Orthologous and heterologous expression of a protein from Saccharomyces cerevisiae: Optimization for a biotechnological application

In this thesis (TFG) the results of the comparison between different methods to obtain a recombinant protein, by orthologous and heterologous expression, are exposed. This study will help us to identify the best way to express and purify a recombinant protein that will be used for biotechnology applications. In the first part of the project the goal was to find the best expression and purification system to obtain the recombinant protein of interest. To achieve this objective, a system expression in bacteria and in yeast was designed. The DNA was cloned into two different expression vectors to create a fusion protein with two different tags, and the expression of the protein was induced by IPTG or glucose. Additionally, in yeast, two promoters where used to express the protein, the one corresponding to the same protein (orthologous expression), and the ENO2 promoter (heterologous expression). The protein of interest is a NAD-dependent enzyme so, in a second time, its specific activity was evaluated by coenzyme conversion. The results of the TFG suggest that, comparing the model organisms, bacteria are more efficient than yeast because the quantity of protein obtained is higher and better purified. Regarding yeast, comparing the two expression mechanisms that were designed, heterologous expression works much better than the orthologous expression, so in case that we want to use yeast as expression model for the protein of interest, ENO2 will be the best option. Finally, the enzymatic assays, done to compare the effectiveness of the different expression mechanisms respect to the protein activity, revealed that the protein purified in yeast had more activity in converting the NAD coenzyme.

Analyzing Carbon Trade Permits in Optimization Framework

The threats caused by global warming motivate different stake holders to deal with and control them. This Master's thesis focuses on analyzing carbon trade permits in optimization framework. The studied model determines optimal emission and uncertainty levels which minimize the total cost. Research questions are formulated and answered by using different optimization tools. The model is developed and calibrated by using available consistent data in the area of carbon emission technology and control. Data and some basic modeling assumptions were extracted from reports and existing literatures. The data collected from the countries in the Kyoto treaty are used to estimate the cost functions. Theory and methods of constrained optimization are briefly presented. A two-level optimization problem (individual and between the parties) is analyzed by using several optimization methods. The combined cost optimization between the parties leads into multivariate model and calls for advanced techniques. Lagrangian, Sequential Quadratic Programming and Differential Evolution (DE) algorithm are referred to. The role of inherent measurement uncertainty in the monitoring of emissions is discussed. We briefly investigate an approach where emission uncertainty would be described in stochastic framework. MATLAB software has been used to provide visualizations including the relationship between decision variables and objective function values. Interpretations in the context of carbon trading were briefly presented. Suggestions for future work are given in stochastic modeling, emission trading and coupled analysis of energy prices and carbon permits.

Ascertaining Key Factors Behind the Coexistence of the Native Ant Species Plagiolepis pygmaea with the Invasive Argentine Ant Linepithema humile (Hymenoptera: Formicidae)

The Argentine ant, Linepithema humile, is a world-wide invasive ant species. Its presence has a strong negative impact on ant diversity. The present study attempts to highlight the reasons for the coexistence of this highly dominant species with Plagiolepis pygmaea, the only native ant species that has proved able to resist the invasion in a natural ecosystem in the north-east of the Iberian Peninsula. To quantify the aggressiveness level of both species we performed aggressiveness tests on workers in different areas: a) Argentine ant workers from areas with P. pygmaea, b) Argentine ant workers from areas without P. pygmaea, c) P. pygmaea from a non-invaded area and d) P. pygmaea from an invaded area. We also confronted Argentine ant workers with P. pallidula and T. nigerrimum. These aggressiveness tests showed that the coexistence of these two species of ants was not due to a habituation process, since the aggressiveness level observed between the four kinds of confrontations were fairly similar. We also found a lack of aggressiveness between Argentine ant workers and P. pygmaea, and highly submissive behavior in the latter when confronted with the invader. The peaceful character of P. pygmaea together with its markedly submissive behavior may be the main factors behind the coexistence of these species in the study area

Effect of Seasonal Dynamics on Queen Densities of the Argentine Ant (Linepithema Humile) in an Invaded Natural Area of the NE Iberian Peninsula

The annual elimination of large numbers of Argentine ant queens near the advance front of an invasion could be a useful tool for weakening the species’ dispersion and, therefore, limiting its establishment in non-invaded areas. However, before carrying out trials to test the effectiveness of this method it would be essential to have sufficient knowledge of the effect of seasonal dynamics acting on the queens’ densities of the species in order to determine the most favourable period of the year to act. We analyzed the seasonal densities and nest dynamics of Argentine ant queens in an invaded Mediterranean natural ecosystem. We observed that the queens’ density varied depending on the season of the year and that this variation was mainly due to the seasonal dynamics of nest aggregations in winter and ant dispersions in summer. The greatest densities per litre of nest soil were observed in winter (December to March, approximately) and the lowest densities were observed in summer ( June to July). This information is essential for improving current knowledge of the Argentine ant’s biology and developing control methods based on the elimination of queens in invaded natural areas

Numerical Dominance of the Argentine Ant vs Native Ants and Consequences on Soil Resource Searching in Mediterranean Cork-Oak Forests (Hymenoptera: Formicidae)

This study is focused on the dominance exerted by the invasive Argentine ant over native ants in a coastal Mediterranean area. Theimpact of this invasive ant on native ant assemblages and its consequences on total ant biomass and on the intensity of habitat explorationwere evaluated. Foraging ants were observed and their trajectories recorded during 5-minute periods in two study zones, one invaded andthe other non-invaded. Ant species detected, ant worker abundance, ant biomass and the intensity of soil surface searching done by antswere compared between the two zones. The Argentine ant invasion provoked a drastic reduction of the ant species richness. Apparentlyonly one native ant species is able to coexist with the Argentine ant, the cryptic Plagiolepis pygmaea. Ant worker abundance was also modified after the invasion: the number of Argentine ant workers detected, which represented 92% of the invaded zone, was two times higher than the number of native ant workers detected in the non-invaded zone. The total ant biomass was inversely affected, becoming four times lower in the invaded zone highly dominated by Linepithema humile. The higher number of Argentine ant workers and their fast tempo of activity implied an alteration of the intensity of soil surface searching: scanning by the Argentine ants in the invaded zone was higher than that done by the native ants in the non-invaded zone, and the estimated time for a complete soil surface scan was 64 minutes in the invaded zone and 108 minutes in the non-invaded zone. Consequently, resources will be discovered faster by ants in the invaded zone than in the non-invaded zone. The increase of the mean temperature and the decrease of the relative humidity from May to August reduced the ant activity in the two study zones but this reduction was greater in the invaded zone

Reduction of the Ant Mandible Gap Range After Biotic Homogenization Caused by an Ant Invasion (Hymenoptera: Formicidae)

After most of the native ant species are displaced by the Argentine ant invasion, it is probable that some ecological processes carried out by natives are not replaced. In some cases this could be due to a morphological difference between the Argentine ant and the displaced native ants. The significant decrease in ant richness after the invasion (only two species detected in the invaded zones vs. 25 species in surrounding non-invaded zones) implies a drastic reduction in the ant mandible gap range (the mandible gap spectra of all the ant species in a community) in the invaded zones. This reduction could explain why some roles that were previously carried out by the displaced native species are not performed by the invasive species. This could be due to a functional inability to carry out these activities. The mandible gap waspositively correlated with the ant body mass in the 26 ant species considered. The functional inability hypothesis could be applied to other invasive ants as well as to the Argentine ant

Swarm Intelligent Selection and Optimization of Machining System Parameters for Microchannel Fabrication in Medical Devices

Current technology trends in medical device industry calls for fabrication of massive arrays of microfeatures such as microchannels on to nonsilicon material substrates with high accuracy, superior precision, and high throughput. Microchannels are typical features used in medical devices for medication dosing into the human body, analyzing DNA arrays or cell cultures. In this study, the capabilities of machining systems for micro-end milling have been evaluated by conducting experiments, regression modeling, and response surface methodology. In machining experiments by using micromilling, arrays of microchannels are fabricated on aluminium and titanium plates, and the feature size and accuracy (width and depth) and surface roughness are measured. Multicriteria decision making for material and process parameters selection for desired accuracy is investigated by using particle swarm optimization (PSO) method, which is an evolutionary computation method inspired by genetic algorithms (GA). Appropriate regression models are utilized within the PSO and optimum selection of micromilling parameters; microchannel feature accuracy and surface roughness are performed. An analysis for optimal micromachining parameters in decision variable space is also conducted. This study demonstrates the advantages of evolutionary computing algorithms in micromilling decision making and process optimization investigations and can be expanded to other applications

Optimization of a heatset dryer

The objective of the thesis was to examine the possibilities in designing better performing nozzles for the heatset drying oven in Forest Pilot Center. To achieve the objective, two predesigned nozzle types along with the replicas of the current nozzles in the heatset drying oven were tested on a pilot-scale dryer. During the runnability trials, the pilot dryer was installed between the last printing unit and the drying oven. The two sets of predesigned nozzles were consecutively installed in the dryer. Four web tension values and four different impingement air velocities were used and the web behavior during the trial points was evaluated and recorded. The runnability in all trial conditions was adequate or even good. During the heat transfer trials, each nozzle type was tested on at least two different nozzle-to-surface distances and four different impingement air velocities. In a test situation, an aluminum plate fitted with thermocouples was set below a nozzle and the temperature measurement of each block was logged. From the measurements, a heat transfer coefficient profile for the nozzle was calculated. The performance of each nozzle type in tested conditions could now be rated and compared. The results verified that the predesigned simpler nozzles were better than the replicas. For runnability reasons, there were rows of inclined orifices on the leading and trailing edges of the current nozzles. They were believed to deteriorate the overall performance of the nozzle, and trials were conducted to test this hypothesis. The perpendicular orifices and inclined orifices of a replica nozzle were consecutively taped shut and the performance of the modified nozzles was measured as before, and then compared to the performance of the whole nozzle. It was found out, that after a certain nozzle-to-surface distance the jets from the two nozzles would collide, which deteriorates the heat transfer.

Optimization of energy in a pulp mill

This study presents examination of ways to increase power generation in pulp mills. The main purpose was to identify and verify the best ways of power generation growth. The literature part of this study presented operation of energy pulp mill departments, energy consumption and generation by the recovery and power boilers. The second chapter of this part described the main directions for increase of electricity generation rise of black liquor dry solid content, increase of main steam parameters, flue gas heat recovery technologies, feed water and combustion air preheating. The third chapter of the literature part presented possible technical, environment and corrosion risks appeared from described alternatives. In the experimental part of this study, calculations and results of possible models with alternatives was presented. The possible combinations of alternatives were generated in 44 `models of energy pulp mill. The target of this part was define extra electricity generation after alternatives using and estimate profitability of generated models. The calculations were made by computer programme PROSIM. In the conclusions, the results were estimated on the basis of extra electricity generation and equipment design data of models. The profitability of cases was verified by their payback periods and additional incomes.

Isolation of compounds attractive to the leaf-cutting ant Atta sexdens rubropilosa forel (hymenoptera: Formicidae) from Mabea fistulifera elaiosome

Mabea fistulifera (Euphorbiaceae)is a pioneer plant species with seeds dispersed by the ant Atta sexdens rubropilosa. Since the ants are attracted to the seeds to use its elaiosome as a source of energy, we investigated its composition. The elaiosomes from 13,000 seeds were extracted with a methanol:chloroform mixture (2:1 v/v) and yielded 22% of a residue. This residue was fractionated by column chromatography and its composition determined by infrared spectroscopy and chromatography/mass spectrometry (GC-MS). The elaiosome lipids are constituted mainly by free fatty acids, triacylglycerols and minor quantities of monoacylglycerols or diacylglycerols.

ContinuousMultimodal Global Optimization with Differential Evolution-Based Methods

Metaheuristic methods have become increasingly popular approaches in solving global optimization problems. From a practical viewpoint, it is often desirable to perform multimodal optimization which, enables the search of more than one optimal solution to the task at hand. Population-based metaheuristic methods offer a natural basis for multimodal optimization. The topic has received increasing interest especially in the evolutionary computation community. Several niching approaches have been suggested to allow multimodal optimization using evolutionary algorithms. Most global optimization approaches, including metaheuristics, contain global and local search phases. The requirement to locate several optima sets additional requirements for the design of algorithms to be effective in both respects in the context of multimodal optimization. In this thesis, several different multimodal optimization algorithms are studied in regard to how their implementation in the global and local search phases affect their performance in different problems. The study concentrates especially on variations of the Differential Evolution algorithm and their capabilities in multimodal optimization. To separate the global and local search search phases, three multimodal optimization algorithms are proposed, two of which hybridize the Differential Evolution with a local search method. As the theoretical background behind the operation of metaheuristics is not generally thoroughly understood, the research relies heavily on experimental studies in finding out the properties of different approaches. To achieve reliable experimental information, the experimental environment must be carefully chosen to contain appropriate and adequately varying problems. The available selection of multimodal test problems is, however, rather limited, and no general framework exists. As a part of this thesis, such a framework for generating tunable test functions for evaluating different methods of multimodal optimization experimentally is provided and used for testing the algorithms. The results demonstrate that an efficient local phase is essential for creating efficient multimodal optimization algorithms. Adding a suitable global phase has the potential to boost the performance significantly, but the weak local phase may invalidate the advantages gained from the global phase.

Experimental analysis of optimization techniques on the road passenger transportation problem

Analyzing the state of the art in a given field in order to tackle a new problem is always a mandatory task. Literature provides surveys based on summaries of previous studies, which are often based on theoretical descriptions of the methods. An engineer, however, requires some evidence from experimental evaluations in order to make the appropriate decision when selecting a technique for a problem. This is what we have done in this paper: experimentally analyzed a set of representative state-of-the-art techniques in the problem we are dealing with, namely, the road passenger transportation problem. This is an optimization problem in which drivers should be assigned to transport services, fulfilling some constraints and minimizing some function cost. The experimental results have provided us with good knowledge of the properties of several methods, such as modeling expressiveness, anytime behavior, computational time, memory requirements, parameters, and free downloadable tools. Based on our experience, we are able to choose a technique to solve our problem. We hope that this analysis is also helpful for other engineers facing a similar problem

Optimization of methodology to analyze ascorbic and dehydroascorbic acid in vegetables

In this study, different solutions to extract vitamin C were tested. High-performance liquid chromatography was chosen and the conditions were based on isocratic elution in reverse phase column. Dehydroascorbic acid was determined indirectly after its reduction using dithiothreitol. The use of metaphosphoric acid to stabilize the vitamin C was shown to be required and it was necessary to neutralize the pH of the extract to apply dithiothreitol. The average recovery was 90% in collard and tomato samples. The presence of oil did not interfere in extraction and the methodology can be used to analyze stir fried vegetables.

Optimization of the carrot leaf dehydration aiming at the preservation of omega-3 fatty acids

The carrot leaf dehydration conditions in air circulation oven were optimized through response surface methodology (RSM) for minimizing the degradation of polyunsaturated fatty acids, particularly alpha-linolenic (LNA, 18:3n-3). The optimized leaf drying time and temperature were 43 h and 70 ºC, respectively. The fatty acids (FA) were investigated using gas chromatography equipped with a flame ionization detector and fused silica capillary column; FA were identified with standards and based on equivalent-chain-length. LNA and other FA were quantified against C21:0 internal standard. After dehydration, the amount of LNA, quantified in mg/100 g dry matter of dehydrated carrot leaves, were 984 mg.