Chromatographic Purification of Recombinant Nucleocapsid Protein of Nipah Virus from Escherichia Coli Homogenate

The nucleocapsid protein (NCp) of Nipah virus (NiV) expressed in Escherichia coli (E. coli) is antigenic and immunogenic. NCp-NiV is a potential serological antigen that can be used in the diagnosis of NiV infections. The yield of NCp expressed in E. coli is low due to the proteolytic degradation by host endogenous proteases. Therefore, it is important to inhibit the endogenous proteolytic degradation activity and shorten the protein recovery process to avoid or reduce the action of protease on the recombinant NCp. A method to predict the type of potential protease that attacks the NCp-NiV and its potential cleavage sites in E. coli to enhance the recovery of NCp was developed. A bioinformatics tool, PeptideCutter was used to identify potential protease and its cleavage sites from the amino acid sequences deduced from the published DNA sequence of the NCp-NiV. The predicted proteases were serine proteases, hence, a range of serine protease inhibitors were tested to improve the yield of NCp. The yield of NCp was increased by 2-fold after the phenylmethylsulphonyl fluoride (PMSF) supplementation. The downstream processing of the NCp-NiV from clarified E. coli homogenate was investigated. Two types of preparative chromatographic purification in a packed bed column; immobilised metal affinity chromatography (IMAC) and hydrophobic interaction chromatography (HIC) were studied and compared. A direct recovery of recombinant NCp-NiV from unclarified E. coli homogenate based on EBA chromatography was then developed by using the type of chromatography that can obtain high yield of the NCp with high antigenicity. In the IMAC system, HisTrapTM 6 Fast Flow was applied to purify the recombinant histidine-tagged NCp. A histidine hexamer tag was placed at the C-terminus of the NCp and this enabled the purification of NCp by IMAC system. The optimal binding was achieved at pH 7.5 and superficial velocity of 75 cm/h. The bound NCp was successfully recovered by a stepwise elution with a range of imidazole concentration (50, 150, 300 and 500 mM). The NCp was captured and eluted from an inlet NCp concentration of 0.4 mg/ml in a scale-up IMAC packed bed column of Nickel SepharoseTM 6 Fast Flow with the optimized conditions obtained from the scouting method. The purification of histidine-tagged NCp using IMAC packed bed column has resulted a 68.3% yield and a purification factor of 7.94. In the HIC system, ammonium sulfate precipitation experiment was performed and it showed that 15% saturation of the salt was the most suitable concentration for the binding buffer. Batch binding of the NCp was performed using Sepharose™ 6 Fast Flow adsorbents coupling separately with four different types of ligand; phenyl low substitution, phenyl high substitution, butyl and octyl. The phenyl low substitution ligand was selected for subsequent optimization process due to its highest yield and purity of the NCp achieved from the batch binding experiment. The HIC for purification of the NCp was further scaled up using a 10 cm column packed with phenyl low substitution Sepharose™ adsorbent. A recovering yield of 81% of the NCp with a purification factor of 9.3 was achieved from this scaled-up HIC operation. Hence, the HIC adsorbent was used to capture the NCp in an EBA column due to its higher yield and purity obtained in the third chapter than the IMAC purification in the second chapter of this study. DNase was added to reduce the viscosity of feedstock and improve the axial mixing prior to the loading of the feedstock to the EBA column packed with the StreamlineTM HIC adsorbent charged with phenyl. The addition of glycerol to the washing buffer has reduced the volume of washing buffer applied, and thus reduced the loss of the NCp during washing stage. The dynamic binding capacity at 10% breakthrough of 3.2 mg/g adsorbent was achieved at a linear flow velocity of 178 cm/h, bed expansion of two and viscosity of 3.4 mPas. The adsorbed NCp was eluted with the buffer containing a step gradient of salt concentration. The purification of hydrophobic NCp using HIC-EBA column has resulted an 80% yield and a purification factor of 12.5

Sensitivity of Arthropod and Microbial Communities Associated with Vertebrate Carrion in Response to Delayed Blow Fly Access: Implication for Carrion Ecology and Forensic Entomology

The objectives of this study were to determine the sensitivity of microbial metabolic community profiles, terrestrial and soil arthropod community structures and function, and soil chemistry dynamics associated with carrion experiencing delayed Diptera colonization. Bacterial metabolism profiles indicate a significant difference between carrion with immediate insect access (Control) and carrion with delayed insect colonization for seven days and 14 days (Treatments). In contrast, soil samples demonstrated no significant change in soil microbial metabolic profiles in 2013, but exhibited significant difference in 2014 trial. These results suggest high sensitivity of microbial community function on pig carrion, but a stochastic response in the soil microbial ecosystem. This phenomenon may be due to the significant abiotic change in the temperatures as well as the differences in the amount of precipitation between trials. Soil chemistry profiles were significantly different between Control and Treatment carcasses. Furthermore, significant differences were found between days of decomposition (temporal sensitive) and soil regions (spatial sensitive). Soil nutrients, such as ammonium, phosphate, non-purgeable organic carbon and total nitrogen were sensitive to treatment effects, but nitrate was not. The treatment effects, community divergence, convergence and resilience for aboveground and belowground arthropods depended on trial, sampling methods (sticky traps, pitfall traps, and sweep nets), taxonomic resolutions (Order, Family, and Genus) and ecological indices (richness, Simpson’s diversity, Shannon-Wiener’s diversity, evenness, and effective number of species) tested. In general, soil arthropod (including acari) community structures were sensitive to treatment effects only at the Family level. The total abundance of acari was not significantly different across treatments in all-sampling days. For aboveground arthropod community structure and function trapped by sticky traps, significant differences in treatments were detected at the Order and Genus levels for both pitfall traps and sweep nets. The present study demonstrated that insect succession on carrion by family level is predictable. However, insect succession by genus level demonstrated stochasticity when dealing with disturbances. Hence, both Clementsian and Gleasonian models explained insect succession and scale matters with regards to these ecological phenomena. These data are valuable for a host of applications, such as forensic sciences, disease ecology, and conservation biology

Cooperative Robots

The topic of this master thesis is cooperative robots. The master and slave configuration is to be used for cooperative tasks and the task chosen is to move a flexible beam together. A trajectory is fed to the master robot to move it to the desired positions. The slave robot then follows the movement of the master robot by force control using force measurements from a force sensor. All experiments are performed in the Robotics Laboratory at the Department of Automatic Control at Lund Institute of Technology. The robots used are an ABB IRB 6 robot and an ABB IRB 2000 robot. The ABB IRB 6 robot is assigned to be the master robot and the ABB IRB 2000 robot as the slave robot. The ABB IRB 2000 robot is equipped with a force and torque sensor of type JR3 for force control. The thesis can be divided broadly into three parts. The first part is a background study of the robot systems and kinematics, on path and trajectory generation and also on the various robot force control schemes. The second part is about optimal trajectory generation. The desired path is first expressed in terms of path index in Cartesian space. It is then converted to joint space with variable step size to ensure that the deviation from the original path is kept within a given limit. The optimal trajectory is then generated using Linear Programming. The third part consists of the application of parallel force/motion control schemes to the slave robot to enable it to follow the master robot. The motion controller is simply the builtin controllers of the IRB 2000 robot. The force controllers used are direct force, impedance and admittance controllers. The schemes are first tested in Matlab Simulink where the robots are simulated by a first order system for each joint. The force measurements are generated by a contact model which simulates the contact forces and torques between the gripper of the IRB 2000 robot and the beam. The resulting joint values are then sent to a Java visualization program written in the platform Eclipse

A Study of the Self-Esteem of Delinquent Male Adolescents and the Perceived Degree of their Parents\u27 Child-Rearing Practices

Problem. Many developmental psychologists are concerned about how par­ents\u27 child-rearing practices influence adolescents\u27 self-esteem, for self-esteem provides one of the foundations for the development of personality. Is it possible that parents\u27 child-rearing practices contribute to the development of a low self-esteem which in turn influences behavior? This study was conducted to determine the relationship of self-esteem of delinquent male adolescents to the perceived degree of their parents\u27 child-rearing practices. Method. The study involved 132 delinquent male adolescents who matriculated during the fall semester of 1979 at Starr Commonwealth, Albion, Michigan. The males ranged from 12 to 18 years of age, with 82 whites, 34 blacks, 4 American Indians, 3 Spanish Americans, and 4 others. One hundred six males came from two-parent homes. Two instruments were used to collect data on the self-esteem of delinquent male adolescents and the perceived degree of their parents\u27 child-rearing practices. The Coopersmith Self-Esteem Inventory (SEI) was employed to assess the self-esteem of delinquent male adolescents. The Parent-Child Relations Questionnaire (PCR) was used to investigate the perceived degree of their parents\u27 child-rearing practices. It was hypothesized that a significant correlation exists between self-esteem of delinquent male adolescents and the perceived degree of their parents\u27 child-rearing practices. It was also hypothesized that significant multiple correlations exist between ten parent-child relations variables and each of four self-esteem variables. Zero-order correlations and multiple regression analysis were used to analyze the data. Results. Among the 132 delinquent male adolescents 95 percent of their fathers were employed while 75 percent of their mothers were home­ makers. Low self-esteem was found among these delinquent male adolescents. They perceived their fathers\u27 child-rearing practices as demanding, casual, loving, and tending to give symbolic-love rewards. They perceived their mothers\u27 child-rearing practices to be demanding, loving, and giving of symbolic-love rewards. A significant correlation exists between social self-esteem of these delinquent male adolescents and the perceived degree of their father\u27s demanding child-rearing practices. A significant correlation exists between general self-esteem of delinquent male adolescents and the perceived degree of symbolic- love rewards given by their mothers. A significant correlation exists between school self-esteem of delinquent male adolescents and the per­ ceived degree of symbolic-love punishment given by their mothers. A significant correlation exists between social self-esteem of delinquent male adolescents and the perceived degree of their mothers\u27 casual child-rearing practices. A significant multiple correlation exists between ten parent- child relations (mothers\u27) variables and school self-esteem. Greater self-esteem tends to be related to delinquent male adolescents\u27 perceptions of less demanding, greater symbolic-love reward and symbolic- love punishment child-rearing practices on the part of their mothers. No significant correlations exist between general self-esteem, school self-esteem, social self-esteem, and home self-esteem of delinquent male adolescents and the perceived degree of their parents\u27 protective, rejecting, neglecting, loving, direct-object reward, and direct-object punishment child-rearing practices. No significant multiple correlations exist between ten parent-child relations variables and general, social, and home self-esteem. Conclusion. In conclusion, delinquent male adolescents have a lower self­ esteem in relation to the perceived degree of their parents\u27 child- rearing practices. The low self-esteem of these delinquent adolescents are related to a pattern of varied chiId-rearing practices as perceived by these youth of their fathers and mothers: demanding fathers, and casual mothers, fathers whose chiId-rearing practices are both demanding and casual, and casual and loving

Testing the Rational Expectations Hypothesis on the Retail Trade Sector Using Survey Data from Malaysia

The rational expectations hypothesis states that when people are expecting things to happen, using the available information, the predicted outcomes usually occur. This study utilized survey data provided by the Business Expectations Survey of Limited Companies to test whether forecasts of the Malaysian retail sector, based on gross revenue and capital expenditures, are rational. The empirical evidence illustrates that the decision-makers expectations in the retail sector are biased and too optimistic in forecasting gross revenue and capital expenditures.REH, Unbiasedness, Non-serial Correlation, Weak-form Efficiency

Possible Barriers Affecting Implementation of ISO28000 for the Supply Chain

This paper is objective to examine the barrier of ISO28000 implementation. As more and more supply chain process faced disruption and delay due to accident, importance of supply chain security management attract the concern from top management of organization. ISO 28000 is a standard that released in 2007 which specify requirement for security management system for the supply chain. However, there are still many organization didnt apply for this standard. Therefore, past research results about barrier for related ISO standards are reviewed to assist determine the barrier of ISO 28000 implementation. After evaluate the possible barrier, 16 barriers for ISO28000 implementation is identified and explained

Mycelial growth and germanium uptake by four species of ganoderma

Four Ganoderma species (G. tsugae, G. subamboinense var. laevisporium, A TCC 52419, G. tropicum and G. lucidum) were incubated in liquid medium containing different concentrations of germanium (Ge) for up to 20 days at 28 C. Increasing the Ge concentration of the medium resulted in a gradual decrease in the growth of the fungal mycelium. However, the Ge content in the mycelium increased with increasing Ge concentration. Different species recorded different levels of tolerance towards the Ge. In each case, the optimum concentration of the incorporated Ge in the medium was established as 100 mg/l for both optimal uptake of Ge by the fungal mycelium and optimal mycelial growth

Creating a GUI solver for linear programming models in MATLAB

The concept of linear programming (LP) was developed to find out the best solution among all feasible solutions in an optimization problem. This technique becomes much popular and attains great attention from researchers due to its wide application in engineering, computer science, marketing, military and industries. Nowadays, there are many commercial software that apply this technique to solve optimization problems, e.g. Excel Solver, TORA, AMPL, LINGO and MATLAB. In this paper, we aim to use MATLAB to develop a Graphical User Interface (GUI) solver for LPs, namely LpSolver. The LP methods that will be included in our solver are the simplex method, the Big-M method, the Two-Phase method and the DualSimplex method. We try to make our solver perform calculations in symbolic form so that the result will be free from rounding errors. Besides that, we added a few features such as creating animated 2-D graphs and generating a detailed tableau showing all intermediate iterative results; in which the user can use it to trace the convergence path that leads to the optimal solution. In the later part of this paper we test our solver with a simple classroom sized problem