Ligand-Induced structural changes in TEM‑1 probed by molecular dynamics and relative binding free energy calculations

The TEM family of enzymes has had a crucial impact on the pharmaceutical industry due to their important role in antibiotic resistance. Even with the latest technologies in structural biology and genomics, no 3D structure of a TEM- 1/antibiotic complex is known previous to acylation. Therefore, the comprehension of their capability in acylate antibiotics is based on the protein macromolecular structure uncomplexed. In this work, molecular docking, molecular dynamic simulations, and relative free energy calculations were applied in order to get a comprehensive and thorough analysis of TEM-1/ampicillin and TEM-1/amoxicillin complexes. We described the complexes and analyzed the effect of ligand binding on the overall structure. We clearly demonstrate that the key residues involved in the stability of the ligand (hot-spots) vary with the nature of the ligand. Structural effects such as (i) the distances between interfacial residues (Ser70−Oγ and Lys73−Nζ, Lys73−Nζ and Ser130−Oγ, and Ser70−Oγ−Ser130−Oγ), (ii) side chain rotamer variation (Tyr105 and Glu240), and (iii) the presence of conserved waters can be also influenced by ligand binding. This study supports the hypothesis that TEM-1 suffers structural modifications upon ligand binding.

Maximizing contention-free executions in multiprocessor scheduling

It is widely assumed that scheduling real-time tasks becomes more difficult as their deadlines get shorter. With deadlines shorter, however, tasks potentially compete less with each other for processors, and this could produce more contention-free slots at which the number of competing tasks is smaller than or equal to the number of available processors. This paper presents a policy (called CF policy) that utilizes such contention-free slots effectively. This policy can be employed by any work-conserving, preemptive scheduling algorithm, and we show that any algorithm extended with this policy dominates the original algorithm in terms of schedulability. We also present improved schedulability tests for algorithms that employ this policy, based on the observation that interference from tasks is reduced when their executions are postponed to contention-free slots. Finally, using the properties of the CF policy, we derive a counter-intuitive claim that shortening of task deadlines can help improve schedulability of task systems. We present heuristics that effectively reduce task deadlines for better scheduability without performing any exhaustive search.

Collision-free beacon scheduling mechanisms for IEEE 802.15.4/Zigbee cluster-tree wireless sensor networks

The recently standardized IEEE 802.15.4/Zigbee protocol stack offers great potentials for ubiquitous and pervasive computing, namely for Wireless Sensor Networks (WSNs). However, there are still some open and ambiguous issues that turn its practical use a challenging task. One of those issues is how to build a synchronized multi-hop cluster-tree network, which is quite suitable for QoS support in WSNs. In fact, the current IEEE 802.15.4/Zigbee specifications restrict the synchronization in the beacon-enabled mode (by the generation of periodic beacon frames) to star-based networks, while it supports multi-hop networking using the peer-to-peer mesh topology, but with no synchronization. Even though both specifications mention the possible use of cluster-tree topologies, which combine multi-hop and synchronization features, the description on how to effectively construct such a network topology is missing. This paper tackles this problem, unveils the ambiguities regarding the use of the cluster-tree topology and proposes two collision-free beacon frame scheduling schemes. We strongly believe that the results provided in this paper trigger a significant step towards the practical and efficient use of IEEE 802.15.4/Zigbee cluster-tree networks.

Collision-free prioritized medium access in the presence of hidden nodes without relying on out-of-band signaling

Consider the problem of sharing a wireless channel between a set of computer nodes. Hidden nodes exist and there is no base station. Each computer node hosts a set of sporadic message streams where a message stream releases messages with real-time deadlines. We propose a collision-free wireless medium access control (MAC) protocol which implements staticpriority scheduling. The MAC protocol allows multiple masters and is fully distributed. It neither relies on synchronized clocks nor out-of-band signaling; it is an adaptation to a wireless channel of the dominance protocol used in the CAN bus. But unlike that protocol, our protocol does not require a node having the ability to receive an incoming bit from the channel while transmitting to the channel. Our protocol has the key feature of not only being prioritized and collision-free but also dealing successfully with hidden nodes. This key feature enables schedulability analysis of sporadic message streams in multihop networks.

Collision-free prioritized medium access control in wireless networks with hidden nodes

We propose a collision-free medium access control (MAC) protocol, which implements static-priority scheduling and works in the presence of hidden nodes. The MAC protocol allows multiple masters and is fully distributed; it is an adaptation to a wireless channel of the dominance protocol used in the CAN bus. But unlike that protocol, our protocol does not require a node having the ability to sense the channel while transmitting to the channel. Our protocol is collision-free even in the presence of hidden nodes and it achieves this without synchronized clocks or out-of-band busy tones. In addition, the protocol is designed to ensure that many non-interfering nodes can transmit in parallel and it functions for both broadcast and unicast transmissions.

Penalty fuzzy function for derivative-free optimization

Penalty and Barrier methods are normally used to solve Nonlinear Optimization Problems constrained problems. The problems appear in areas such as engineering and are often characterised by the fact that involved functions (objective and constraints) are non-smooth and/or their derivatives are not know. This means that optimization methods based on derivatives cannot net used. A Java based API was implemented, including only derivative-free optimizationmethods, to solve both constrained and unconstrained problems, which includes Penalty and Barriers methods. In this work a new penalty function, based on Fuzzy Logic, is presented. This function imposes a progressive penalization to solutions that violate the constraints. This means that the function imposes a low penalization when the violation of the constraints is low and a heavy penalisation when the violation is high. The value of the penalization is not known in beforehand, it is the outcome of a fuzzy inference engine. Numerical results comparing the proposed function with two of the classic penalty/barrier functions are presented. Regarding the presented results one can conclude that the prosed penalty function besides being very robust also exhibits a very good performance.

Improvement of a filters method in a derivative free optimization

Constraints nonlinear optimization problems can be solved using penalty or barrier functions. This strategy, based on solving the problems without constraints obtained from the original problem, have shown to be e ective, particularly when used with direct search methods. An alternative to solve the previous problems is the lters method. The lters method introduced by Fletcher and Ley er in 2002, , has been widely used to solve problems of the type mentioned above. These methods use a strategy di erent from the barrier or penalty functions. The previous functions de ne a new one that combine the objective function and the constraints, while the lters method treat optimization problems as a bi-objective problems that minimize the objective function and a function that aggregates the constraints. Motivated by the work of Audet and Dennis in 2004, using lters method with derivative-free algorithms, the authors developed works where other direct search meth- ods were used, combining their potential with the lters method. More recently. In a new variant of these methods was presented, where it some alternative aggregation restrictions for the construction of lters were proposed. This paper presents a variant of the lters method, more robust than the previous ones, that has been implemented with a safeguard procedure where values of the function and constraints are interlinked and not treated completely independently.

Characterisation of noisy speech channels in 2G and 3G mobile networks

As the wireless cellular market reaches competitive levels never seen before, network operators need to focus on maintaining Quality of Service (QoS) a main priority if they wish to attract new subscribers while keeping existing customers satisfied. Speech Quality as perceived by the end user is one major example of a characteristic in constant need of maintenance and improvement. It is in this topic that this Master Thesis project fits in. Making use of an intrusive method of speech quality evaluation, as a means to further study and characterize the performance of speech codecs in second-generation (2G) and third-generation (3G) technologies. Trying to find further correlation between codecs with similar bit rates, along with the exploration of certain transmission parameters which may aid in the assessment of speech quality. Due to some limitations concerning the audio analyzer equipment that was to be employed, a different system for recording the test samples was sought out. Although the new designed system is not standard, after extensive testing and optimization of the system's parameters, final results were found reliable and satisfactory. Tests include a set of high and low bit rate codecs for both 2G and 3G, where values were compared and analysed, leading to the outcome that 3G speech codecs perform better, under the approximately same conditions, when compared with 2G. Reinforcing the idea that 3G is, with no doubt, the best choice if the costumer looks for the best possible listening speech quality. Regarding the transmission parameters chosen for the experiment, the Receiver Quality (RxQual) and Received Energy per Chip to the Power Density Ratio (Ec/N0), these were subject to speech quality correlation tests. Final results of RxQual were compared to those of prior studies from different researchers and, are considered to be of important relevance. Leading to the confirmation of RxQual as a reliable indicator of speech quality. As for Ec/N0, it is not possible to state it as a speech quality indicator however, it shows clear thresholds for which the MOS values decrease significantly. The studied transmission parameters show that they can be used not only for network management purposes but, at the same time, give an expected idea to the communications engineer (or technician) of the end-to-end speech quality consequences. With the conclusion of the work new ideas for future studies come to mind. Considering that the fourth-generation (4G) cellular technologies are now beginning to take an important place in the global market, as the first all-IP network structure, it seems of great relevance that 4G speech quality should be subject of evaluation. Comparing it to 3G, not only in narrowband but also adding wideband scenarios with the most recent standard objective method of speech quality assessment, POLQA. Also, new data found on Ec/N0 tests, justifies further research studies with the intention of validating the assumptions made in this work.

Disposable immunosensor using a simple method for oriented antibody immobilization for label-free real-time detection of an oxidative stress biomarker implicated in cancer diseases

This work proposes a novel approach for a suitable orientation of antibodies (Ab) on an immunosensing platform, applied here to the determination of 8-hydroxy-2′-deoxyguanosine (8OHdG), a biomarker of oxidative stress that has been associated to chronic diseases, such as cancer. The anti-8OHdG was bound to an amine modified gold support through its Fc region after activation of its carboxylic functions. Non-oriented approaches of Ab binding to the platform were tested in parallel, in order to show that the presented methodology favored Ab/Ag affinity and immunodetection of the antigen. The immunosensor design was evaluated by quartz-crystal microbalance with dissipation, atomic force microscopy, electrochemical impedance spectroscopy (EIS) and square-wave voltammetry. EIS was also a suitable technique to follow the analytical behavior of the device against 8OHdG. The affinity binding between 8OHdG and the antibody immobilized in the gold modified platform increased the charge transfer resistance across the electrochemical set-up. The observed behavior was linear from 0.02 to 7.0 ng/mL of 8OHdG concentrations. The interference from glucose, urea and creatinine was found negligible. An attempt of application to synthetic samples was also successfully conducted. Overall, the presented approach enabled the production of suitably oriented Abs over a gold platform by means of a much simpler process than other oriented-Ab binding approaches described in the literature, as far as we know, and was successful in terms of analytical features and sample application.

Chitosan/AuNPs Modified Graphene Electrochemical Sensor for Label-Free Human Chorionic Gonadotropin Detection

A new immunosensor is presented for human chorionic gonadotropin (hCG), made by electrodepositing chitosan/gold-nanoparticles over graphene screen-printed electrode (SPE). The antibody was covalently bound to CS via its Fc-terminal. The assembly was controlled by electrochemical Impedance Spectroscopy (EIS) and followed by Fourier Transformed Infrared (FTIR). The hCG-immunosensor displayed linear response against the logarithm-hCG concentration for 0.1–25 ng/mL with limit of detection of 0.016 ng/mL. High selectivity was observed in blank urine and successful detection of hCG was also achieved in spiked samples of real urine from pregnant woman. The immunosensor showed good detection capability, simplicity of fabrication, low-cost, high sensitivity and selectivity.

Disposable immunosensor with simple antibody orientation for label-free real-time detection of a cancer biomarker

This work proposes a novel approach for a suitable orientation of antibodies (Ab) on an immunosensing platform, applied here to the determination of 8-hydroxy-2’-deoxyguanosine (8OHdG), a biomarker of oxidative stress that has been associated to chronic diseases, such as cancer. The Anti-8OHdG was bound to an amine modified gold support through its Fc region after activation of its carboxylic functions. Non-oriented approaches of Ab binding to the platform were tested in parallel, in order to show that the presented proposal favored Ab/Ag affinity. The immunosensor design was evaluated by Quartz-Crystal microbalance with Dissipation, Atomic Force Microscopy, Electrochemical Impedance Spectroscopy (EIS) and Square-Wave Voltammetry. EIS was also a suitable technique to follow the analytical behavior of the device against 8OHdG. The affinity binding between 8OHdG and the antibody immobilized in the gold modified platform increased the charged transfer resistance across the electrochemical sep-up. The observed behavior was linear from 0.02 to 7.0 ng/mL of 8OHdG concentrations. The interference from Glucose, Urea and Creatinine was found negligible. An attempt of application to synthetic samples was also successfully conducted. Overall, the presented approach enabled the production of suitably oriented Abs over a gold platform by means of a much simpler process than other oriented-Ab binding approaches described in the literature, as far as we know, and was successful in terms of analytical features and sample application.

Label-free human chorionic gonadotropin detection at picogram levels using oriented antibodies bound to graphene screen-printed electrodes

Human chorionic gonadotropin (hCG) is a key diagnostic marker of pregnancy and an important biomarker for cancers in the prostate, ovaries and bladder and therefore of great importance in diagnosis. For this purpose, a new immunosensor of screen-printed electrodes (SPEs) is presented here. The device was fabricated by introducing a polyaniline (PANI) conductive layer, via in situ electropolymerization of aniline, onto a screen-printed graphene support. The PANI-coated graphene acts as the working electrode of a three terminal electrochemical sensor. The working electrode is functionalised with anti-hCG, by means of a simple process that enabled oriented antibody binding to the PANI layer. The antibody was attached to PANI following activation of the –COOH group at the Fc terminal. Functionalisation of the electrode was analysed and optimized using Electrochemical Impedance Spectroscopy (EIS). Chemical modification of the surface was characterised using Fourier transform infrared, and Raman spectroscopy with confocal microscopy. The graphene–SPE–PANI devices displayed linear responses to hCG in EIS assays from 0.001 to 50 ng mL−1 in real urine, with a detection limit of 0.286 pg mL−1. High selectivity was observed with respect to the presence of the constituent components of urine (urea, creatinine, magnesium chloride, calcium chloride, sodium dihydrogen phosphate, ammonium chloride, potassium sulphate and sodium chloride) at their normal levels, with a negligible sensor response to these chemicals. Successful detection of hCG was also achieved in spiked samples of real urine from a pregnant woman. The immunosensor developed is a promising tool for point-of-care detection of hCG, due to its excellent detection capability, simplicity of fabrication, low-cost, high sensitivity and selectivity.

Specific label-free and real-time detection of oxidized low density lipoprotein (oxLDL) using an immunosensor with three monoclonal antibodies

Increased levels of plasma oxLDL, which is the oxidized fraction of Low Density Lipoprotein (LDL), are associated with atherosclerosis, an inflammatory disease, and the subsequent development of severe cardiovascular diseases that are today a major cause of death in modern countries. It is therefore important to find a reliable and fast assay to determine oxLDL in serum. A new immunosensor employing three monoclonal antibodies (mAbs) against oxLDL is proposed in this work as a quick and effective way to monitor oxLDL. The oxLDL was first employed to produce anti-oxLDL monoclonal antibodies by hybridoma cells that were previously obtained. The immunosensor was set-up by selfassembling cysteamine (Cyst) on a gold (Au) layer (4 mm diameter) of a disposable screen-printed electrode. Three mAbs were allowed to react with N-hydroxysuccinimide (NHS) and ethyl(dimethylaminopropyl)carbodiimide (EDAC), and subsequently incubated in the Au/Cys. Albumin from bovine serum (BSA) was immobilized further to ensure that other molecules apart from oxLDL could not bind to the electrode surface. All steps were followed by various characterization techniques such as electrochemical impedance spectroscopy (EIS) and square wave voltammetry (SWV). The analytical operation of the immunosensor was obtained by incubating the sensing layer of the device in oxLDL for 15 minutes, prior to EIS and SWV. This was done by using standard oxLDL solutions prepared in foetal calf serum, in order to simulate patient's plasma with circulating oxLDL. A sensitive response was observed from 0.5 to 18.0 mg mL 1 . The device was successfully applied to determine the oxLDL fraction in real serum, without prior dilution or necessary chemical treatment. The use of multiple monoclonal antibodies on a biosensing platform seemed to be a successful approach to produce a specific response towards a complex multi-analyte target, correlating well with the level of oxLDL within atherosclerosis disease, in a simple, fast and cheap way.

A label-free DNA aptamer-based impedance biosensor for the detection of E. coli outer membrane proteins

A label-free DNA aptamer-based impedance biosensor for the detection of E. coli outer membrane proteins (OMPs) was developed. Two single stranded DNA sequences were tested as recognition elements and compared. The aptamer capture probes were immobilized, with and without 6-mercapto-1-hexanol (MCH) on a gold electrode. Each step of the modification process was characterized by Faradaic impedance spectroscopy (FIS). A linear relationship between the electron-transfer resistance (Ret) and E. coli OMPs concentration was demonstrated in a dynamic detection range of 1 × 10−7–2 × 10−6 M. Moreover, the aptasensor showed selectivity despite the presence of other possible water contaminates and could be regenerated under low pH condition. The developed biosensor shows great potential to be incorporated in a biochip and used for in situ detection of E. coli OMPs in water samples.

Label-free Detection of Microcystin-LR in Waters Using Real-Time Potentiometric Biosensors Based on Single-Walled Carbon Nanotubes Imprinted Polymers

Microcystin-LR (MC-LR) is a dangerous toxin found in environmental waters, quantified by high performance liquid chromatography and/or enzyme-linked immunosorbent assays. Quick, low cost and on-site analysis is thus required to ensure human safety and wide screening programs. This work proposes label-free potentiometric sensors made of solid-contact electrodes coated with a surface imprinted polymer on the surface of Multi-Walled Carbon NanoTubes (CNTs) incorporated in a polyvinyl chloride membrane. The imprinting effect was checked by using non-imprinted materials. The MC-LR sensitive sensors were evaluated, characterized and applied successfully in spiked environmental waters. The presented method offered the advantages of low cost, portability, easy operation and suitability for adaptation to flow methods.