Development of screening assays to test novel integrin antagonists in allergic inflammation

Aim of the research: to develop a prototype of homogeneous high-throughput screening (HTS) for identification of novel integrin antagonists for the treatment of ocular allergy and to better understand the mechanisms of action of integrin-mediated levocabastine antiallergic action. Results: This thesis provides evidence that adopting scintillation proximity assay (SPA) levocabastine (IC50=406 mM), but not the first-generation antihistamine chlorpheniramine, displaces [125I]fibronectin (FN) binding to human a4b1 integrin. This result is supported by flow cytometry analysis, where levocabastine antagonizes the binding of a primary antibody to integrin a4 expressed in Jurkat E6.1 cells. Levocabastine, but not chlorpheniramine, binds to a4b1 integrin and prevents eosinophil adhesion to VCAM-1, FN or human umbilical vein endothelial cells (HUVEC) cultured in vitro. Similarly, levocabastine affects aLb2/ICAM-1-mediated adhesion of Jurkat E6.1 cells. Analyzing the supernatant of TNF-a-treated (24h) eosinophilic cells (EoL-1), we report that levocabastine reduces the TNF-a-induced release of the cytokines IL-12p40, IL-8 and VEGF. Finally, in a model of allergic conjunctivitis, levocastine eye drops (0.05%) reduced the clinical aspects of the early and late phase reactions and the conjunctival expression of a4b1 integrin by reducing infiltrated eosinophils. Conclusions: SPA is a highly efficient, amenable to automation and robust binding assay to screen novel integrin antagonists in a HTS setting. We propose that blockade of integrinmediated cell adhesion might be a target of the anti-allergic action of levocabastine and may play a role in preventing eosinophil adhesion and infiltration in allergic conjunctivitis.

Development and evaluation of a 44 Ti, 44 Sc radionuclide generator and labeling of biomolecules with 44 Sc and 68 Ga for PET imaging

Non-invasive molecular-imaging technologies are playing a key role in drug discovery, development and delivery. Positron Emission Tomography (PET) is such a molecular imaging technology and a powerful tool for the observation of various deceases in vivo. However, it is limited by the availability of vectors with high selectivity to the target and radionuclides with a physical half-life which matches the biological half-life of the observed process. The 68Ge/68Ga radionuclide generator makes the PET-nuclide anywhere available without an on-site cyclotron. Besides the perfect availability 68Ga shows well suited nuclide properties for PET, but it has to be co-ordinated by a chelator to introduce it in a radiopharmaceuticals.rnHowever, the physical half-life of 68Ga (67.7 min) might limit the spectrum of clinical applications of 68Ga-labelled radiodiagnostics. Furthermore, 68Ga-labelled analogues of endoradiotherapeuticals of longer biological half-live such as 90Y- or 177Lu-labeled peptides and proteins cannot be used to determine individual radiation dosimetry directly. rnThus, radionuclide generator systems providing positron emitting daughters of extended physical half-life are of renewed interest. In this context, generator-derived positron emitters with longer physical half-life are needed, such as 72As (T½ = 26 h) from the 72Se/72As generator, or 44Sc (T½ = 3.97 h) from the 44Ti/44Sc generator.rnIn this thesis the implementation of radioactive gallium-68 and scandium-44 for molecular imaging and nuclear medical diagnosis, beginning with chemical separation and purification of 44Ti as a radionuclide mother, investigation of pilot generators with different elution mode, building a prototype generator, development and investigation of post-processing of the generator eluate, its concentration and further purification, the labeling chemistry under different conditions, in vitro and in vivo studies of labeled compounds and, finally, in vivo imaging experiments are described.

Technology Development and Consumer Acceptance of Innovation in Fruit Production

Precision Agriculture (PA) and the more specific branch of Precision Horticulture are two very promising sectors. They focus on the use of technologies in agriculture to optimize the use of inputs, so to reach a better efficiency, and minimize waste of resources. This important objective motivated many researchers and companies to search new technology solutions. Sometimes the effort proved to be a good seed, but sometimes an unfeasible idea. So that PA, from its birth more or less 25 years ago, is still a “new” management, interesting for the future, but an actual low adoption rate is still reported by experts and researchers. This work aims to give a contribution in finding the causes of this low adoption rate and proposing a methodological solution to this problem. The first step was to examine prior research about Precision Agriculture adoption, by ex ante and ex post approach. It was supposed as important to find connections between these two phases of a purchase experience. In fact, the ex ante studies dealt with potential consumer’s perceptions before a usage experience occurred, therefore before purchasing a technology, while the ex post studies described the drivers which made a farmer become an end-user of PA technology. Then, an example of consumer research is presented. This was an ex ante research focused on pre-prototype technology for fruit production. This kind of research could give precious information about consumer acceptance before reaching an advanced development phase of the technology, and so to have the possibility to change something with the least financial impact. The final step was to develop the pre-prototype technology that was the subject of the consumer acceptance research and test its technical characteristics.

Development of a microgrid with renewable energy sources and electrochemical storage system integration

Beside the traditional paradigm of "centralized" power generation, a new concept of "distributed" generation is emerging, in which the same user becomes pro-sumer. During this transition, the Energy Storage Systems (ESS) can provide multiple services and features, which are necessary for a higher quality of the electrical system and for the optimization of non-programmable Renewable Energy Source (RES) power plants. A ESS prototype was designed, developed and integrated into a renewable energy production system in order to create a smart microgrid and consequently manage in an efficient and intelligent way the energy flow as a function of the power demand. The produced energy can be introduced into the grid, supplied to the load directly or stored in batteries. The microgrid is composed by a 7 kW wind turbine (WT) and a 17 kW photovoltaic (PV) plant are part of. The load is given by electrical utilities of a cheese factory. The ESS is composed by the following two subsystems, a Battery Energy Storage System (BESS) and a Power Control System (PCS). With the aim of sizing the ESS, a Remote Grid Analyzer (RGA) was designed, realized and connected to the wind turbine, photovoltaic plant and the switchboard. Afterwards, different electrochemical storage technologies were studied, and taking into account the load requirements present in the cheese factory, the most suitable solution was identified in the high temperatures salt Na-NiCl2 battery technology. The data acquisition from all electrical utilities provided a detailed load analysis, indicating the optimal storage size equal to a 30 kW battery system. Moreover a container was designed and realized to locate the BESS and PCS, meeting all the requirements and safety conditions. Furthermore, a smart control system was implemented in order to handle the different applications of the ESS, such as peak shaving or load levelling.

Development of a highly potent bispecific antibody format targeting the novel tumor-specific antigen CLDN18.2

Due to multiple immune evasion mechanisms of cancer cells, novel therapy approaches are required to overcome the limitations of existing immunotherapies. Bispecific antibodies are potent anti-cancer drugs, which redirect effector T cells for specific tumor cell lysis, thus enabling the patient’s immune system to fight cancer cells. The antibody format used in this proof of concept study–bispecific ideal monoclonal antibodies termed BiMAB–is a tailor-made recombinant protein, which consists of two fused scFv antibodies recognizing different antigens. Both are arranged in tandem on a single peptide chain and the individual variable binding domains are separated by special non-immunogenic linkers. The format is comprised of a scFv targeting CLDN18.2–a gastric cancer tumor associated antigen (TAA) –while the second specificity binds the CD3 epsilon (CD3ε) subunit of the T cell receptor (TCR) on T cells. For the first time, we compared in our IMAB362-based BiMAB setting, four different anti-CD3-scFvs, respectively derived from the mAbs TR66, CLB-T3, as well as the humanized and the murine variant of UCHT1. In addition, we investigated the impact of an N- versus a C-terminal location of the IMAB362-derived scFv and the anti-CD3-scFvs. Thus, nine CLDN18.2 specific BiMAB proteins were generated, of which all showed a remarkably high cytotoxicity towards CLDN18.2-positive tumor cells. Because of its promising effectiveness, 1BiMAB emerged as the BiMAB prototype. The selectivity of 1BiMAB for its TAA and CD3ε, with affinities in the nanomolar range, has been confirmed by in vitro assays. Its dual binding depends on the design of an N-terminally positioned IMAB362 scFv and the consecutive C-terminally positioned TR66 scFv. 1BiMAB provoked a concentration and target cell dependent T cell activation, proliferation, and upregulation of the cytolytic protein Granzyme B, as well as the consequent elimination of target cells. Our results demonstrate that 1BiMAB is able to activate T cells independent of elements that are usually involved in the T cell recognition program, like antigen presentation, MHC restriction, and co-stimulatory effector molecules. In the first in vivo studies using a subcutaneous xenogeneic tumor mouse model in immune incompetent NSG mice, we could prove a significant therapeutic effect of 1BiMAB with partial or complete tumor elimination. The initial in vitro RIBOMAB experiments correspondingly showed encouraging results. The electroporation of 1BiMAB IVT-RNA into target or effector cells was feasible, while the functionality of translated 1BiMAB was proven by induced T cell activation and target cell lysis. Accordingly, we could show that the in vitro RIBOMAB approach was applicable for all nine BiMABs, which proves the RIBOMAB concept. Thus, the CLDN18.2-BiMAB strategy offers great potential for the treatment of cancer. In the future, administered either as protein or as IVT-RNA, the BiMAB format will contribute towards finding solutions to raise and sustain tumor-specific cellular responses elicited by engaged and activated endogenous T cells. This will potentially enable us to overcome immune evasion mechanisms of tumor cells, consequently supporting current solid gastric cancer therapies.

PROOF OF CONCEPT PROTOTYPE FOR A RAILROAD PEDESTRIAN WARNING SYSTEM USING WIRELESS SENSOR NETWORKS

Wireless sensor network is an emerging research topic due to its vast and ever-growing applications. Wireless sensor networks are made up of small nodes whose main goal is to monitor, compute and transmit data. The nodes are basically made up of low powered microcontrollers, wireless transceiver chips, sensors to monitor their environment and a power source. The applications of wireless sensor networks range from basic household applications, such as health monitoring, appliance control and security to military application, such as intruder detection. The wide spread application of wireless sensor networks has brought to light many research issues such as battery efficiency, unreliable routing protocols due to node failures, localization issues and security vulnerabilities. This report will describe the hardware development of a fault tolerant routing protocol for railroad pedestrian warning system. The protocol implemented is a peer to peer multi-hop TDMA based protocol for nodes arranged in a linear zigzag chain arrangement. The basic working of the protocol was derived from Wireless Architecture for Hard Real-Time Embedded Networks (WAHREN).

Development of a rapid bioassessment method and index of biotic integrity for estuaries located along the northwest Gulf of Mexico

Recently it has been proposed that the evaluation of effects of pollutants on aquatic organisms can provide an early warning system of potential environmental and human health risks (NRC 1991). Unfortunately there are few methods available to aquatic biologists to conduct assessments of the effects of pollutants on aquatic animal community health. The primary goal of this research was to develop and evaluate the feasibility of such a method. Specifically, the primary objective of this study was to develop a prototype rapid bioassessment technique similar to the Index of Biotic Integrity (IBI) for the upper Texas and Northwestern Gulf of Mexico coastal tributaries. The IBI consists of a series of "metrics" which describes specific attributes of the aquatic community. Each of these metrics are given a score which is then subtotaled to derive a total assessment of the "health" of the aquatic community. This IBI procedure may provide an additional assessment tool for professionals in water quality management.^ The experimental design consisted primarily of compiling previously collected data from monitoring conducted by the Texas Natural Resource Conservation Commission (TNRCC) at five bayous classified according to potential for anthropogenic impact and salinity regime. Standardized hydrological, chemical, and biological monitoring had been conducted in each of these watersheds. The identification and evaluation of candidate metrics for inclusion in the estuarine IBI was conducted through the use of correlation analysis, cluster analysis, stepwise and normal discriminant analysis, and evaluation of cumulative distribution frequencies. Scores of each included metric were determined based on exceedances of specific percentiles. Individual scores were summed and a total IBI score and rank for the community computed.^ Results of these analyses yielded the proposed metrics and rankings listed in this report. Based on the results of this study, incorporation of an estuarine IBI method as a water quality assessment tool is warranted. Adopted metrics were correlated to seasonal trends and less so to salinity gradients observed during the study (0-25 ppt). Further refinement of this method is needed using a larger more inclusive data set which includes additional habitat types, salinity ranges, and temporal variation. ^

The Software Prototype as Digital Boundary Object – A Revelatory Longitudinal Innovation Case

With the availability of lower cost but highly skilled software development labor from offshore regions, entrepreneurs from developed countries who do not have software development experience can utilize this workforce to develop innovative software products. In order to succeed in offshored innovation projects, the often extreme knowledge boundaries between the onsite entrepreneur and the offshore software development team have to be overcome. Prior research has proposed that boundary objects are critical for bridging such boundaries – if they are appropriately used. Our longitudinal, revelatory case study of a software innovation project is one of the first to explore the role of the software prototype as a digital boundary object. Our study empirically unpacks five use practices that transform the software prototype into a boundary object such that knowledge boundaries are bridged. Our findings provide new theoretical insights for literature on software innovation and boundary objects, and have implications for practice.

DEVELOPMENT AND IMPLEMENTATION OF A REMOTE AUDIT TOOL FOR HIGH DOSE RATE (HDR) 192IR BRACHYTHERAPY USING OPTICALLY STIMULATED LUMINESCENCE DOSIMETRY

This work aimed to create a mailable and OSLD-based phantom with accuracy suitable for RPC audits of HDR brachytherapy sources at institutions participating in NCI-funded cooperative clinical trials. An 8 × 8 × 10 cm3 prototype with two slots capable of holding nanoDot Al2O3:C OSL dosimeters (Landauer, Glenwood, IL) was designed and built. The phantom has a single channel capable of accepting all 192Ir HDR brachytherapy sources in current clinical use in the United States. Irradiations were performed with an 192Ir HDR source to determine correction factors for linearity with dose, dose rate, and the combined effect of irradiation energy and phantom construction. The uncertainties introduced by source positioning in the phantom and timer resolution limitations were also investigated. It was found that the linearity correction factor was where dose is in cGy, which differed from that determined by the RPC for the same batch of dosimeters under 60Co irradiation. There was no significant dose rate effect. Separate energy+block correction factors were determined for both models of 192Ir sources currently in clinical use and these vendor-specific correction factors differed by almost 2.6%. For Nucletron sources, this correction factor was 1.026±0.004 (99% Confidence Interval) and for Varian sources it was 1.000±0.007 (99% CI). Reasonable deviations in source positioning within the phantom and the limited resolution of the source timer had insignificant effects on the ability to measure dose. Overall measurement uncertainty of the system was estimated to be ±2.5% for both Nucletron and Varian source audits (95% CI). This uncertainty was sufficient to establish a ±5% acceptance criterion for source strength audits under a formal RPC audit program. Trial audits of eight participating institutions resulted in an average RPC-to-institution dose ratio of 1.000 with a standard deviation of 0.011.

Prioritization of Policies: A Prototype Model of a Flowchart Method

This paper builds a prototype model of how to prioritize policies by using a flowchart. We presented the following six steps to decide priorities of policies: Step 1 is to attain the social subsistence level (primary education, health care, and food sufficiency); Step 2 is to attain macroeconomic stability; Step 3 is to liberalize the economy by structural adjustment programs; Step 4 is capacity building specific to a growth strategy by facilitating sufficient infrastructure (physical infrastructure and institutions); Step 5 is to initiate a growth strategy; and Step 6 is to narrow income inequalities. We illustrated the effectiveness of our "flowchart method" in case studies of Morocco, Laos, Vietnam, and China. The first priority of reforms in Morocco was given to social sectors of primary education and health care, particularly in the rural areas at Step 1. Laos should not put much emphasis on growth strategy before educational reform, attainment of macroeconomic stability, and institutional capacity building at Steps 1, 2, and 3. Vietnam can focus on reforming the state-run enterprises and developing the stock markets at Step 5 of growth strategies. We found that we should apply our flowchart method to China not nation-wide but province-wide.

Development of Efficient FPGA-Based Multi-Channel Phase Meters for IR-Interferometers

Infrared (IR) interferometry is a method for measuring the line-electron density of fusion plasmas. The significant performance achieved by FPGAs in solving digital signal processing tasks advocates the use of this type of technology in two-color IR interferometers of modern stellarators, such as the TJ-II (Madrid, Spain) and the future W7-X (Greifswald, Germany). In this work the implementation of a line-average electron density measuring system in an FPGA device is described. Several optimizations for multichannel systems are detailed and test results from the TJ-II as well as from a W7-X prototype are presented.

Evaluation of a PET Prototype Using LYSO:Ce Monolithic Detector Block

We have analyzed the performance of a PET demonstrator formed by two sectors of four monolithic detector blocks placed face-to-face. Both front-end and read-out electronics have been evaluated by means of coincidence measurements using a rotating 22Na source placed at the center of the sectors in order to emulate the behavior of a complete full ring. A continuous training method based on neural network (NN) algorithms has been carried out to determine the entrance points over the surface of the detectors. Reconstructed images from 1 MBq 22Na point source and 22Na Derenzo phantom have been obtained using both filtered back projection (FBP) analytic methods and the OSEM 3D iterative algorithm available in the STIR software package [1]. Preliminary data on image reconstruction from a 22Na point source with Ø = 0.25 mm show spatial resolutions from 1.7 to 2.1 mm FWHM in the transverse plane. The results confirm the viability of this design for the development of a full-ring brain PET scanner compatible with magnetic resonance imaging for human studies.

NetCDF based data archiving system applied to ITER Fast Plant System Control prototype

EURATOM/CIEMAT and Technical University of Madrid (UPM) have been involved in the development of a FPSC [1] (Fast Plant System Control) prototype for ITER, based on PXIe (PCI eXtensions for Instrumentation). One of the main focuses of this project has been data acquisition and all the related issues, including scientific data archiving. Additionally, a new data archiving solution has been developed to demonstrate the obtainable performances and possible bottlenecks of scientific data archiving in Fast Plant System Control. The presented system implements a fault tolerant architecture over a GEthernet network where FPSC data are reliably archived on remote, while remaining accessible to be redistributed, within the duration of a pulse. The storing service is supported by a clustering solution to guaranty scalability, so that FPSC management and configuration may be simplified, and a unique view of all archived data provided. All the involved components have been integrated under EPICS [2] (Experimental Physics and Industrial Control System), implementing in each case the necessary extensions, state machines and configuration process variables. The prototyped solution is based on the NetCDF-4 [3] and [4] (Network Common Data Format) file format in order to incorporate important features, such as scientific data models support, huge size files management, platform independent codification, or single-writer/multiple-readers concurrency. In this contribution, a complete description of the above mentioned solution is presented, together with the most relevant results of the tests performed, while focusing in the benefits and limitations of the applied technologies.

Development of an adequate model for verification of design safety-margins of the HTTR nuclear test facility

This work is based on the prototype High Engineering Test Reactor (HTTR) of the Japan Agency of Energy Atomic (JAEA). Its objective is to describe an adequate deterministic model to be used in the assessment of its design safety margins via damage domains. The concept of damage domain is defined and it is shown its relevance in the ongoing effort to apply dynamic risk assessment methods and tools based on the Theory of Stimulated Dynamics (TSD). To illustrate, we present results of an abnormal control rod (CR) withdrawal during subcritical condition and its comparison with results obtained by JAEA. No attempt is made yet to actually assess the detailed scenarios, rather to show how the approach may handle events of its kind