In each individual cell, there are many signaling pathways that may interact or cross talk with each other. Especially, some can sense the same signal and go through different pathways but eventually converge at some points. Therefore repetitive signal stimulations may result in intricate cell responses, among which the priming effect has been extensively studied in monocytes and macrophages as it plays an unambiguously crucial role in immunological protection against pathogen infection. Priming basically describes the phenomena that host cells can launch a dramatically enhanced response to the second higher dose of stimulus if cells have been previously treated with a lower dose of identical stimulus. It was reported to be associated with many human immune diseases (such as rheumatoid arthritis and hepatitis) that are attracting more and more researches on the priming effect. It is undoubtable that many genes are involved in this complicated biological process. Microarray is one of the standard techniques that are applied to do the transcriptome profiling of cells under repetitive stimuli and reveal gene regulatory networks. Therefore a well-established pipeline to analyze microarray data is of special help to investigate the underlying mechanism of priming effect. In this research, we aimed to design a strategy that can be used to interpret microarray data and to propose gene candidates that potentially participate in priming effect. To confirm our analysis results, we used a detailed mathematical model to further demonstrate the mechanism of a specific case of priming effect in a computational perspective. / Master of Science

Due to the increasing resistance demonstrated by insects to conventional insecticides, the need for compounds with novel modes of action is becoming more urgent. Also, the discovery and production of new insecticides is vital as regulations and restrictions on conventional insecticides become increasingly stringent (Casida and Quistad 1998). Research in this area requires screening of many candidate compounds which is costly and time-consuming. The goal of this research was to produce in vitro insect neurons from Sf21 insect ovarian cell lines, which could lead to new high throughput screening methods and a way to mass produce insect material for basic research. This study used a culture of Sf21 cells and a mixture of differentiation agents to produce viable neuron-like cells. In the presence of the molting hormone 20-hydroxyecdysone (20-HE), or insulin, in the growth medium, Sf21 cells began to express neuronal morphology, or the production of elongated, axon-like processes within 2-3 days. Maximal differentiation occurred when in the presence of 42 μM 20-HE or 10 μM insulin. Effects were maximal on day 2 for 20-E and day 3 for insulin. Insulin was more potent at day 2 for inducing differentiation (EC₅₀ = 247 nM) than 20-HE (EC₅₀ = 13 μM). In combination, 20-HE and insulin produced apparent synergistic effects on differentiation. Caffeine, a central nervous system (CNS) stimulant, inhibited induction of elongated processes by 20-HE and/or insulin. Caffeine was a potent inhibitor of 42 μM 20-HE, with an IC50 of 9 nM, and the inhibition was incomplete, resulting in about one quarter of the differentiated cells remaining, even at high concentrations (up to 1 mM). The ability to induce a neural phenotype simplifies studies with of insect cells, compared to either the use of primary nervous tissue or genetic engineering techniques. The presence of ion channels or receptors in the differentiated cells remains to be determined. If they are present, high throughput screening for new insecticides will be accelerated and made more economical by the utility of this method. / Master of Science

This thesis shows advancements towards the development of engineered bacteria for sensing and responding to environmental pollutants by exploring the use of UDP-glycosyltransferases (UGTs) for their metabolism of toxins, along with the use of engineered tetracycline repressor protein (TetR) based transcriptional regulators as sensors for environmental toxins. The importance and applicability of UGTs as well as the adaptability of TetR systems for future developments are shown through a function-based review of UGTs, the development of high-throughput fluorescent UGT assay technique, and the creation of novel TetR transcription regulatory sequences. The assays effectively measured UGT71G1 activity based on the presence of reaction byproducts, leading to the identification of several new substrates including the toxin bisphenol A. Next, hybrid TetRs were assembled from complementary DNA-binding and ligand-binding domains of TetR homologs. The ability to interchange these domains while retaining their function multiplies the unique TetR systems available for use in cellular systems. In future, novel TetR and UGT71G1 systems may be developed to detect and respond to substrates like bisphenol A.

Conventional pre-clinical drug screening, reliant on 2D cell cultures and animal studies, faces challenges—the former lacks biological complexity, and the latter lacks predictability due to differences between animals and humans from genetic to functional levels. Organ-on-chip technologies have evolved to bridge the gap between preclinical and clinical trials, necessitating human cells for precise predictions of human responses. Considering the significance of the vascular system in various diseases, incorporating vascular units into organ-on-chip devices is critical. For effective drug discovery using vessels-on-chips, achieving high-throughput and consistency between samples is crucial. However, many vessels-on-chips are manually handled during preparation and data collection, reducing throughput and increasing sample-to-sample variations. The conventional closed microfluidic chip format further impedes accessibility, hindering automation. This thesis focuses on two high-throughput micro-vessel models replicating vascular functions under perfusion in a 384-well plate format. These open-top models allow automated preparation and examination, enhancing efficiency in compound screening. The first model features a self-assembled perfusable micro-vascular network on a 384-well plate, co-culturing endothelial cells (EC) with stromal cells in a hydrogel. Automated using a robotic system and a fluorescent plate reader, it supports organ-specific functions and enables nanoparticle transport to target tissues. Utilized for testing cancer therapeutic drugs, it demonstrates dose-related responses in vascular permeability and architectures. The second model is dedicated to crafting micro-vessels of consistent quality for biological testing and disease modeling. It employs a sacrificial material for pre-designed tubular shapes for EC seeding. The integration of automated processes and a straight channel design minimizes sample discrepancies. Furthermore, a tri-culture system enhances barrier integrity, enabling effective drug screening that distinguishes between vasculotoxic and non-vasculotoxic agents with notable sensitivity and specificity. Looking ahead, there is potential to further refine these models to encompass a broader range of vascular diseases, which could lead to novel insights and therapeutic targets. / Thesis / Doctor of Philosophy (PhD) / In clinical trials, a staggering 90% of drugs fail during testing in people. Traditional preclinical drug screening methods rely on culturing human cells on flat surfaces or using animal models, both fraught with limitations such as lacking structural complexity or having DNA differences from humans. Addressing this issue could notably reduce efforts and costs. This thesis is dedicated to advancing preclinical drug testing through micro-vessel models. It focuses on constructing 3D vessels using human cells, offering a more accurate representation of human physiology. Two models are discussed: one with self-assembled vessels featuring complex structures, and another emphasizing sacrificial materials to design simpler vascular shapes, ensuring consistency in testing. By leveraging these innovative models, researchers can subject various drugs to micro-vessels constructed in vitro, enabling them to predict their effects in humans. This approach has the potential to transform drug testing methodologies, moving towards the utilization of artificial human organ models.

Thesis (Ph.D.)--Indiana University, 2009. / Title from screen (viewed on February 2, 2010). Department of Biochemistry and Molecular Biology, Indiana University-Purdue University Indianapolis (IUPUI). Advisor(s): Mark R. Kelley, Millie M. Georgiadis, John J. Turchi, Martin L. Smith. Includes vitae. Includes bibliographical references (leaves 114-133).

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.

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.