The bacterial phosphotriesterase (PTE) from Pseudomonas diminuta possess very broad substrate specificity for organophosphorus compounds. It is capable of hydrolyzing several insecticides including paraoxon and various chemical warfare agents such as sarin (GB), soman (GD), cyclosarin (GF) and VX. The catalytic ability of PTE for the hydrolysis of paraoxon is close to the limit of diffusion of the reactant in solution. However, the catalytic activity of PTE for the organophosphate nerve agents is lower than that for paraoxon. It was reported that the wild-type PTE preferentially catalyze the hydrolysis of the less toxic Rp- enantiomers of organophosphate nerve agents and their analogues than the more toxic Sp- enantiomers. The first generation of PTE mutants that contains a modified substrate binding pocket was identified and it was observed that their catalytic activity towards the more toxic Sp- enantiomers organophosphate nerve agent analogues was enhanced. The H254G/H257W/L303T mutant was shown to have a reversed stereoselectivity. The kcat/Km values of this mutant towards the hydrolysis of the SpRc- and SpSc-enantiomers of the GD analogue and the Sp-enantiomer of the GF analogue were enhanced by 73-, 543-, and 1340-fold relatively to the wile-type enzyme, respectively. The second generation of PTE mutants were isolated and shown to have higher activity toward the Sp-enantiomers of the GD and GF analogues than the first generation mutants. Saturation mutagenesis, in vitro screening and in vivo selection were conducted using the gene for the mutants from the first generation. The GWT-d3 mutant was identified as the most active PTE mutant towards the hydrolysis of the Spenantiomers of the GD analogue, the kcat/Km values were 780- and 3530-fold higher than the wild-type enzyme toward the SpRc- and SpSc-enantiomers of the GD analogues. The GWT-f5 mutant was the best PTE mutant towards the Sp-enantiomer of the GF analogue, the kcat/Km values were 15500-fold higher than the wild-type enzyme. The X-ray crystal structures of the wild-type PTE and the G60A mutant were determined in the presence of the hydrolysis product diethyl phosphate and a product analogue cacodylate, respectively. This result supports the reaction mechanism previously proposed by Dr. Sarah Aubert.

Phosphotriesters, also known as organophosphates (OP), represent a class of toxic compounds first synthesized in Germany. Enzymatic removal of harmful insecticides and breakdown products is a promising alternative to skimming or dredging. Wild type bacterial phosphotriesterase (PTE) was screened against 7 agricultural organophosphates: coumaphos, chlorpyrifos, fenitrothion, temephos, profenofos, pirimiphosmethyl and diazinon. The initial results laid the groundwork for a mutagenesis study to investigate the determining factors in enzyme reactivity. Coumaphos is hydrolyzed more efficiently than any other target by the wild type cobalt enzyme (kcat/Km = 2 x 10^7 M^-1s^-1). Coumaphos, fenitrothion and chlorpyrifos had the lowest Km values from the initial screen and were targets for steady state kinetic characterization of active site mutants. Site directed mutagenesis of binding sites was conducted and the most reactive point mutants, F132G, F132V and S308G, were used as backgrounds for subsequent mutation. Seven active site double mutants: F132G/S308G, F132G/S308T, F132V/S308G, F132V/S308T, F132G/I106T, F132V/I106T and G308/W309 were purified to homogeneity for kinetic characterization. The double mutant G308/F132V enhanced chlorpyrifos reactivity relative to the wild type enzyme. This enhancement of reactivity is proposed to result from conformational rearrangement following substrate bond hydrolysis.

Phosphotriesterase (PTE) from Pseudomonas diminuta is a zinc metalloenzyme found in soil bacteria capable of organophosphate hydrolysis at rates approaching the diffusion controlled limit. Interest in PTE for degradation of chemical warfare agents and disposal of pesticides supports the need to understand the mechanism by which it performs hydrolysis. For further mechanistic clarity, this work will provide direct confirmation of the solvent bridge identity and the protonated species resulting in loss of catalytic identity. Inhibitor and product binding to the metal center will also be addressed; as well as the evaluation of the catalytic activity of Fe(II)-substituted PTE. This work has determined that the Mn/Mn-PTE electron paramagnetic resonance (EPR) spectrum exhibits exchange coupling that is facilitated through a hydroxide bridge. Protonation of the bridging hydroxide results in the loss of the exchange coupling between the two divalent cations and the loss of catalytic activity. The reversible protonation of the bridging hydroxide has an apparent pKa of 7.3 based upon changes in the EPR spectrum of Mn/Mn-PTE with alterations in pH. The pH-rate profile for the hydrolysis of paraoxon by Mn/Mn-PTE shows the requirement of a single function group that must be unprotonated with a pKa of 7.1. The comparable pKa values are proposed to result from the protonation of the same ionizable species. The effects of inhibitor and product binding on the magnetic properties of the metal center and the hydroxyl bridge are investigated by accessing new EPR spectral features. This work concludes that the binding of inhibitor occurs at the metal center and results in an increase of non-bridged hydroxyl species. These results, in conjunction with kinetic and crystallographic data, suggest that substrate binding via the phosphoryl oxygen at the ?-metal weakens the hydroxyl bridge coordination to the ?-metal. This loss of coordination would increase the nucleophilic character of the bridge, and binding of the substrate to the metal center would result in a stronger nucleophile for hydrolysis. Lastly, Fe(II) binding and activation of apoenzyme is evaluated under anaerobic conditions. This work concludes Fe/Fe-PTE is not catalytically active, but can bind up to 2 equivalent Fe(II) ions per active site.

Greenhouse food production has intensive fertilizer practices, and it leads to contamination in the soil and food chains. This study investigated the potentially toxic elements (As, Pb, Zn, Cd, Cr, Cu, and Ni) (PTE) in agriculture soil and their bioaccumulation by dragon fruit and banana. Different fertility tests are conducted to see if there is a correlation between PTEs and fertilizers. CHNS/O analyzer was used to determine organic carbon and nitrogen concentration in the soil. Besides, organic and inorganic phosphorus amounts were analyzed to detect the amount of fertilizer input. Elemental analysis was conducted with ICP-MS. The amount of N, P, and K and PTEs compared and discussed to source the PTEs. Excessive fertilizer practices were acquired, and possible reasons and results were discussed. Besides, the investigations have revealed that concentrations of potentially toxic elements in greenhouse and open-field farms in Gazipaşa/Turkey are below the environmental standards in the soil and fruit samples. Organic farming practices are more dominant than inorganic farming practices in the sampling stations. / Seralarda yapılan gıda üretimi yoğun gübre uygulamalarına sahip olup hem toprakta hem de besin zincirlerinde kontaminasyona yol açmaktadır. Bu çalışma, tarım toprağındaki potansiyel olarak toksik elementleri (As, Pb, Zn, Cd, Cr, Cu ve Ni) (PTE) ve bunların ejder meyvesinde ve muzda olan biyolojik birikimlerini araştırmıştır. PTE'ler ve gübreler arasında bir korelasyon olup olmadığını görmek için farklı verimlilik testleri yapılmıştır. Topraktaki organik karbon ve azot konsantrasyonunu belirlemek için CHNS/O analiz cihazı kullanılmıştır. Ayrıca gübre girdi miktarının tespiti için organik ve inorganik fosfor miktarları analiz edilmiştir. Özellikle PTE tespiti için ICP-MS ile element analizi yapılmıştır. N, P ve K ve PTE'lerin miktarı, PTE'lerin kaynağını tespit edebilmek için karşılaştırıldı ve tartışıldı. Aşırı gübre kullanımı tespit edildi. Bu durumun olası nedenleri ve sonuçları tartışıldı. Ayrıca yapılan araştırmalar, Gazipaşa’daki sera ve açık tarla çiftliklerinde potansiyel toksik element konsantrasyonlarının toprak ve meyve örneklerinde çevre standartlarının altında olduğunu ortaya koymuştur. Örnekleme istasyonlarında organik tarım uygulamalarının inorganik tarım uygulamalarına göre daha baskın olduğu da tespit edilmiştir.

For widespread use of biochar in agriculture and horticulture, it must be ensured that application will neither adversely affect soil and plants, nor exceed legislated contaminant concentrations. The most relevant groups of contaminants in biochar are potentially toxic elements (PTEs), polycyclic aromatic hydrocarbons (PAHs) and volatile organic compounds (VOC). In this thesis, the concentrations of these groups of contaminants were analysed in 90 different biochars produced by slow pyrolysis. Subsequently, the concentrations were compared to legislation/guideline threshold values and linked to production conditions. The risk these contaminants pose to plant growth was also assessed, to give recommendations on production of safe biochar. PTEs can neither be formed nor destroyed, which means their presence in biochar is predominantly determined by feedstock type. However, significant levels of Cr, Fe and Ni were introduced into biochar from the furnace steel, whilst PTEs with low boiling points, such as As, Cd and Zn, partially evaporated during pyrolysis. PTEs were not responsible for phytotoxic effects observed for PTE-rich biochars despite biochar’s exceedance of available and total PTE threshold values for soil and soil amendments. Although initial tests were promising, the risk that PTE-rich biochars as amendment for soil and growing media pose, needs further investigation. The PAH concentration in biochar was markedly reduced by increasing carrier gas flow rate, and the type of feedstock also influenced the PAH content. However, there was no clear dependence of pyrolysis temperature on PAH concentrations, which was attributed to PAHs being increasingly formed and evaporated at higher pyrolysis temperatures. Ultimately, condensation of pyrolysis vapours and deposition on biochar was identified as the main risk for biochar contamination with PAHs, as this resulted in elevated concentrations of high-risk, higher molecular weight PAHs. Weaknesses in the pyrolysis unit design, such as cold zones, resulted in elevated concentrations of VOCs, as well as PAHs, in biochar. Comparing concentrations and phytotoxic potential of both compound groups, it was concluded that observed toxic effects were much more likely caused by VOCs in biochars containing both contaminants. Overall, formation of VOCs and PAHs cannot be prevented, but their presence in biochar resulting from retention and deposition can be minimised.

La gestion des connaissances dans les petites et moyennes entreprises a toujours été un défi. Ces entreprises ont des caractéristiques particulières qui sont liés à la taille, la structure et la coordination et la collaboration de leurs membres. L’enjeu scientifique de ce travail est de mieux appréhender les spécificités de la gestion des connaissances et de l’apprentissage organisationnel dans ces petites entreprises. / Knowledge management in small and medium enterprises has always been a challenge. These companies have special features that are related to the size, structure and coordination and cooperation between members. The scientific challenge of this work is to better understand the specifics of knowledge management and organizational leaning in these small businesses.

Data processing for Big Data plays a vital role for decision-makers in organizations and government, enhances the user experience, and provides quality results in prediction analysis. However, many modern data processing solutions make a significant investment in hardware and maintenance costs, such as Hadoop and Spark, often neglecting the well established and widely used relational database management systems (RDBMS's). In this dissertation, we study three fundamental graph problems in RDBMS. The first problem we tackle is computing shortest paths (SP) from a source to a target in large network graphs. We explore SQL based solutions and leverage the intelligent scheduling that a RDBMS performs when executing set-at-a-time expansions of graph vertices, which is in contrast to vertex-at-a-time expansions in classical SP algorithms. Our algorithms perform orders of magnitude faster than baselines and outperform counterparts in native graph databases. Second, we studied the PageRank problem which is vital in Google Search and social network analysis to determine how to sort search results and identify important nodes in a graph. PageRank is an iterative algorithm which imposes challenges when implementing it over large graphs. We study computing PageRank using RDBMS for very large graphs using a consumer-grade machine and compare the results to a dedicated graph database. We show that our RDBMS solution is able to process graphs of more than a billion edges in few minutes, whereas native graph databases fail to handle graphs of much smaller sizes. Last, we present a carefully engineered RDBMS solution to the problem of triangle enumeration for very large graphs. We show that RDBMS's are suitable tools for enumerating billions of triangles in billion-scale networks on a consumer grade machine. Also, we compare our RDBMS solution's performance to a native graph database and show that our RDBMS solution outperforms by orders of magnitude. / Graduate

This thesis investigates improved estimators of the parameters of the linear regression models with normal errors, under sample and non-sample prior information about the value of the parameters. The estimators considered are the unrestricted estimator (UE), restricted estimator (RE), shrinkage restricted estimator (SRE), preliminary test estimator (PTE), shrinkage preliminary test estimator (SPTE), and shrinkage estimator (SE). The performances of the estimators are investigated with respect to bias, squared error and linex loss. For the analyses of the risk functions of the estimators, analytical, graphical and numerical procedures are adopted. In Part I the SRE, SPTE and SE of the slope and intercept parameters of the simple linear regression model are considered. The performances of the estimators are investigated with respect to their biases and mean square errors. The efficiencies of the SRE, SPTE and SE relative to the UE are obtained. It is revealed that under certain conditions, SE outperforms the other estimators considered in this thesis. In Part II in addition to the likelihood ratio (LR) test, the Wald (W) and Lagrange multiplier (LM) tests are used to define the SPTE and SE of the parameter vector of the multiple linear regression model with normal errors. Moreover, the modified and size-corrected W, LR and LM tests are used in the definition of SPTE. It is revealed that a great deal of conflict exists among the quadratic biases (QB) and quadratic risks (QR) of the SPTEs under the three original tests. The use of the modified tests reduces the conflict among the QRs, but not among the QBs. However, the use of the size-corrected tests in the definition of the SPTE almost eliminates the conflict among both QBs and QRs. It is also revealed that there is a great deal of conflict among the performances of the SEs when the three original tests are used as the preliminary test statistics. With respect to quadratic bias, the W test statistic based SE outperforms that based on the LR and LM test statistics. However, with respect to the QR criterion, the LM test statistic based SE outperforms the W and LM test statistics based SEs, under certain conditions. In Part III the performance of the PTE of the slope parameter of the simple linear regression model is investigated under the linex loss function. This is motivated by increasing criticism of the squared error loss function for its inappropriateness in many real life situations where underestimation of a parameter is more serious than its overestimation or vice-versa. It is revealed that under the linex loss function the PTE outperforms the UE if the nonsample prior information about the value of the parameter is not too far from its true value. Like the linex loss function, the risk function of the PTE is also asymmetric. However, if the magnitude of the scale parameter of the linex loss is very small, the risk of the PTE is nearly symmetric.

This dissertation is particularly focused on a novel approach of a wirelessly powered neuromodulation system for chronic patients. The inductively coupled transmitter (TX) and receiver (RX) coils are designed through optimization to achieve maximum efficiency. A power management unit (PMU) consisting of a voltage rectifier, voltage regulator along with a stimulation circuitry is also designed to provide pulse stimulation to genetically modified neurons. For continuous health monitoring purposes, the response from the brain due to stimulation needs to be recorded and transmitted wirelessly outside the brain for analysis. A low-power high-data duty-cycled impulse-radio ultra-wideband (IR-UWB) transmitter is designed and implemented using the standard CMOS process. Another focus of this dissertation is the design of a reverse electrowetting-on-dielectric (REWOD) based energy harvesting circuit for wearable sensor applications which is capable of generating a very low-frequency signal from motion activity such a walking, running, jogging, etc. A commercial off-the-shelf (COTS) based and on-chip based energy harvesting circuit is designed for very low-frequency signals. The experimental results show promising progress towards the advancement in the wirelessly powered neuromodulation system and building the self-powered wearable sensor.

This thesis describes the characterization of SsoPox, a lactonase with promiscuous phosphotriesterase activity from the hyperthermophilic archaeon, Sulfolobus solfataricus, and the potential of the phosphotriesterase from Brevundimonas diminuta (PTEBd) to function as an organophosphate sensor. Arg-223 and Tyr-99 of SsoPox are not essential for lactonase activity, however substitution of a phenylalanine in place of Tyr-97 abolished lactonase activity while reducing paraoxonase activity by 20-fold. Substrate specificity of SsoPox can be modulated through the partial blockage of the hydrophobic binding tunnel adjacent to the active site. The specificity constant for N-(3-oxo-decanoyl)-L-homoserine lactone decreased 37-fold when a phenylalanine was introduced in place of Leu-226. PTEBd was expressed and purified from Pseudomonas putida and, like SsoPox, can be immobilized to Disruptor paper. The immobilized enzyme can be used to detect five organophosphates at concentrations as low as 50 μM. Incubation of PTEBd-immobilized sensors at different temperatures proved that the enzyme is stable for at least 40 days at 23.5 degrees Celsius without any detectable change in activity.