This thesis presents the development of soft- and hardware for a high-throughput prism-based Total Internal Reflection Fluorescence (pTIRF) microscope. By re-purposing components from various sources, most notably from an Illumina sequencer, the project details the build and integration of necessary components for the microscopy platform. This pTIRF setup aims to enable high-throughput quantitative binding measurements between biomolecules by combining the sensitivity of TIRF microscopy with the scalability of sequencing technology. The project details the construction process, experimental setup, and potential applications of the developed platform. / Projektet beskriver utvecklingen av mjuk- och hårdvara för ett high-troughput prismabaserat Total Internal Reflection Fluorescence (pTIRF) mikroskop. Genom att återanvända olika komponenter, framför allt från en Illuminasekvenserare, behandlar projektet tillverkning och integrering av nödvändiga komponenter för mikroskopiplattformen. Denna pTIRF-uppställning ämnar utföra high-throughput kvantitativa bindningsmätningar mellan biomolekyler genom att kombinera känsligheten hos TIRF-mikroskopi med skalbarheten hos sekvenseringsteknologi. Uppsatsen beskriver byggprocessen, det experimentella upplägget och de potentiella tillämpningarna av den utvecklade plattformen.

Receptores nucleares controlam a transcrição em células eucarióticas quando ativados por ligantes e, além do sítio de interação com ligantes, há outros sítios alternativos em sua superfície que podem ser alvo de compostos capazes de interferir com as interações proteína-proteína desativando o RN. A ativação do Receptor X de Pregnano (RXP) e do Receptor Constitutivo de Androstano (RCA) resulta na indução do metabolismo e efluxo de fármacos. Portanto, RXP/RCA sao responsáveis por causar reações adversas ou falhar terapias. Uma abordagem combinando a triagem experimental à nível cellular, em uma biblioteca de fármacos, e validação com ensaios in vitro e in silico, conseguimos identificar três novos antagonistas de RXP e cinco novos contra RCA, cada um com um perfil único de interação. / Nuclear receptors can control transcription in eukaryotic cells in a ligand-dependent manner and, besides the ligand-binding pocket there is evidence of the existence of alternative ligand-binding sites on the surface, which can be addressed by small organic molecules that disrupt specific protein-protein interactions and thereby may antagonise NR function. Activation of pregnane X receptor (PXR) and constitutive androstane receptor (CAR) results in the induction of first-pass metabolism and drug efflux. Hereby PXR/CAR may cause adverse drug reactions or therapeutic failure of drugs. Therefore, PXR and/or CAR antagonists can minimise adverse effects or improve therapeutic efficiencies. Combination of cellular high-throughput screen identified CAR and PXR potent antagonists in a library of approved and investigational drugs. Further validated by cellular and in vitro assays, as well as molecular docking, suggesting additional or exclusive binding outside the classical ligand binding pocket. In conclusion, we here have identified three approved drugs as novel potent PXR antagonists and five potential CAR inverse agonists with differential receptor interaction profiles.

The interferon (IFN) response is a crucial component of cellular innate immunity, vital for controlling virus infections. Dysregulation of the IFN response however can lead to serious medical conditions including autoimmune disorders. Modulators of IFN induction and signalling could be used to treat these diseases and as tools to further understand the IFN response and viral infections. We have developed cell-based assays to identify modulators of IFN induction and signalling, based on A549 cell lines where a GFP gene is under the control of the IFN-β promoter (A549/pr(IFN-β).GFP) and the ISRE containing MxA promoter (A549/pr(ISRE).GFP) respectively. The assays were optimized, miniaturized and validated as suitable for HTS by achieving Z' Factor scores >0.6. A diversity screen of 15,667 compounds using the IFN induction reporter assay identified 2 hit compounds (StA-IFN-1 and StA-IFN-4) that were validated as specifically inhibiting IFNβ induction. Characterisation of these molecules demonstrated that StA-IFN-4 potently acts at, or upstream, of IRF3 phosphorylation. We successfully expanded this HTS platform to target viral interferon antagonists acting upon IFN-signalling. An additional assay was developed where the A549/pr(ISRE).GFP.RBV-P reporter cell line constitutively expresses the Rabies virus phosphoprotein. A compound inhibiting viral protein function will restore GFP expression. The assay was successfully optimized for HTS and used in an in-house screen. We further expanded this assay by placing the expression of RBV-P under the control of an inducible promoter. This demonstrates a convenient approach for assay development and potentiates the targeting of a variety of viral IFN antagonists for the identification of compounds with the potential to develop a novel class of antiviral drugs.

This work is focused on the issue of wireless networks according to the IEEE 802.11 standard. The main subject of research is the Rate Adaptation Algorithms (RAA). The work also contains a description of simulation environments NS-3 and OMNeT in terms of adaptation algorithms. At the end of the work there is a summary of wireless network simulations in NS-3 and OMNeT environments and their comparison with the actual measurement of network parameters.

Indiana University-Purdue University Indianapolis (IUPUI) / The DNA Base Excision Repair (BER) pathway repairs DNA damaged by endogenous and exogenous agents including chemotherapeutic agents. Removal of the damaged base by a DNA glycosylase creates an apurinic / apyrimidinic (AP) site. AP endonuclease1 (Ape1), a critical component in this pathway, hydrolyzes the phosphodiester backbone 5’ to the AP site to facilitate repair. Additionally, Ape1 also functions as a redox factor, known as Ref-1, to reduce and activate key transcription factors such as AP-1 (Fos/Jun), p53, HIF-1α and others. Elevated Ape1 levels in cancers are indicators of poor prognosis and chemotherapeutic resistance, and removal of Ape1 via methodology such as siRNA sensitizes cancer cell lines to chemotherapeutic agents. However, since Ape1 is a multifunctional protein, removing it from cells not only inhibits its DNA repair activity but also impairs its other functions. Our hypothesis is that a small molecule inhibitor of the DNA repair activity of Ape1 will help elucidate the importance (role) of its repair function in cancer progression as wells as tumor drug response and will also give us a pharmacological tool to enhance cancer cells’ sensitivity to chemotherapy. In order to discover an inhibitor of Ape1’s DNA repair function, a fluorescence-based high-throughput screening (HTS) assay was used to screen a library of drug-like compounds. Four distinct compounds (AR01, 02, 03 and 06) that inhibited Ape1’s DNA repair activity were identified. All four compounds inhibited the DNA repair activity of purified Ape1 protein and also inhibited Ape1’s activity in cellular extracts. Based on these and other in vitro studies, AR03 was utilized in cell culture-based assays to test our hypothesis that inhibition of the DNA repair activity of Ape1 would sensitize cancer cells to chemotherapeutic agents. The SF767 glioblastoma cell line was used in our assays as the chemotherapeutic agents used to treat gliobastomas induce lesions repaired by the BER pathway. AR03 is cytotoxic to SF767 glioblastoma cancer cells as a single agent and enhances the cytotoxicity of alkylating agents, which is consistent with Ape1’s inability to process the AP sites generated. I have identified a compound, which inhibits Ape1’s DNA repair activity and may have the potential in improving chemotherapeutic efficacy of selected chemotherapeutic agents as well as to help us understand better the role of Ape1’s repair function as opposed to its other functions in the cell.

Master of Science / Department of Agronomy / William T. Schapaugh / Remote sensing technologies might serve as indirect selection tools to improve phenotyping to differentiate genotypes for yield in soybean breeding program as well as the assessment of soybean cyst nematode (SCN), Heterodera glycines. The objective of these studies were to: i) investigate potential use of spectral reflectance indices (SRIs) and canopy temperature (CT) as screening tools for soybean grain yield in an elite, segregating population; ii) determine the most appropriate growth stage(s) to measure SRI’s for predicting grain yield; and iii) estimate SCN population density among and within soybean cultivars utilizing canopy spectral reflectance and canopy temperature. Experiment 1 was conducted at four environments (three irrigated and one rain-fed) in Manhattan, KS in 2012 and 2013. Each environment evaluated 48 F4- derived lines. In experiment 2, two SCN resistant cultivars and two susceptible cultivars were grown in three SCN infested field in Northeast KS, in 2012 and 2013. Initial (Pi) and final SCN soil population (Pf) densities were obtained. Analyses of covariance (ANCOVA) revealed that the green normalized vegetation index (GNDVI) was the best predictive index for yield compared to other SRI’s and differentiated genotype performance across a range of reproductive growth stages. CT did not differentiate genotypes across environments. In experiment 2, relationships between GNDVI, reflectance at single wavelengths (675 and 810 nm) and CT with Pf were not consistent across cultivars or environments. Sudden death syndrome (SDS) may have confounded the relationships between remote sensing data and Pf. Therefore, it would be difficult to assess SCN populations using remote sensing based on these results.

ITC/USA 2008 Conference Proceedings / The Forty-Fourth Annual International Telemetering Conference and Technical Exhibition / October 27-30, 2008 / Town and Country Resort & Convention Center, San Diego, California / The integrated Network Enhanced Telemetry effort (iNET) was launched to create a telemetry network that will enhance the traditional point-to-point telemetry link from test articles (TAs) to ground stations (GS). Two of the critical needs identified by the Central Test and Evaluation Investment Program (CTEIP) are, "the need to be able to provide reliable coverage in potentially high capacity environments, even in Over-The-Horizon (OTH) settings", and "the need to make more efficient use of spectrum resources through dynamic sharing of said resources, based on instantaneous demand thereof". Research conducted at Morgan State University (MSU) has focused on providing solutions for both critical problems. The Mixed Network architecture developed by MSU has shown that a hybrid network can be used to provide coverage for TAs that are beyond the coverage area of the GS. The mixed network uses clustering techniques to partition the aggregate network into clusters or sub-networks based on properties of each TA, which currently include signal strengths, and location. The paper starts with a detailed analysis of two parameters that affect the performance of each sub-network: contention between the TAs in the mobile ad-hoc network, and queuing at the Gateway TAs that serve as the link between the mobile ad-hoc and the Cellular networks. Contention and queuing will be used to evaluate two performance (distance) measures for each sub-network: throughput and delay. We define a new distance measure known as "power", which is equal to the ratio of throughput over delay, and is used as a measure of performance of the mixed network for Quality of Service (QOS). This paper describes the analytical foundation used to prove that the "power" performance measure is an excellent tool for optimizing the clustering of a mixed network to provide QOS.

The increased use of mass spectrometry in the clinical setting has led to a demand for high sample throughput. Developments such as ultra high performance liquid chromatography and the ambient ionization techniques enable high sample throughput by reducing chromatographic run times or by removing the requirement for sample preparation and fractionation prior to analysis. This thesis assesses the reproducibility and robustness of these high throughput techniques for the analysis of clinical and pharmaceutical samples by ion mobility-mass spectrometry. The rapid quantitative analysis of the urinary biomarkers of chronic obstructive pulmonary disease, desmosine and isodesmosine has been performed by ultra high performance liquid chromatography combined with ion mobility-mass spectrometry. The determination of health status based on the free unbound fraction rather than the total bound and unbound desmosine and isodesmosine, significantly reduces the time taken in sample preparation. The potential for direct analysis of the urinary metabolites from undeveloped TLC plates using a solvent extraction surface sample probe is demonstrated. The use of a solvent gradient for the extraction separates urinary metabolites from salts and other matrix components and allows fractionation of the sample as a result of differential retention on the undeveloped RP-TLC plate. This separation, combined with ion mobility-mass spectrometry provides a rapid ambient ionization method for urinary profiling. The combination of a thermal desorption probe with extractive electrospray ionization has been applied to the direct detection of a known genotoxic impurity from a surrogate active pharmaceutical ingredient. The volatility of the impurity compared to the matrix, allowed selective thermal desorption of the analyte, which was ionized by extractive electrospray and detected by mass spectrometry. The use of a rapid on-probe derivatisation reaction, combined with thermal desorption is demonstrated for the direct determination of urinary creatinine. The aqueous acylation of creatinine significantly increases the volatility of the analyte enabling separation from the urine matrix and analysis by thermal desorption extractive electrospray combined with ion mobility-mass spectrometry.

This project presents a mechanism for instrumentation of secure VoIP calls. The experiments were run under different network conditions and security systems. VoIP services such as Google Talk, Express Talk and Skype were under test. The project allowed analysis of the voice quality of the VoIP services based on the Mean Opinion Score (MOS) values generated by Perceptual valuation of Speech Quality (PESQ). The quality of the audio streams produced were subjected to end-to-end delay, jitter, packet loss and extra processing in the networking hardware and end devices due to Internetworking Layer security or Transport Layer security implementations. The MOS values were mapped to Perceptual Evaluation of Speech Quality for wideband (PESQ-WB) scores. From these PESQ-WB scores, the graphs of the mean of 10 runs and box and whisker plots for each parameter were drawn. Analysis on the graphs was performed in order to deduce the quality of each VoIP service. The E-model was used to predict the network readiness and Common vulnerability Scoring System (CVSS) was used to predict the network vulnerabilities. The project also provided the mechanism to measure the throughput for each test case. The overall performance of each VoIP service was determined by PESQ-WB scores, CVSS scores and the throughput. The experiment demonstrated the relationship among VoIP performance, VoIP security and VoIP service type. The experiment also suggested that, when compared to an unsecure IPIP tunnel, Internetworking Layer security like IPSec ESP or Transport Layer security like OpenVPN TLS would improve a VoIP security by reducing the vulnerabilities of the media part of the VoIP signal. Morever, adding a security layer has little impact on the VoIP voice quality.

9 pp. / As today's dairy industry consolidates, cows are being milked more rapidly through larger milking parlors on larger dairies than ever before. Because milk is the primary commodity and source of income for producers, the harvesting of milk is the single most important job on any dairy. Producing high-quality milk to maximize yields and economic value requires effective parlor management, an enormous challenge for producers. Managing large parlors includes managing labor, milking equipment, as well as monitoring and evaluating parlor performance. Decisions concerning the milking center are some of the most complicated decisions a dairy producer has to make. Milking procedures, herd size, milking interval, the milk market, and the equity position of a producer influence these decisions. Producers will have to make the following decisions before they can select or develop management protocols for a milking parlor: 1. How many cows will be milked through the parlor? 2. What milking procedure will be used (minimal or full)? 3. If a full milking routine; how much contact time do you want (strips per teat)? 4. Which milking routine will be used (sequential, grouping, or territorial)? 5. Are you willing to train teams of milkers to operate large parlors?