Thesis advisor: Tim van Opijnen / Thesis advisor: Michelle Meyer / Although bacteria are often studied as planktonic or free-living organisms, they frequently grow in complex surface-attached communities known as biofilms. Biofilms are communities of microorganisms attached to surfaces and embedded in a self-produced extracellular matrix. Biofilms are dynamic structures analogous to human settlements shaped by space and environment. These microbial communities fulfill critical roles in multiple infections in the human body. Streptococcuspneumoniae is a human pathogen that can cause biofilm-associated infections in various tissues and organs. This thesis offers a unique outlook for the study of S. pneumoniae biofilms by combining in vitro, genome-wide, and in vivo experiments to elucidate the complex population dynamics of S. pneumoniae biofilms. Existing methods to cultivate S. pneumoniae biofilms fail to fully capture the complexity of these communities, and most studies are limited to short periods of time. We developed a robust in vitro assay to grow S. pneumoniae biofilms. This assay can be maintained forever rather than days. We then use this robust assay to study their behavior in vivo and monitor disease outcomes. After establishing clear differences in biofilm and dispersal samples, we monitor population dynamics using genome-wide techniques (Tn-seq, RNA-seq and WGS) to provide some insights into this complex mode of growth. This work includes the first global identification of genetic requirements during biofilm establishment in two different S. pneumoniae strains using Tn-Seq. Coupled with our transcriptomic analysis, we found that genes involved in multiple pathways, such as capsule biosynthesis, nucleotide metabolism, and stress response, contributed to biofilm growth. Lastly, we studied the development of antibiotic resistance to three different types of antibiotics under S. pneumoniae biofilm conditions. We revealed common adaptive pathways to achieve biofilm growth and antibiotic resistance (antibiotic target genes), as well as novel routes of adaptation to develop resistance. Our findings add to the growing body of knowledge in the field of bacterial genetics and antimicrobial resistance, paving the way for future research and therapeutic advancement. / Thesis (PhD) — Boston College, 2023. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Malaria is vectored by the mosquito Anopheles gambiae (Ag) in Sub-Saharan Africa and infects approximately 500 million people annually. The increasing prevalence of pyrethroid-resistant mosquitoes has amplified the need for development of new, selective mosquitocides for use on insecticide-treated nets. We have developed several phenyl-substituted N-methylcarbamates producing a high degree of selectivity for Anopheles gambiae acetylcholinesterase (AgAChE) over human AChE. Molecular models suggest alternate conformations (flexibility) of W84 and W431 (Ag numbering) at the hydrophobic subpocket of the AgAChE active site and poor flexibility within human AChE, allowing for the high selectivity of our novel carbamates. Initial selectivity data was obtained through screening of these insecticides while using ethanol as a solvent. Re-screening of these carbamates in the presence of 0.1% DMSO (v/v) resulted in antagonism of inhibition for AgAChE, thus reducing the AgAChE-selectivity by at least 10-fold. However, the presence of 0.1% DMSO did not antagonize the inhibition of human, Drosophila melanogaster, or Musca domestica AChE. Non-selective carbamates also displayed no solvent-dependent antagonism of inhibition in any species studied, including AgAChE. Molecular models provide an explanation for antagonism of inhibition when DMSO is present. I, and collaborators, propose that W84 and W431 in AgAChE comprise an allosteric pocket that is stabilized by DMSO and is responsible for the solvent-dependent antagonism of inhibition observed with AgAChE. / Master of Science in Life Sciences

<p dir="ltr">Phospholipase C (PLC) enzymes are essential for normal cardiovascular function. These enzymes hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP₂) at the inner leaflet of the plasma membrane, producing diacylglycerol (DAG) and inositol phosphates (IP₃). IP₃ increases intracellular Ca²⁺, a key secondary messenger in cardiovascular activity. Changes in PLC expression and activity, specifically PLCβ3, have been found to play a critical role in cardiac hypertrophy and contractility. Cardiac hypertrophy, especially left ventricular hypertrophy, is a primary cause of ischemic heart disease, the leading cause of mortality worldwide. Despite the importance of these enzymes, a selective inhibitor for studying their function in cells and animal models has not yet been discovered. To address this unmet need, a lentiviral system for expressing human PLCβ3 and its two major activators, the heterotrimeric G protein subunits Ga_q and Gβγ was developed. These constructs were then utilized to establish a high-throughput screening methodology with the aim of identifying a novel allosteric inhibitor of PLCβ3, and ultimately other PLCs.</p>

Wireless Local Area Networks (WLAN) have proliferated in the last 5 years. The IEEE 802.11b products have become commonplace both in the residential and business places for untethered Internet access. However the end user experience has often been less satisfactory than what the technology can offer. The degradation in the performance of the system is mainly attributed to the poor network design. The current network design is primarily RF centric. There are two factors that need to be in the incorporated in the design. Firstly a clear understanding of the traffic sources in the network such as the peak load of the system is necessary. Secondly the design should account for the limitations of the indoor propagation such as interference and multipath. The goal of this thesis is to develop a simulator which will predict the performance (throughput) of an end user. The throughput is predicted for a given topology and traffic source. The simulator is built on object oriented design. To validate the simulator a measurement campaign was conducted. The campaign was conducted in two different channel conditions, office space and open hall. The channel measurements were also performed at these locations to understand the multipath. Comparative studies indicate that the choice of the rate adaptation algorithm hugely influences the predicted throughput. The simulator results match very well with the measurement results for the open space scenario. For the office space scenario the simulator varied by roughly 20% from the measurement results. This was due to existence of multipath leading to Inter Symbol Interference. / Master of Science

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

Die Blut-Hirn-Schranke (BHS) stellt eine selektiv durchlässige Barriere dar, die den Austausch von Stoffen zwischen Blut und ZNS kontrolliert und so neuroprotektiv wirkt. Sie verhindert allerdings nicht nur die Passage toxischer Metaboliten, sondern verwehrt auch vielen therapeutischen Wirkstoffen den Zugang zum Gehirn. Die Forschung an Methoden zum Erreichen höherer Arzneimittelkonzentrationen im Gehirn ist deshalb essenziell für die Behandlung zerebraler Erkrankungen wie dem zerebral metastasierten Mammakarzinom. Ziel dieser Arbeit war es deshalb, Wirkstoffe zu identifizieren, die die Permeabilität der BHS erhöhen. Die Substanzdatenbank LO1208 von Sigma-Aldrich wurde im Rahmen eines HTS auf ihre permeabilitätsbeeinflussenden Eigenschaften untersucht. Hierbei konnten 31 Substanzen identifiziert werden, welche die Permeabilität von BLECs um mindestens 50 % erhöhen. Aus diesen wurden 4-Amino-1,8-naphthalimid (PARP-Inhibitor) und GW2974 (TKI) für eine genauere Analyse ausgewählt. Als dritter Wirkstoff wurde Ibuilast (Inhibitor der PDE4, des MIF sowie des Toll-like-Rezeptor-4) untersucht, wobei dieser keine signifikante Veränderung der Permeabilität bewirkt. Die Messung des TEERs und der Permeabilität für Fluorescein bestätigten die Ergebnisse aus dem HTS, welches demnach zukünftig für Permeabilitätstests eingesetzt werden kann. Die Zellviabilität wird durch 4 Amino-1,8-naphthalmid nicht beeinflusst. GW2974 und Ibudilast zeigen bei 500 µM einen toxischen Einfluss auf MCF-7-Zellen. BLECs werden durch 100 µM GW2974 gehemmt. Es konnte gezeigt werden, dass die erhöhte Permeabilität mit einer Veränderung der TJ-Proteinexpression einhergeht. 4-Amino-1,8-naphthalimid senkt die Expression von Occludin auf mRNA- und Proteinebene. GW2974 vermindert zusätzlich die Expression von VE-Cadherin, Claudin-5 und ZO-1. Darüber hinaus wurde die Wirkung auf Effluxpumpen untersucht. Die Ergebnisse der mRNA- und Protein-expression weichen voneinander ab, weshalb eine genauere Untersuchung der Translationsvorgänge sinnvoll erscheint. Glut-1 wird in GW2974 behandelten Zellen überexprimiert, was auf eine erhöhte Aktivität der BLECs hinweist. GW2974 und 4-Amino-1,8-naphthalimid könnten durch ihre permeabilitätssteigernde Wirkung die Ansprechrate einer systemischen Behandlung von PatientInnen mit einem zerebral metastasierten Mammakarzinom erhöhen und somit ihre Prognose verbessern. Detaillierte Studien zu Kombinationstherapien, den notwendigen Wirkstoff-konzentrationen und eventuellen negativen neurologischen Wirkungen sollten erwogen werden. / The Blood-Brain Barrier (BBB) represents a selectively permeable barrier that controls the exchange of substances between the blood and the brain and thus has a neuroprotective effect. However, it not only prevents the passage of toxic metabolites, but also limits the access of therapeutic agents to the brain. Further research into methods to achieve higher drug concentrations in the brain is essential for the treatment of cerebral diseases such as cerebral metastatic breast cancer. The goal of this study was to identify drugs that increase the permeability of the BBB. The substance database LO1208 from Sigma-Aldrich was examined for its permeability-influencing properties as part of a high throughput drug screening (HTS). 31 of the examined substances showed an increase of the permeability on brain-like endothelial cells (BLECs) by at least 50%. Thereof 4-amino-1,8-naphthalimide (PARP inhibitor) and GW2974 (TKI) were selected for a more detailed analysis. Ibudilast (inhibitor of PDE4, MIF and Toll-like receptor-4) was found to be the third most active substance, although it did not cause any significant change in permeability. The measurement of the trans endothelial electrical resistance (TEER) and the permeability for fluorescein confirmed the results from the HTS and therefore is suggested to be used in further permeability tests in the future. Cell viability is not affected by 4 amino-1,8-naphthalmide. GW2974 and Ibudilast have a toxic effect on MCF-7 cells at a concentration of 500 µM, whereas BLECs are inhibited at a concentration of 100 µM of GW2974. The results show that the increased permeability is associated with a change in tight junction protein expression. 4-Amino-1,8-naphthalimide decreases the expression of occludin at mRNA and protein level. GW2974 also reduces the expression of VE-cadherin, claudin-5 and ZO-1. In addition to the abovementioned analysis, also the effect on efflux pumps was investigated. As the results of the mRNA and protein expression differ from each other, a more detailed analysis will be necessary to investigate the translation process. Glut-1 is overexpressed in GW2974-treated cells, which indicates an increased activity of the BLECs. GW2974 and 4-amino-1,8-naphthalimide could increase the response rate to systemic therapy of patients with cerebral metastatic breast cancer through their permeability-enhancing effect and thereby improve their prognosis. Detailed studies on combination therapies, the necessary drug concentrations and possible negative neurological effects are recommended to gain further insight.

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.

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.

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).