Medical and Health Sciences

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3D surface reconstruction for lower limb prosthetic model using modified radon transform
(Universiti Teknologi Malaysia, 2017) Mohd. Sobani, Siti Syazalina
Computer vision has received increased attention for the research and innovation on three-dimensional surface reconstruction with aim to obtain accurate results. Although many researchers have come up with various novel solutions and feasibility of the findings, most require the use of sophisticated devices which is computationally expensive. Thus, a proper countermeasure is needed to resolve the reconstruction constraints and create an algorithm that is able to do considerably fast reconstruction by giving attention to devices equipped with appropriate specification, performance and practical affordability. This thesis describes the idea to realize three-dimensional surface of the residual limb models by adopting the technique of tomographic imaging coupled with the strategy based on multiple-views from a digital camera and a turntable. The surface of an object is reconstructed from uncalibrated two-dimensional image sequences of thirty-six different projections with the aid of Radon transform algorithm and shape-from-silhouette. The results show that the main objective to reconstruct three-dimensional surface of lower limb model has been successfully achieved with reasonable accuracy as the starting point to reconstruct three-dimensional surface and extract digital reading of an amputated lower limb model where the maximum percent error obtained from the computation is approximately 3.3 % for the height whilst 7.4%, 7.9% and 8.1% for the diameters at three specific heights of the objects. It can be concluded that the reconstruction of three-dimensional surface for the developed method is particularly dependent to the effects the silhouette generated where high contrast two-dimensional images contribute to higher accuracy of the silhouette extraction. The advantage of the concept presented in this thesis is that it can be done with simple experimental setup and the reconstruction of three-dimensional model neither involves expensive equipment nor require any service by an expert to handle sophisticated mechanical scanning system
Anti-inflammatory and cytoprotective effects of 5, 7-dimethoxyflavone and 4', 5-7 - trimethoxyflavone on human umbilical vein endothelial cells
(Universiti Teknologi Malaysia, 2016) Nadri, Muhammad Helmi
Previous cell culture-based studies have shown potential health benefits of polyphenolic compounds conveyed by fruit and vegetables. However, most of these studies have rather tested higher concentrations of polyphenols than those maximum plasma concentrations, which is rarely exceed 10 µM, attained after a consumption of polyphenol-rich diet. Therefore, the present in vitro study investigates the antiinflammatory and cytoprotective effects of 5,7-dimethoxyflavone (DMF) and 4’,5,7- trimethoxyflavone (TMF) at both physiological and supraphysiological concentrations. DMF and TMF were tested for inhibitory activities on cyclooxygenase-2 (COX-2) and 15-lipoxygenase (15-LOX), both play important role in inflammation, using direct enzyme inhibition assay. DMF at concentrations of 0.01-10 µM were also used to treat oxidative stress-induced human umbilical vein endothelial cells (HUVEC), to assess effects on the expression of markers related to increased vasodilation and inflammation. Particularly, markers for vasodilation including endothelial nitric oxide synthase (eNOS) and endothelin-1 (ET-1), and markers for inflammation such as intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), were assayed using qRT-PCR. DMF and TMF strongly inhibit prostaglandins production through direct suppression of COX-2 activity, nearly equivalent to indomethacin with the mechanism mimicked current used drug, diclofenac based on molecular docking. Physiological and supraphysiological concentrations of DMF and TMF have been found to effectively abolish the effect of hydrogen peroxide (H2O2)-induced cell death in HUVEC independent of antioxidant activities. Furthermore, DMF at supraphysiological concentration was found to regulate eNOS expression at transcription rather than translational level in H2O2-induced HUVEC. In addition, H2O2 was found to increase ET-1, ICAM-1 and VCAM-1 mRNA expression in HUVEC culture and these negative effects was reversed by DMF at supraphysiological concentration. Since tested markers are the key molecules involved in the early atherogenic process, the present study emphasize a novel mechanism by which DMF may exert antiatherogenic activities under oxidative stress condition.
Biomechanical evaluation and new improvement on ankle external fixator
(Universiti Teknologi Malaysia, 2015) Ramlee, Muhammad Hanif
An ankle external fixator is a medical device that can be used for temporary fixation in order to limit movement during treatment. This device has been effectively proven in clinical studies to promote the healing process. The stability of the construct could also be attributed to clinical outcomes. However, this knowledge is limited in the literature. Therefore, the main purpose of this study was to biomechanically evaluate and optimise the external fixator with the aim of producing a better construct for the improvement of stress distribution. In this project, the biomechanical study using a finite element method involved several analyses of the effects of external fixator designs as well as its material properties. In order to do that, first and foremost, a three-dimensional ankle model was reconstructed using CT data images which consisted of tibia, fibula, talus, calcaneus, navicular, three cuneiform, cuboid and five metatarsal bones. The cartilages were developed with an estimated uniform thickness of 1 mm. A total of 34 ligaments and 3 plantar fascias were also modelled. Two pathological conditions of ankle problems were simulated with the external fixator by applying axial compression loads based on the swing and stance phase. The results of the finite element study showed that the Delta frame configuration had better stability in terms of relative micromovement, displacement and von Mises stress as compared to the Mitkovic and Unilateral external fixators. In addition, the use of 6 mm pin and 11 mm connecting bar were more favourable options to provide a stable construction. However, the use of extra pins at cuboid and medial cuneiform bone did not contribute to enhance stability. For better improvement of the external fixator, additional hollow cylinder fitted at the pin was considered to decrease stress.
Characterisation of Malaysian honeys and electrochemical detection of gallotannin for pure honey identification
(Universiti Teknologi Malaysia, 2017) Ismail, Norjihada Izzah
Seventeen samples (n = 17) of Malaysian gelam, acacia, nanas, tualang and kelulut honeys were analysed for their physicochemical, biochemical and phytochemical properties to evaluate their influence on floral source and bee type. Comparisons were also made with synthetic honeys to determine a suitable measure for fast identification of pure honey from synthetic honey. Solid phase extraction (SPE) was utilised for isolation of phenolic compounds in honey samples. The phenolic compounds present in the samples were analysed using high performance liquid chromatography-diode array detector (HPLC-DAD) and liquid chromatography tandem mass spectrometry (LC-MS/MS). Three electrode systems were utilised for rapid identification of pure Malaysian honeys. Properties of honey were shown to be influenced by the floral source and bee type to the lesser extent. Kelulut honeys were observed of having lower pH, higher free acid, moisture and ash contents as well as higher electrical conductivity (EC), the properties that distinguish Trigona honey from the common Apis honey. Antioxidant properties were different for the five types of honey with Trigona honey dominating most of the antioxidant tests. Up to 16 phenolic compounds were identified using HPLC-DAD system. Similar dominant compounds were observed between tualang and acacia honeys, and between kelulut and gelam honeys, suggesting that the floral source of unifloral honey is an equally important food source for the analysed multifloral honey. More phenolic compounds were detected spectrometrically using full scan method and multiple reaction monitoring (MRM). Plant gallotannin, penta-ο-galloyl-β-D-glucose (PGG) was successfully detected at low potential 0.173 V vs Ag/AgCl in pH 7 phosphate buffer solution using glassy carbon electrode (GCE) without any prior electrode activation, chemical modification and pre-concentration at the GCE. The PGG detection in blank pure honey and via standard addition approach in the Malaysian honeys revealed its presence only in the pure honeys. The present study suggested that electrochemical detection of PGG using GCE could be used as a tool for pure honey identification through a rapid and simple method rather than other conventional, highly-technical, expensive and time-consuming analytical techniques
Characterization of a novel bacterial dehalogenase isolated from cow dung
(Universiti Teknologi Malaysia, 2018) Ismail, Siti Nurul Fasehah
The large quantities of the halogenated compound for example 2,2- dichloropropionic acid (2,2DCP) in the environment may lead to health problems in humans and pollution due to their toxicity and recalcitrance, respectively. Interestingly, previous studies have indicated that cow dung was proven to degrade pollutants. Hence, such animals feeding on a daily diet of halogen contaminated forage may influence the microflora in their digestive tract. Bacterial species from cow dung able to utilize 2,2DCP is yet to be reported. Therefore, the purpose of this study was to isolate, identify and characterize dehalogenase bacteria from cow dung. Four bacteria were isolated which are SN1, SN2, SN3, and SN4. Strain SN1 was observed with rapid growth in 20 mM 2,2DCP liquid minimal media, and was used for further experiments such as growth in different concentration of 2,2DCP, High-performance liquid chromatography (HPLC), Biolog GENIII, 16S rRNA analysis, characterization of purified enzyme, kinetic analysis, Liquid Chromatography-Mass Spectrometry (LC-MS/MS) and amplification of dehalogenase gene. The growth of strain SN1 in various concentrations (10 mM, 20 mM, 30 mM and 40 mM) of the substance was evaluated. The study found the bacteria grew particularly well in 20 mM 2,2DCP with the highest chloride ion released (39.5 μmolCIˉ/mL) while exhibiting a remarkably short doubling time of 3.85 h. The utilization of 2,2DCP was also confirmed by detection of 20 mM 2,2DCP depletion in the growth medium containing strain SN1 measured using HPLC. The result showed 98.6 % utilization of 2,2DCP in the growth medium. Species identification via Biolog GENIII system and 16S rRNA analysis was performed and identified strain SN1 as Bacillus cereus. Further investigations on dehalogenase enzyme were done by using purified enzyme of Bacillus cereus SN1. The molecular weight of the purified enzyme was 25 kDa by SDS-PAGE. The enzyme characteristics revealed it was optimum at pH 6 and 30 °C. It also has low Km value of 0.2 mM. The dehalogenase peptide was identified by LC-MS/MS with 18% sequence coverage to haloacid dehalogenase, Bacillus cereus (strain 03BB102). Moreover, Group I and Group II dehalogenase primers were used to amplify dehalogenase gene and the band only appeared for Group I. The dehalogenase gene fragment amplified was designated “DehSN1” and belongs to Group I dehalogenase since it has 75 % similarity with Group I dehalogenase (DehE). Five conserved residues were identified as Asn33, Tyr117, Cys42, Ala120 and Asp136. As a conclusion, this is the first reported case of a Bacillus sp. isolated from cow dung capable of utilizing 2,2DCP. Therefore, further assessment of its ability to degrade other types of haloalkanoic acids merits special consideration
Characterization of arsenate reduction by arsenic tolerant microbacterium foliorum strain SZ1 isolated from gold ores
(Universiti Teknologi Malaysia, 2016) Mohd. Bahari, Zaratulnur
Arsenic is a metalloid of global concern that primarily exists in two inorganic forms of severe toxicity, As (III) and As (V). The reduction of As (V) to As (III) increases toxicity, mobility and bioavailability of arsenic. Understanding how microorganisms reduce As (V) is important to elucidate As (V) reduction mechanism and inevitably, discover approaches to minimise its toxic impact on the environment. This study was aimed at investigating the capability of arsenic tolerant Microbacterium foliorum strain SZ1 isolated from gold ores to undergo As (V) reduction to As (III). This strain demonstrated complete reduction of 1 mM As (V) achieved within 120 hours under aerobic condition indicating a possible mechanism of detoxification through regulation of ars operon. Further optimization of factors enhancing As (V) reduction capacity of strain SZ1 resulted in complete reduction of 1 mM As (V) achieved within 36 hours in Tris minimal medium supplemented with 10 mM sucrose and 0.1 % (w/v) tryptone at pH 7. The effect of cell adaptation or acclimation towards As (V) reduction was investigated. Well-adapted strain SZ1 recorded complete reduction of 0.5 mM As (V) to 3 mM As (V) within 18 hours to 42 hours incubation. Exopolysaccharides (EPS) was observed to be secreted during reduction of As (V) and subjected to further characterization through chemical analysis of neutral carbohydrate and protein contents and Fourier transform infra-red (FT-IR) analysis. As As (V) concentration increased, so did the protein and carbohydrates concentration of EPS, indicating that EPS played an important role in enabling strain SZ1 to resist and reduce arsenic. Haldane inhibition model was used to fit the reduction rate at different initial As (V) concentrations and the parameters µmax, Ks and Ki were determined to be 0.14 h-1, 0.39 mM and 35.3 mM, respectively. In addition, presence of As (III) as the final product was further confirmed by detection through high performance liquid chromatography (HPLC) analysis. Field emission scanning electron microscopy analysis (FESEM) showed that cells grown in the presence of As (V) exhibited distinct changes in cell morphology and presence of EPS. Exploration of the draft genome of M. foliorum SZ1 identified the presence of ars operon (arsC-arsC-ACR3-arsT-arsC-arsR-arsC) and another two stand-alone genes, arsC and arsB which further confirmed SZ1’s tolerance towards high concentration of arsenic. From the screening of plant growth promoting (PGP) traits, strain SZ1 was able to produce siderophores and indole acetic acid which highlighted its potential use in microbe-assisted arsenic phytoremediation. This is the first study that elucidates the characterization of As (V) reduction by M. foliorum SZ1.
Characterization of dehalogenase for the degradation of 3-chloropropionic acid
(Universiti Teknologi Malaysia, 2017) Hassan Muslem, Wafaa
The use of herbicides that contain halogenated compounds, for example 3-chloropropionic acid (3-CP) poses significant environmental hazards as well as detrimental to human. The research detailed here investigated the isolation and identification of bacteria strains that could degrade 3-CP as its sole carbon source. Dehalogenase that can degrade 3-CP is rare in nature. In this study, two strains of dehalogenase producing bacteria capable of utilizing 3-CP were successfully isolated from abandoned agricultural land in Universiti Teknologi Malaysia. These bacteria were characterized by using 16S rRNA as well as biochemical analysis. Strain WH1 showed a 98 % sequence identity to Burkholderia cepacia with (accession number KU318403) whereas strain WH2 showed a 99% sequence identity to Bacillus cereus with (accession number KU721999). The results have shown that these bacteria were capable to grow in liquid minimal media supplied with 10 mM 3-CP as sole carbon source with doubling time of 43.62 h for WH1 and 14.75 h for WH2. Utilization of 3-CP was confirmed by detection of chloride ion released using halide ion assay technique for both strains which indicate their ability to degrade 3-CP. For further confirmation of 3-CP consumption, analysis by high performance liquid chromatography (HPLC) revealed that both B. cepacia WH1 and B. cereus WH2 effectively utilized ~100% of 10 mM 3-CP. This is the first report detailing both strains able to competently utilize 3-CP as their sole carbon source. Cell free extract of B. cereus strain WH2 was further characterized due to its faster growth on 3-CP compared to B. cepacia strain WH1. The intracellular dehalogenase from B. cereus WH2 was purified to homogeneity to afford a 2.5-fold (50 % yield) concentration with an estimated molecular mass of 37 kDa by SDS-PAGE analysis. Its highest enzyme activity was achieved at conditions of 30 oC and pH 7. While the activity of WH2 dehalogenase was substantially repressed by both Hg2+ and Ag2+, the enzyme was not inhibited by DTT and EDTA. Pertinently, kinetics evaluation revealed a higher affinity of the WH2 dehalogenase towards 3-CP than 3-chlorobutyric acid (3-CB), affording Km values of 0.32 mM (kcat 3.97 s-1) and 0.52 mM (kcat 4.35 s-1), respectively. The WH2 dehalogenase was ~1.6-fold catalytically more efficient (kcat/Km) in dehalogenating the three-carbon, 3-CP (12.4 mM-1 s-1) over the four-carbon, 3-CB (8.27 mM-1 s-1). From the data, it was identified that 3-CP degradation was not stimulated by co-factors, such as NAD+, NADH, NADP+, NADPH, FAD and CoA that did not affect the enzyme activity by demonstrating activities of <0.1 unite (g protein)-1. The amplified dehalogenase gene fragment was designated “deh-wh2” and subsequent analysis showed it belongs to Group II dehalogenase. Eight conserved residues that line the active site were identified: Asp10, Thr14, Ser117, Lys150, Tyr156, Ser174, Asn176 and Asp179. These residues are consistent with the residues found in the active site of DhlB, DehIVa and L-DEX. The product of 3-CP degradation was 3-hydroxypropionic acid based on HPLC. In conclusion, this study confirmed the presence of new dehalogenase isolated from various bacteria that have potential to utilize 3-CP, especially from contaminated environment
Characterization of novel marine bacterium vitellibacter aquimaris using polyphasic approach and genomic analyses
(Universiti Teknologi Malaysia, 2018) Thevarajoo, Suganthi
The discovery of novel marine bacteria provides an opportunity in exploring new bioactive compounds and their significance. Extensive industrial applications of enzymes promote the search of the enzymes from the new source. In this study, a yellow-orange strain designated as D-24 was isolated from Desaru, Johor. The study aimed to taxonomically and genomically characterize strain D-24 through polyphasic approach and genome analyses, respectively. The 16S rRNA gene sequence of strain D-24 indicated that it belongs to genus Vitellibacter. Strain D-24 was distinct from the other Vitellibacter spp. in hydrolyse Tween 60 and tyrosine, composition of fatty acids, polar lipid profile and DNA–DNA relatedness. Hence, strain D-24 was proposed as a new type strain with the name of Vitellibacter aquimaris. Following, strain D-24 was characterized on its protease activity and extracellular protease encoding genes. The characterization of crude protease demonstrated optimum activity at 60 °C, 5 % (w/v) NaCl and pH 7. The draft genome sequence of V. aquimaris (3.1 Mbp) was generated using an Illumina MiSeq sequencer. Mining of genes revealed the presence of three metalloproteases, two serine proteases and a cysteine protease. Recently, genus Vitellibacter was suggested as the same genus of Aequorivita based on phylogenomic data with no further analyses. Therefore, the detailed comparative analyses were performed based on their phenotypic and genomic features. Analyses of 16S rRNA gene sequences, housekeeping genes, percentage of conserved proteins and phenotypic features supported both of them as same genus. Furthermore, the presence of proteases, glycoside hydrolases and denitrification genes in genome of Vitellibacter/Aequorivita revealed their role in detergent industry, food industry and in carbon mineralization. In a nutshell, this study has provided new knowledge on V. aquimaris and also the useful genes such as serine protease, beta-mannosidase and nitrous oxide reductase that could be further characterized
Corrosion behavior and mechanical properties of silicon and zinc-oxide coated magnesium-based bionanocomposite with hydroxyapatite and titania additives
(Universiti Teknologi Malaysia, 2016) Khalajabadi, Shahrouz Zamani
Magnesium (Mg)-based alloys were emerged as potential biodegradable material for temporary implants. However, their fast degradation in the high chloride environment of the physiological solution is detrimental unless inhibited. These Mg-based implants lose their mechanical integrity before the tissue is being sufficiently healed. Furthermore, the accumulation of hydrogen gas upon fast degradation in a physiological solution restricted their clinical applications. In this view, the present research is targeted to improve the biocorrosion behavior and mechanical properties of pure Mg by alloying it with nanostructured hydroxyapatite (HA), MgO, and TiO2 through milling-pressing-sintering powder metallurgy route. Four different Mg-based bionanocomposites including Mg/HA, Mg/HA/MgO, Mg/HA/TiO2 and Mg/HA/TiO2/MgO were synthesized to evaluate their bioimplantation efficacy. The Mg/HA/TiO2/MgO bionanocomposite was further coated with nano-Si, nano-ZnO single-layer, and nano-Si/ZnO double-layers using radio frequency magnetron sputtering technique to achieve such goal. The influence of varying amounts of the additives, the ball milling duration, the annealing temperature, and the coating agents on the biocorrosion and mechanical properties of these bionanocomposites were evaluated using electrochemical, immersion and compression tests. The phase evolution of the synthesized bionanocomposites before and after immersing in the simulated body fluid (SBF) solution was characterized via X-ray diffraction, Fourier-transform infrared and X-ray photoelectron spectroscopy. The detailed microstructures were determined using field-emission scanning electron, transmission electron, and atomic force microscopies. The bionanocomposites wettability was measured via video contact angle method. Thermal gravimetric and differential thermal analysis were performed to evaluate the activation energy and the reaction kinetics of the prepared powder bionanocomposites. In vitro corrosion resistance was analysed using potentiodynamic polarization, immersion, pH variation, and hydrogen evolution tests. After 8 h of ball milling the corrosion resistance of Mg/xHA/10TiO2/10MgO (wt%) bionanocomposite for two different compositions of HA such as 12.5wt% and 27.5wt% was found to increase from 1.35 and 2.19 kΩ.cm2 to 2.25 and 4.78 kΩ.cm2, respectively. Meanwhile, by annealing at 630°C, these two bionanocomposites demonstrated reduced corrosion rates compared to those annealed at 500°C. Interestingly, the compression failure strain (ductility) of HA incorporated Mg was decreased by increasing milling time. The corrosion rate of Mg/12.5HA/10TiO2/10MgO coated with nano-Si/ZnO exhibited a significant reduction from 5.82 (uncoated) to 0.25 mm/year. The cell culture test authenticated that the Mg-based bionanocomposites appeared biocompatible in the presence of HA, MgO, TiO2 additives and nano-Si/ZnO coating. It is noticed that the synthesized Mg/12.5HA/10TiO2/10MgO coated with nano-Si/ZnO has a great potential to become a candidate for biodegradable implants
Detection of onset muscle fatigue based on joint analysis of surface electromyography spectrum and amplitude
(Universiti Teknologi Malaysia, 2017) Mohamad Ishak, Nurul Ain
Many studies have been conducted to track muscle fatigue and to understand the mechanisms that contribute to the deterioration of muscle performance. Electromyography fatigue threshold (EMGFT) and Integrated Electromyography (IEMG) are two techniques that have been applied to determine the Onset of Muscle Fatigue (OMF) by depending on the percentage force output and amplitude respectively. Nevertheless, force and amplitude are correlated with one another during fatigue. Joint Analysis of EMG Spectrum and Amplitude (JASA) is commonly used to discriminate force-related from fatigue induced EMG changes. However, the length of signal affects the performance of JASA in discriminating fatigue signal. Apart from that, JASA has not been used to detect OMF. Thus, the purpose of this study is to determine the OMF region by applying JASA on the segmented EMG signal. Surface EMG signals were recorded from 30 college students while they were performing isometric contractions of Biceps Brachii muscles for 2 minutes. Each recorded signal was segmented into 15-second time interval. Root Mean Square (RMS) and Mean Frequency (MNF) were used as the muscle fatigue indicators. The indicators were extracted from 3-second epoch length within each segment. A polynomial regression model was applied to describe the trends of the indicators in a segment. The first segment that simultaneously showed a decrease in the frequency and an increase in the amplitude of a sEMG signal with correlation coefficient r = 0.7 was classified as the region where the OMF occurred. Out of 30 subjects, 20 subjects (67%) either admitted to experience muscle discomfort and at the same time the OMF region was also detected or vice-versa. For the other 10 subjects, the OMF region was able to be detected in 90% of them but due to better endurance levels, they required longer time to experience muscle discomfort. The temporal-spectral fatigue indicator (Instantaneous Mean Frequency (iMNF)) was used to determine the reliability of the developed technique. The decrement of iMNF on the detected OMF region showed high correlation coefficient (r > 0.6). The subjects were also asked to perform dynamic contractions for 2 minutes. The proposed technique was applied to the recorded signals and the OMF was detected in 24 subjects. Eighteen of them (72%) acknowledged that they had experienced muscle discomfort. Fourteen out of 18 subjects felt muscle discomfort after OMF was detected. The results indicate that muscle discomfort develops gradually after the onset of muscle fatigue. For handwriting activity, 4 subjects were asked to write for 5 minutes while the sEMG signals were captured from Flexor Carpi Radialis muscle (small muscle). Out of 4 subjects, all of them showed an increment in pen pressure, and 75% of them showed an increment in the writing speed after detecting OMF region. This study concludes that the proposed technique is feasible to detect the OMF; not only during isometric contraction but also during dynamic contraction. The technique also has the potential to be applied to small muscle contraction
Enzymatic analysis of native and recombinant fibrinolytic enzyme from bacillus cereus 13BN isolated from belacan
(Universiti Teknologi Malaysia, 2016) Zakaria, Zarita
Fibrinolytic enzymes from natural sources have now attracted more attention than ever because they are less costly and have less undesirable side-effects as compared to modern chemical thrombolytic agents. A bacterial isolate designated as Bacillus cereus 13BN having excellent fibrinolytic enzyme was isolated from fermented shrimp paste, belacan. The enzyme has a dual mechanism of fibrin degradation; it directly degrades fibrin plus it can also act as a tissue type plasminogen activator (t-PA), which converts plasminogen to plasmin and consecutively degrades fibrin. T-PA also participates in a wide variety of connective tissue matrix alterations and upregulated by proinflammatory cytokines which can act as antidepressants. In silico analyses indicated that the enzyme belonged to the typical subtilisin-like serine protease group with a conserved region of a catalytic triad; Asp226, His298 and Ser658. Subtilisin-like 13BN gene was successfully cloned into pET22b (+) vector and then transformed into E. coli BL21 (DE3) to give recombinant BL-21(DE)-(pET22b(+)(SL-13BN). The vector carries an N-terminal pelB signal sequence for periplasmic translocation and a C-terminal His•Tag® for easy purification of the protein. Recombinant enzyme RSL-13 BN was over-expressed as extracellular enzyme (EX) at 41% of total fibrinolytic activity, besides been expressed as periplasmic enzyme (PE) at 56 % and solubilized inclusion bodies (SIB) at 3 %. Under optimized condition where cell mass was propagated by induction with 1 mM IPTG at 30 ºC for 16 hours, 14,616 UL-1 total activity by RSL-13BN could possibly be achieved. This is four fold higher than the extracellular native enzyme SL-13BN (3,468 UL-1). Purification of extracellular SL-13BN enzyme was preceded with ammonium sulphate precipitation, followed by anion exchange and gel filtration chromatography. Alternatively, following ammonium sulphate precipitation, single-step purification was performed for extracellular RSL-13BN enzyme using affinity chromatography utilizing His????-TrapTM Ni-NTA Sepharose column. Both native and recombinant enzymes have equivalent biochemical characteristics. These enzymes are serine proteases with plasminogen activator potential and have a molecular weight of 80kDa. Their strong fibrinolytic activity was verified through fibrin clot degradation pattern on SDS-PAGE gel. The proteolytic action of SL-13BN and RSL-13BN differ from the typical human plasmin in that they can completely hydrolyze whole fibrinogen and fibrin clots without the aid of other proteolytic enzymes.The optimum temperature for fibrinolysis for both was 50°C but their optimum pH differed slightly; pH 7.4 for SL-13BN and pH 7 for RSL-13BN. The activity of both enzymes was enhanced by Ca2+, Na+, K+, Mg2+ and Mn2+, but inhibited by Ag+, Co2+, Cu2+, Fe2+, Fe3+, Hg+, Zn2+, PMSF, EDTA and SDS. Purified SL-13BN had a Vmax of 2.665 U mg-1min-1and Km of 0.5722 mg mL-1 and purified RSL-13BN had a Vmax of 2.642 U mg-1min-1and Km of 0.5776 mg mL-1 towards fibrin. In conclusion, this study showed that RSL-13BN enzyme can be over-produced, acting as t-PA which can provide many medical benefits, easily purified as well as can hydrolyze fibrin and fibrinogen as a whole without the help of other proteolytic enzymes. This makes the enzyme a potential anticoagulant substitute for the treatment of thrombosis-linked diseases
Evaluation of Justicia gendarussa crude leaf extract for enhancement of flavonoids production via adventitious root culture and genetic modifications
(Universiti Teknologi Malaysia, 2017) Ayob, Zahidah
Justicia gendarussa extract possesses various bioactivities associated with the availability of flavonoids. Low availability of flavonoids could limit or even hinder the bioactivities effects. Therefore, attempts to enhance the flavonoids production via tissue culture approaches are being studied. This study aimed to optimize flavonoids contents in J. gendarussa using different tissue culture systems (in vitro plant regeneration and adventitious root culture) and genetic transformation methods. The cytotoxicity of plant extracts against various cancer cell lines was also evaluated. Detection and quantification of naringenin and kaempferol were performed using GC-FID. Cytotoxicity tests of crude extract against cancer cell lines (MCF-7, MDA-MB-231, MDA-MB-468, HT-29, HeLa and BxPC-3) were determined by MTT assay. The optimization of elicitors used including yeast extracts (YE), casein hydrolysate (CH) and proline (P) at various concentrations (0, 0.2, 0.4, 0.6, 0.8 or 1.0 mg/L) were examined using nodal explants from in vitro plants. Adventitious roots were inoculated into MS liquid medium supplemented with IBA (2.0-4.0 mg/L). For genetic transformation studies, plasmids pCAMBIA 1305.2, which harbour the PKS gene and plant selectable marker, HPT for hygromycin resistance was used to transform nodal explants of J. gendarussa under the optimized transformation protocol using biolistic and Agrobacterium tumefaciens-mediated transformation. Results showed that mature leaves extract, JG1 had the highest naringenin (444.35 ± 81.43 mg/kg) and kaempferol (1591.80 ± 94.91 mg/kg), while the cytotoxicity against BxPC-3 cell was the strongest (IC50~16 µg/mL). The highest naringenin and kaempferol contents were obtained in leaf crude extracts when treated with 0.6 g/L of CH (1180.30 ± 50.23 mg/kg) and 0.6 g/L of P (385.01 ± 13.10 mg/kg), respectively. Adventitious root culture produced high naringenin (97.54 ± 5.47 mg/kg) and kaempferol (853.82 ± 56.52 mg/kg) when treated with 2.0 mg/L IBA. The optimal parameters for biolistic method were established at 1100 psi helium pressure and 12 cm target distance with 95% of transformation efficiency. Meanwhile, the optimal transformation condition of A. tumefaciens method was bacterial concentration at OD600nm ~ 0.8, 20 minutes of inoculation time, 500 µM AS and 1 cm explant size with 90% transformation efficiency. Even though A. tumefaciens method produced lower percentage of transient GUS expression than biolistic method, a few transformed explants were successfully produced. The integration of the PKS gene with band size of 1200 bp into the genome of transgenic plants were verified by PCR, sequencing and subsequently confirmed by Southern blot analysis. The content of kaempferol were found to be higher in stem extracts of transgenic plants (450.40 ± 7.82 mg/kg) than non-transgenic plants (197.13 ± 2.29 mg/kg). In conclusion, addition of elicitors, establishment of adventitious root culture and A. tumefaciens-mediated transformation could enhance flavonoid contents in J. gendarussa
Genetic tolerance to ferrous and aluminium toxicities for seed germination in oryza sativa L.
(Universiti Teknologi Malaysia, 2017) Jahan, Nusrat
Direct seeded culture is famous for rice cultivation due to its low inputs and less labour demand. However seeds are exposed to various abiotic stresses in direct seeding culture. Ferrous (Fe2+) and aluminium (Al3+) are toxic metals that severely affect seed germination and growth traits. Genetic tolerance to metal toxicity would be one of the possible solutions to combat with these challenges. The genetic study on Fe2+ and Al3+ for seed germination can hardly be found in literature. The present study was conducted to identify the potential loci linked with genes controlling tolerance to Fe2+ and Al3+ for seed germination. A segregating mapping population of F2:3 was developed from the cross between selected Fe2+ and Al3+ tolerant parent Pokkali and sensitive parent, Pak Basmati. Parental DNA was used for polymorphic survey and F2 DNA was used for genotyping. A molecular linkage map was constructed using 84 markers data. The molecular linkage map covered 3435.5cM with an average distance of 7.63cM except 4 larger gaps on chromosome 1, 2, and 4. F3 progenies (129) were evaluated against the optimized toxic level of Fe2+ and Al3+ under controlled environment. Germination traits such as final germination percentage (FG%), germination energy (GE), germination rate (GR), germination speed (GS), germination index (GI), mean time of germination (MGT), germination value (GV), germination velocity (GVe), peak value of germination (PV), germination capacity (GC) and growth traits such as root length (RL), shoot length (SL), total dry biomass (TDB), and germination vigour index (GVI) were measured. In the present study, screening of six rice varieties showed significant differences for seed germination, however Pokkali exhibited the minimum and Pak Basmati showed the maximum influence in seed germination. A 20mM (at pH4.0) of both Fe2+ and Al3+ was found to be the optimized toxic level, as most germination and growth parameters were found to significantly affected at this concentration. Phenotypic data showed significant variations in germination and growth parameters. Total of 39 QTLs (Quantitative Trait Loci) for germination traits and 8 QTLs for growth parameters linked with Fe2+ toxicity tolerance were determined by Simple Interval Mapping (SIM) at 0.06% to 39.9% of phenotypic variations, respectively. Thirty four QTLs for germination parameters and 8 QTLs for growth traits with phenotypic variations 0 to 47% traits linked with Fe2+ toxicity tolerance were mapped by Composite Interval Mapping (CIM). Forty nine QTLs for germination and 23 putative QTLs for growth parameters with phenotypic variations 0.01% to 69% were determined using Multiple Interval Mapping (MIM). Epistasis analysis revealed that the QTLs are mostly dependent on the alleles at other loci. For Al3+ toxicity tolerance 40 markers linked with germination and growth parameters with phenotypic variation 0 to 62.64% were identified by Simple Interval Mapping. Forty-six putative QTLs with phenotypic variation 0 to 28.1% were detected for germination and growth parameters for Al3+ toxicity tolerance using Composite Interval Mapping. Sixty five markers linked with germination and growth traits, at 0 to 72.7% phenotypic variations were mapped by Multiple Interval Mapping. Epistasis analysis showed that Al3+ toxicity tolerance is polygenic and controlled by additive effect. The results suggested that the QTL regions (18cM) and (72cM) were tightly linked to Al3+ tolerance genes could be used for marker assisted selection programme using fine mapping. Moreover, this study also provides an understanding to exclusion tolerance mechanism of Al3+ toxicity as known in the Triticeae within sub-family Pooideae. The identified major QTLs of this research would be useful for rice hybridization programs to induce tolerance against these toxic metals
Lactic acid production from solid pineapple waste using Rhizopus Oryzae NRRL 395
(Universiti Teknologi Malaysia, 2017) Aziman, Siti Nurbalqis
Solid pineapple waste (SPW) is one of the most abundant agricultural wastes found in tropic region. It was reported about 40-50% of the wastes generated from pineapple canning industry are from solid wastes. Their disposal poses a serious environmental pollution problem. This study looks into the potential of utilizing a mixture of solid pineapple waste including residual pulp, peels, cores, stems and leaves for the production of lactic acid by Rhizopus oryzae NRRL 395 in solid state fermentation (SSF). Characterization of SPW using Fourier transform infrared (FT-IR) and Scanning electron microscope (SEM) indicate that the structural and chemical composition of autoclaved SPW was suitable for use as SSF substrate. Screening studies through 2-level factorial (2LF) design revealed that R. oryzae NRRL 395 was best suited for lactic acid metabolism under the conditions of SSF system. The optimum SSF condition in shake flasks conducted based on central composite design was obtained at 67.53%, 3 days of incubation, at temperature of 32.2°C, pH of 5.6, and inoculum size of 1×107 spores/g, with 1.21 fold increment of lactic acid yield compared to that produced in one-factor-at-a-time experiment. This study has successfully designed a novel modified Memmert-tray bioreactor to analysed the effects of lactic acid production in larger scale (1kg) SSF of SPW. The highest concentration of lactic acid in the bioreactor was obtained at condition variables of 70 ± 2% of humidity chamber with 2 Liters per minute (LPM) aeration rate, incubation temperature of 30°C, pH 6 and 70% of initial moisture content of SPW bed, where the SSF was run for 3 days.The lactic acid yield (Yp/s), maximum and overall lactic acid productivity of R. oryzae NRRL 395 in modified Memmert-tray bioreactor were 1.03, 1.05, 1.14 fold higher than those under optimum condition performed in shake flask system. As a conclusion, a significant lactic acid production from SPW by Rhizopus oryzae NRRL 395 has proved that it could be contributed towards the sustainability of agricultural industry by creating wealth from waste and promoting economic biotechnology for future development
Lignocellulolytic enzymes by aspergillus sp. A1 and bacillus sp. B1 isolated from gut of bulbitermes sp. in solid state fermentation using sawdust as substrate
(Universiti Teknologi Malaysia, 2017) Kamsani, Noratiqah
Sawdust is one of the common lignocellulosic waste biomass produced during the process of planning mills, moulding plants and furniture manufacturing. In practice, the sawdust is discarded in landfill areas, causing dust and dirt pollution in nearby localities. Therefore, the need to find an efficient and practical approach to revalorize sawdust as a starting raw material in the production of lignocellulolytic enzymes is essential as a way to manage and turn the residues into value added products. Prospecting for efficient degrading lignocellulose microorganisms is crucial to facilitate the process of lignocellulolytic enzymes production from the lignocellulosic biomass. This study aimed to exploit microorganisms isolated from gut of termite Bulbitermes sp. in producing lignocellulolytic enzymes under solid-state fermentation (SSF) system by using untreated sawdust as substrate. Seventeen bacterial and five fungal with positive lignocellulolytic enzymes activities were successfully isolated from the gut of two hundred termites. Four isolates identified as Aspergillus sp. A1, Bacillus sp. B1, Bacillus sp. B2 and Brevibacillus sp. Br3 were selected for further characterization. Among the isolates, Aspergillus sp. A1 showed highest activities of lignin peroxidase (LiP) (729.12 U/g) and ß-glucosidase (22.97 U/g). The highest activities of endoglucanase (138.77 U/g) and manganese peroxidase (MnP) (47.73 U/g) were recorded in Bacillus sp. B1. The Bacillus sp. B2 produced the highest activities of exoglucanase (32.16 U/g) and laccase (71.18 U/g). The highest xylanase activity (104.96 U/g) was observed in Brevibacillus sp. Br3. The production of endoglucanase, ß-glucosidase, xylanase, LiP and laccase were approximated 17?93% higher in co-culture compared to individual culture. Compared to other di-, tri- and quad-mixed culture, Aspergillus sp. A1 (A1) and Bacillus sp. B1 (B1) co-culture produced the highest lignocellulolytic enzymes activities (endoglucanase, 190.1; exoglucanase, 13.5; ß- glucosidase, 33.7; xylanase, 202.5; LiP, 713.5; MnP, 23.3 and laccase, 52.1 U/g). The interaction between A1 and B1 is not antagonistic. Study on the effect of SSF operational variables showed that the use of unsieved sawdust produced significantly higher activities of exoglucanase, xylanase, LiP and laccase compared to that of sieved sawdust. In addition, temperature, pH and moisture content significantly impacted lignocellulolytic enzymes production. In comparing to control, moistening the unsieved sawdust with Mandel basal medium (pH 8) to 1:2.5 (solid:moisture) ratio, and incubation at 35 °C for 9 days produced 1.2?49.4 fold higher lignocellulolytic enzymes activities. Endoglucanase, ß-glucosidase and xylanase could be classified as moderately thermostable enzymes with better stability in acidic pH range. Meanwhile, ligninases possessed thermophilic and alkaliphilic characteristics. The co-culture produced 1.9?11.8 fold higher reducing sugars than those yielded by single cultures in the enzymatic degradation of sawdust. The use of co-culture enzymes also produced 3.6?85.4% higher reducing sugars as well as 1.3?2.3 times higher raffinose, cellobiose, maltose, glucose and xylose concentrations compared to that of commercial cellulase (Celluclast) solution. As conclusion, this work has generated a microbial co-culture that could be used for improved lignocellulolytic enzymes and reducing sugars production using untreated sawdust as substrate
Management information system for hajj pilgrim's total wellness
(Universiti Teknologi Malaysia, 2017) Idris, Muhammad Iqbal Tariq
Hajj is a spiritual journey which require physical and mental preparation since pilgrims need to face hectic activity, extreme temperature and exhaustive environment during Hajj. Recently, there are few instruments and models that relate to wellness however they are too general and not specific for certain event or religious rituals. Besides, existing management system only focuses on treatment and emphasize on physical, physiological and medical history only. Thus, the purpose of this study was to develop instrument, model, prescription and management information system specific for Hajj Pilgrim’s Wellness. Sequential exploratory design were used trough out this research. Eight construct were established from the interview conducted with 5 panel of expert consist of physical activity, physical care, healthy eating, intrapersonal, interpersonal, knowledge, mental toughness and relationship with Creator and creatures. Items for each construct were determine based on past study and need analysis. A survey was conducted to 300 respondents from six mosques in Johor Bahru district. The data gathered were analyzed using Rasch Measurement analysis. The findings showed instrument fit the model in terms of construct validity, item and person reliability, rating scale, dimensional and item fit. Besides, there were significant differences between wellness based on demographic characteristics including age, health status and occupation except gender. Next, a model was developed using average of item logit to determine the contribution factors hierarchy towards wellness level. Then, prescription was developed based on previous research and content validity were gathered from three panel of experts. Finally, a web based system was developed and the usability of the developed system was measured using IsoMetricS questionnaire. Thus, it was recommended that the Ministry of Health and Tabung Haji used and promote awareness among hajj pilgrims by referring to the model in the success of Hajj practices.
Microfluidic fabric-based electrochemical device for detection of clinical analytes in physiological fluids
(Universiti Teknologi Malaysia, 2014) Malon Marugan, Radha Swathe Priya
In recent years, inexpensive alternative materials (paper, yarn/thread and fabric) have been proposed for the construction of new generation of point-of-care (POC) microfluidic diagnostic devices. The purpose of this study is to investigate the integration of electrochemical detection in fabric-based microfluidic device for quantitative measurement of biomarkers in physiological fluids. The device was fabricated using two methods, template for patterning the electrodes and wax-patterning for creating the hydrophilic/hydrophobic contrast. The electrodes incorporated within the fabricated device were evaluated using cyclic voltammetry. The feasability of the device to determine clinical analytes such as glucose, lactate and ascorbic acid (AA) in control serum, saliva and artificial urine samples, respectively was demonstrated using chronoamperometry at the optimal detection potential (-0.2 V vs. Ag/AgCl for glucose and lactate; 0.28 V vs. Ag/AgCl for AA). The levels of the analytes measured were within the margin of error of the actual concentrations. The sensitivity of the device for the determination of glucose, lactate and AA were 0.294 ± 0.082, 0.3169 ± 0.099 and 0.4202 ± 0.229 |iA/mM, respectively, which are within the physiological range (2 to 25, 0.1 to 5 and 0.5 to 10 mM, respectively) of these analytes. The influence of mechanical stress towards the morphology and electrochemical behaviour were examined for both microfluidic fabric-based electrochemical device (^FED) and microfluidic paper-based electrochemical device (^PED). The results implied that the proposed |iFED was more durable under mechanical stress (sensitivity drop of ~ 21.6%) in comparison to the |iPED (sensitivity drop of ~ 48.6%). A three dimensional (3D) |iFED prototype for sample collection and continuous assessment (t = 170 min) of dynamic electrochemical measurement was also developed and evaluated. The |iFED provided an efficient sample delivery towards the reaction chamber, allowing dynamic electrochemical measurement in real-time without interruption unlike the |iPED. This study shows the potential of the proposed |iFED as a novel microfluidic sensing platform for a variety of assays that require simplicity, low-cost, portability, flexibility and continuous real-time monitoring
Molecular cloning and characterization of a new fibrinolytic enzyme from acinetobacter baumannII TU04
(Universiti Teknologi Malaysia, 2016) Krishnan, Renuka P.
In Malaysia, the occurrence of cardiovascular diseases has increased for the past thirty years and has remained as the number one killer. Fibrinolytic enzymes play a vital role in treating this disorder. However, their high production cost and undesirable side-effects circumscribe their widespread commercial use. This study aimed to clone, over-express, purify and characterize a new microbial fibrinolytic protease from Asian traditional fermented foods. A potent subtilisin-like serine protease gene encoding fibrinolytic enzyme from a newly isolated Acinetobacter baumannii TU04 was successfully cloned and expressed. The nucleotide sequence of the cloned gene revealed a single open reading frame of 2,184 bp coding for 736 amino acids and the deposited GenBank accession number is KP204011. The recombinant clone was expressed in the cytoplasm of E. coli Lemo 21 (DE3) as soluble and active enzyme. The resulting enzyme, SERpro was successfully purified via an immobilized nickel cation affinity chromatography column. SERpro was purified to homogeneity with a purification factor of 18-fold and recovery yield of 5%. SERpro exhibited maximal activity at 37 ºC and at pH 7.4, respectively. The molecular weight of the purified SERpro was about 82 kDa as determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fibrinogenolysis peptide sequence analysis revealed that SERpro degraded Bß chain of fibrin at a much lower rate but cleaved Aa and ?- chain extensively. The clotting time of human blood serum i.e; relative partial thromboplastin time increased by 1.14-fold increase (13.9 %) in the presence of 1U SERpro. SERpro exhibited analogous sequence similarity with other established fibrinolytic enzymes. As a conclusion, data suggests that SERpro from Acinetobacter baumannii TU04 is a potent protease with anti-thrombotic activity
Penggunaan sisa udang untuk penghasilan kitinase oleh 'trichoderma virens' menggunakan fermentasi keadaan pepejal
(Universiti Teknologi Malaysia, 2015) Rachmawaty
The chitinase production by Trichoderma virens using shrimp waste as a substrate was studied in solid state fermentation with 70% of moisture content. Six different pretreatment methods namely oven pretreatment, microwave pretreatment, boiling and crushing pretreatment, sun-dried pretreatment and chemical pretreatment were conducted on shrimp waste with non-treated shrimp waste as a control. The highest chitinase activity was obtained from microwave pretreatment on the third day of fermentation with chitinase activity of 0.194 U/g IDS, 3.2 fold higher than the untreated shrimp waste (0.06 U/g IDS). Study on the effect of nitrogen source on chitinase production using general factorial design showed that ammonium sulphate with 30.29 mM nitrogen gave significant effect compared toyeast extract with 7.43 mM nitrogen. Two level factorial design, incubation time, temperature, and substrate moisture also have a significant impact on the production of chitinase. Central composite design (CCD) was used in optimizing the conditions for chitinase production of shrimp waste by solid-state fermentation. Chitinase production was found to have increased 2.46 times (0.487 U/g IDS) at optimum condition: temperature of 27.9 ° C, 54.5% of substrate moisture and six days of incubation time. The optimal degradation showed an improvement of chitinase production of 2.46 fold as compared to before optimization using CCD. For partial characterization of chitinase, the optimum temperature and pH are at 60 °C and pH 3.0, respectively. Chitinase retained 72% of its activity at 70 °C. However, the loss of the chitinase activity occurred after 60 minutes of incubation at 70 °C and 80 °C with residual activity are 48% and 28%, respectively. Chitinase was more stable in acidic than in alkaline pH. The molecular weight of chitinase was 50 and 42 kDa for endochitinase, 33 and 25 kDa for eksokitinase and 18 kDa for protease. Extraction of crude chitinase from Trichoderma virens can inhibit the growth of Ganoderma boninense
Polyvinyl alcohol-grafted-multiwalled carbon nanotubes as a delivery system for curcumin in H2O2-induced damaged neuroblastoma SH-SY5Y cells
(Universiti Teknologi Malaysia, 2017) Zawawi, Nurliyana Ahmad
Treating neurodegenerative disease using Curcumin, a pigment from turmeric is found difficult due to its low bioavailability. To overcome this problem, polyvinyl alcohol multi-walled carbon nanotubes (PVA-MWCNT) was developed to improve its delivery and uptake by the brain cells. It was first prepared by oxidizing pristine MWCNT (p-MWCNT) in 3:1 sulfuric and nitric acid mixture. Three methods were employed to optimize production of oxidized MWCNT (ox-MWCNT); which is stirring and sonication for 2 and 6 hours. The selected ox-MWCNT with minimal structural damage was then functionalized with PVA via carbodiimide esterification, and confirmed by field-emission scanning electron microscopy (FESEM), Fourier transform infra-red (FTIR) spectroscopy, dispersion test and thermal gravimetric analysis (TGA). Next, Curcumin was loaded onto PVA-MWCNT, p-MWCNT and ox-MWCNT, and evaluated their adsorption capacity and behaviour using adsorption kinetics, isotherm and thermodynamic studies. Percentage of Curcumin desorbed from the MWCNT was analyzed in physiological buffers of pHs 7.4 and 5.5. Lastly, potential of Curcumin loaded on PVA-MWCNT (Cur-PVA-MWCNT) to protect neurons was screened in neuroblastoma SH-SY5Y cells, including other Cur-loaded MWCNT samples. The cells were pre-incubated with hydrogen peroxide (H2O2) at half the maximal inhibitory concentration (IC50) for 1 hour, before concurrent treatment of the samples. Cell survival was compared to controls treated with Curcumin-unloaded MWCNT, i.e. PVA-MWCNT, ox-MWCNT and p-MWCNT. From the results, MWCNT was oxidized with minimal structural damage using stirring method. The evidence of PVA grafting was confirmed through the presence of matrix polymer embedded on ox-MWCNT in FESEM, high stability in water, identification C=O stretching of ester group at 1736 cm-1 in FTIR and its stable structure compared to ox-MWCNT and p-MWCNT in TGA. PVA-MWCNT adsorbed Curcumin at only 5.1 mg/g, which follows the Freundlich isotherm model (physisorption), while the highest amount was loaded on ox-MWCNT at 714 mg/g that follows the Langmuir model (chemisorption). Although Curcumin adsorption on PVA-MWCNT was only at minimal amount, it showed the most efficient desorption occurred at pH 5.5 (25%) rather than pH 7.4 (3%) with sustained release over a 3-day incubation. This suggests Curcumin weak binding through physisorption to the PVAMWCNT facilitated its release at lower pH. Cur-PVA-MWCNT also protected SHSY5Y cells from H2O2-induced oxidative stress most significantly at 100 ng/ml, 1 ìg/ml and 10 ìg/ml compared to PVA-MWCNT. Cur-ox-MWCNT and Cur-p- MWCNT indicated no obvious difference as compared to their controls. The change in the cell environment after damage perhaps encouraged the pH to become acidic which may facilitate Curcumin release from PVA-MWCNT. Overall, PVA-MWCNT was considered promising for loading and the release of Curcumin. The efficacy of the system in in vitro cell lines was also enhanced, demonstrating it as a prospective carrier for Curcumin in the treatment of neurodegenerative disease.

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