable cells in a population. BEAS-2B was cultured for three days on sterilised TPU, PLA, and TPU/PLA scaffolds to test their biocompatibility and toxicity of the material. Both pure TPU and PLA indicated biocompatibility towards BEAS-2B cells. The result showed that the viability of BEAS-2B was greater than 80% in all compositions up until day 3, indicating that none of the compositions was toxic to the cells. The study was demonstrated that the degradation and pH of the TPU/PLA did not affect cell proliferation and attachment. Conclusion: This study concluded that 3D printed TPU and PLA composite had a promising property and were suitable for future applications in tracheal tissue engineering

Mechanical properties and in vitro evaluation of thermoplastic polyurethane and polylactic acid blend for fabrication of 3D filaments for tracheal tissue engineering

Surgical reconstruction of extensive tracheal lesions is challenging. It requires a mechanically stable, biocompatible, and nontoxic material that gradually degrades. One of the possible solutions for overcoming the limitations of tracheal transplantation is a three-dimensional (3D) printed tracheal scaffold made of polymers. Polymer blending is one of the methods used to produce material for a trachea scaffold with tailored characteristics. The purpose of this study is to evaluate the mechanical and in vitro properties of a thermoplastic polyurethane (TPU) and polylactic acid (PLA) blend as a potential material for 3D printed tracheal scaffolds. Both materials were meltblended using a single screw extruder. The morphologies (as well as the mechanical and thermal characteristics) were determined via scanning electron microscopy (SEM), Fourier Transform Infrared (FTIR) spectroscopy, tensile test, and Differential Scanning calorimetry (DSC). The samples were also evaluated for their water absorption, in vitro biodegradability, and biocompatibility. It is demonstrated that, despite being not miscible, TPU and PLA are biocompatible, and their promising properties are suitable for future applications in tracheal tissue engineering

Preliminary study on reactive compatibilisation of poly-lactic acid with maleic anhydride and dicumyl peroxide for fabrication of 3D printed filaments

Biodegradable poly-lactic acid (PLA) has been extensively used in various fields including biomedical applications and tissue engineering. However, its inherent brittleness, less flexibility and miscibility limit its uses when blended with other polymers. Grafting of maleic anhydride (MAH) onto PLA using dicumyl peroxide (DCP) as radical initiator had been advocated in an attempt to produce functional groups which would improve the interfacial adhesion of PLA polymer blends, therefore enhance the mechanical properties of the products. In this preliminary work, the physical properties of grafted PLA and the effects of MAH on the grafting percentage of the PLA were investigated. A series of maleic anhydride grafted poly-lactic acid (PLA-g-MAH) was prepared by mixing PLA and MAH with constant DCP (0.2 phr) at 180°C in an internal mixer. Effects of DCP and MAH concentration on the grafting percentage were deduced by Fourier Transform Infrared (FTIR) spectroscopy, thermal and titration analyses. The molecular weight changes were analysed using gel permeation chromatography (GPC). Grafting was confirmed and the degree of grafting was found to be dependent on the MAH concentration. The thermal properties of PLA-g-MAH were affected due to formation of new functional groups after grafting and there were changes in the molecular weight of the grafted samples. This study concluded that addition of predetermined concentration of MAH in the presence of minimal DCP was effective for reactive compatibilisation of PLA

Targeting Lung Cancer Stem Cells: Research and Clinical Impacts

Lung cancer is the most common cancer worldwide, accounting for 1.8 million new cases and 1.6 million deaths in 2012. Non-small cell lung cancer (NSCLC), which is one of two types of lung cancer, accounts for 85–90% of all lung cancers. Despite advances in therapy, lung cancer still remains a leading cause of death. Cancer relapse and dissemination after treatment indicates the existence of a niche of cancer cells that are not fully eradicated by current therapies. These chemoresistant populations of cancer cells are called cancer stem cells (CSCs) because they possess the self-renewal and differentiation capabilities similar to those of normal stem cells. Targeting the niche of CSCs in combination with chemotherapy might provide a promising strategy to eradicate these cells. Thus, understanding the characteristics of CSCs has become a focus of studies of NSCLC therapies

Investigation of the in vitro and in vivo biocompatibility of a three-dimensional printed Thermoplastic Polyurethane/Polylactic Acid blend for the development of Tracheal Scaffolds

Tissue-engineered polymeric implants are preferable because they do not cause a significant inflammatory reaction in the surrounding tissue. Three-dimensional (3D) technology can be used to fabricate a customised scaffold, which is critical for implantation. This study aimed to investigate the biocompatibility of a mixture of thermoplastic polyurethane (TPU) and polylactic acid (PLA) and the effects of their extract in cell cultures and in animal models as potential tracheal replacement materials. The morphology of the 3D-printed scaffolds was investigated using scanning electron microscopy (SEM), while the degradability, pH, and effects of the 3D-printed TPU/PLA scaffolds and their extracts were investigated in cell culture studies. In addition, subcutaneous implantation of 3D-printed scaffold was performed to evaluate the biocompatibility of the scaffold in a rat model at different time points. A histopathological examination was performed to investigate the local inflammatory response and angiogenesis. The in vitro results showed that the composite and its extract were not toxic. Similarly, the pH of the extracts did not inhibit cell proliferation and migration. The analysis of biocompatibility of the scaffolds from the in vivo results suggests that porous TPU/PLA scaffolds may facilitate cell adhesion, migration, and proliferation and promote angiogenesis in host cells. The current results suggest that with 3D printing technology, TPU and PLA could be used as materials to construct scaffolds with suitable properties and provide a solution to the challenges of tracheal transplantation