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This research addresses the challenges associated with traditional antimicrobial peptide (AMP) anchoring by introducing an innovative artificial biomaterial platform, thereby enhancing AMP stability and efficacy. In Chapter 1, we establish the importance of studying lipid diversity for comprehending AMP-membrane interactions and underline our objectives: the creation of a smart biopolymer with scaffolds, spacers, and AMPs, and exploring bacterial versus mammalian lipid composition in AMP selectivity through solid-state NMR spectroscopy. Chapter 2 details the methodologies employed, ranging from biomaterial synthesis using the E. coli expression system to characterization techniques like dynamic light scattering and solid-state NMR methods. Chapter 3 introduces our unique biopolymer tether approach for augmenting AMP activity, supported by results from genetic modifications. Chapter 4 delves into the impact of lamellar and nonlamellar lipid compositions on bilayer properties, offering novel methodologies to study unsaturated lipid systems, which were previously challenging. Chapter 5 focuses on the quantitative analysis of the role of cholesterol in lipid bilayer rigidity, permeability, and micro-mechanics, revealing a correlation between bending rigidity and lipid packing. Building on these insights, Chapter 6 examines the effects of the Cathelicidin antimicrobial peptide LL37 and cholesterol on DOPE and DOPC membranes. Combining the probe lipid approach with molecular dynamics simulations, this research offers unprecedented atomic-level insights into the interactions between AMPs, cholesterol, and lipid bilayers. Collectively, the study provides a comprehensive understanding of AMP activity and selectivity in various lipid environments.

Viroporins are a class of viral membrane proteins that play diverse roles in the viral infection cycle. Viroporins are known to play roles in ion transport, inducing membrane curvature, as well as participating in a variety of protein-protein interactions. However, there is little known about the oligomeric state of viroporins. The lack of quantitative data on the oligomeric state of viroporins is due to key analytical challenges associated viroporins, including that they are 1) hydrophobic, and require a membrane mimetic for analysis, 2) small, making viroporins unamenable to some structural biology techniques such as cryo-EM, and 3) contain intrinsically disordered regions, which also poses challenges for structural characterization. To overcome these analytical challenges, this dissertation outlines the application of the unique combination of native mass spectrometry (MS) and nanodiscs for the oligomeric state determination of viroporins. The unique combination of native MS and nanodiscs can be used to determine the oligomeric state and lipid specificities of membrane proteins (such as viroporins) while embedded within a lipid bilayer using mass defect analysis. This dissertation reviews how to perform mass defect analysis, provides examples of how mass defect analysis has been applied in the past, as well as providing tips for overcoming some of the technical limitations associated with mass defect analysis. This dissertation also outlines a structure-activity relationship study performed to identify novel charge reducing agents for native MS. Charge reducing agents can be useful for the preservation of fragile complexes, such as viroporins, during native MS. Here, we found improved charge reducing agents that were effective on a wide range of analytes. We also uncovered the chemical principles governing charge reduction, which may provide a basis for the development of even better novel charge reagents in the future. The first viroporin characterized with nati

Lipids play critical roles in modulating membrane protein structure, interactions, and activity. Changes in lipid composition and their organization are distinguishing features for study membrane protein. Nanodiscs provide a tunable membrane mimetic that can model these endogenous protein-lipid interactions in a nanoscale lipid bilayer. Lipid exchange is a technique for measuring how lipids partition around membrane proteins.2 The rationale behind our interest in studying lipid exchange is to define annular lipid environment preferences, including electrostatic properties and hydrophobic lengths. Through the application of lipid exchange, specific lipids with properties will undergo preferential phase partitioning to remodel the membrane and optimize the lipid environment. However, most studies of membrane proteins with nanodiscs use simple synthetic lipids that lack the head group and fatty acyl diversity of natural extracts. Prior research has successfully used natural lipid extracts in nanodiscs that more accurately mimic natural environments, but it is not clear how nanodisc assembly may bias the incorporated lipid profiles. Here, we applied lipidomics to investigate how nanodisc assembly conditions affect the profile of natural lipids in nanodiscs. Specifically, we tested the effects of assembly temperature, nanodisc size, and lipidome extract complexity. Collectively, these practices then enabled our lipidomics lipid exchange application of nanodiscs to study protein-lipid interactions in complex natural lipid environments. In particular, the bulk effects were accessed within the lipid classes that differ from the headgroup, while specific interactions were examined based on both the statistical significance and the magnitude of the fold change. In summary, incorporating nanodiscs into analytical methods with advanced lipid exchange lipidomics approaches can aid in identifying and localize essential lipids to membrane proteins and characterize the influence of

The Colorado River is the main water source in the southern United States serving seven states and Mexico. Overallocation of the Colorado river in the original compact governing Colorado River water allocation and drought conditions over the past two decades have left the river’s reservoirs (primarily Lakes Powell and Mead) at historically low water levels, requiring the need for new administrative measures. To help maintain higher Lake Mead elevations, an initial set of strategies was established under the 2007 Interim Guidelines (2007-IG) outlined a set of penalties to Arizona and Nevada once Lake Mead levels reached 1075 ft amsl. Mexico later joined the conservation effort under Minute 319 also accepting reductions in deliveries when Lake Mead reached1075ft amsl. Rapidly decreasing lake elevations and drought conditions after the adoption of the 2007-IG motivated new legislation even before Lake Mead hit the 1075ft critical mark. The new agreement, the Drought Contingency Plan (DCP) of 2019, was developed and adopted by all seven Colorado River basin states and Mexico. The DCP increased penalties beyond those established in the 2007-IG. The DCP also added new penalty tiers beginning with reductions at Lake Mead elevations of 1090ft amsl. New penalties included reductions to California which was exempt from reductions under the 2007-IG; it also added measures relative to conservation and allowed state to recover reductions specific to the DCP in later years if lake levels recovered. To assess the effectiveness of the DCP in comparison to the 2007-IG. sustainability, resilience, and robustness (SRR) metrics applied under conditions specific to either the 2007-IG and the DCP. Four metrics are used here as measures of DCP performance. These include functionality, sustainability, mean time to repair (MTTR) and mean time between failures (MTBF). Functionality is calculated annually and measures fractional demand delivered over the period analyzed. Similarly, sustainabi

The work detailed in this dissertation investigates the challenges of chronic neurochemical measurements in vivo and expands the toolbox of the neuroanalytical chemist working to overcome those challenges. Following the introduction in Chapter 1, Chapter 2 investigates the effects that biofouling has on chronically implanted electrodes, specifically for long-term FSCV. The effects on impedance and reference electrode polarization are explored, and a three-electrode FSCV configuration is designed and employed to mitigate the effects on impedance. Chapter 3 describes the fabrication, characterization, and utilization of an iridium oxide (IrOx) reference electrode. The IrOx reference electrode is shown to perform as well as the conventional Ag/AgCl-wire reference electrode in vitro and in vivo, with the additional benefit of biocompatibility. IrOx has the capability to provide a stable reference potential for chronic FSCV in animals and eventually humans. Chapter 4 details the design and employment of an Ommaya-reservoir-based probe for chronic, minimally invasive collection of cerebrospinal fluid (CSF) from non-human primates (NHPs). The development and optimization of an ion-pair HPLC method with electrochemical detection for neurochemical analysis of the collected CSF is described. The use of this novel collection strategy to investigate the effects of vagus nerve stimulation on CSF neurotransmitter levels is explored. Additionally, the behavioral characteristics of NHPs during the completion of custom-written visual learning tasks involving social hierarchy are studied. Combination of these behavioral observations with CSF neurotransmitter analysis via Ommaya reservoir is feasible and would allow for novel insight into the relationships between neurotransmission, social hierarchy, and learning. Together, the work presented in this dissertation offers novel insights, tools, and strategies toward making accurate and meaningful neurochemical measurements in vivo and p

Antimicrobial peptides (AMPs) are an important part of the innate immune system and demonstrate promising applications in the fight against antibiotic resistant infections due to their unique mechanism of targeting bacterial membranes. Nevertheless, it is challenging to study the interactions of these peptides within lipid bilayers, making it difficult to understand their mechanisms of toxicity and selectivity. Lipoprotein nanodiscs are ideally suited for the study of AMPs with native mass spectrometry because they provide a relatively monodisperse nanoscale lipid bilayer environment for delivering membrane peptides into the gas phase. However, native mass spectrometry of nanodiscs produces complex spectra that can be challenging to assign unambiguously. This dissertation first describes a method to simplify interpretation of nanodisc spectra using an engineered series of mutant membrane scaffold proteins (MSP) that do not affect nanodisc formation but shift the masses of nanodiscs in a controllable way, thus eliminating isobaric interference from the lipids. Moreover, by mixing two different belts before assembly, the stoichiometry of MSP is encoded in the peak shape, which allows the stoichiometry to be assigned unambiguously from a single spectrum. Next, this dissertation describes the use of fast photochemical oxidation of peptides (FPOP), an irreversible footprinting technique that labels solvent accessible residues, in combination with LC-MS-MS and native charge detection-mass spectrometry to study AMP-lipid interactions and surface exposed residues within different lipid bilayer nanodiscs. The findings herein provide complementary information on the potential modes of action and lipid selectivity of various AMPs.

The field of bioconjugate chemistry is expansive and rapidly evolving, perennially introducing new strategies for labeling biological entities with high specificity. Advancement of bioconjugate protocols provides researchers with new tools to study complex biological systems, which ultimately expedites the rate of scientific discoveries, and even provides new insights important to development of novel therapeutics. One of the most popular sub-fields of bioconjugate chemistry pertains to modification and functionalization of proteins via small molecular probes for various applications including protein purification, fluorescent imaging, and therapeutic delivery. As such, our group has been dedicated to the development of molecular probes capable of versatile, orthogonal modification of proteins. Furthermore, we aimed to develop systems with reactivity that is triggered under specific chemical environments, which provides additional temporal and proximal selectivity. Herein, we provide an overview of bioconjugate and bioorthogonal protein labeling strategies and their associated applications. Furthermore, we highlight the development of several novel bioconjugate systems capable of site-selective protein modification of either tyrosine, or lysine. More specifically, we note the utility of aryl diazonium ions within bioconjugation, and the evolution and applications of protected aryl diazonium systems including triazabutadienes and triazenes. To that end, we first report the ability to fluorescently label proteins via a one-step protocol using our novel coumarin triazabutadienes in the presence of either mild acid, or UV irradiation. We next introduce a cyclooctyne triazabutadienes capable of rapid modification of proteins with a bioorthogonal handle for bifurcated azide functionalization. Inspired by our work with triazabutadiene, we show the repurposing of phenyldiazenyl triazenes to label proteins in an analogous fashion. Importantly, we reported for the first time

Neurological disorders may cause various disabilities. The mechanism of neurologicaldisorders may attribute to neuronal, structural, electrical, or molecular abnormalities. Many therapies such as surgery, deep brain/spinal cord stimulation are invasive treatments but with amazing treatment results. More common therapy of various types of medicine is the main method for clinical application. This work focuses on a few types of therapeutic neuroactive molecules and aims to investigate the In vitro and In vivo properties. Chapter 1 will introduce the principles of classic neurological disorders and the current common detection methods for neurotransmitters and peptides. Chapter 2 will introduce the method development of nano liquid chromatography coupled to tandem mass spectrometry for a better analysis of low abundance neurotherapeutic molecules. Chapter 3 will investigate the In vitro stability improvement of four types of glycosylated peptide-based drugs. Chapter 4 will investigate the improvement of In vivo stability and blood brain barrier (BBB) transmission of glycosylated neuroactive peptide-based drugs with developed online-preservation microdialysis coupled to liquid chromatography tandem mass spectrometry technique. We also studied the impact of micelle-assisted drug delivery formula for improved bioavailability. Chapter 5 will introduce the small neuroactive molecule, ketamine and its application in the treatment of LID. We studied the pharmacokinetic profile of single injection and infusion injection in the rat model and human model respectively. Chapter 6 will conclude the work and introduce the future directions of more therapeutic neuroactive peptides.

To better understand the brain and how it communicates, we need to build tools that can quantify neurochemical signaling. By understanding brain function, new treatments can be developed for neurodegenerative diseases and disorders. In addition, by gaining insight into the inner workings of the brain, we can answer fundamental questions regarding learning, memory, and behavior. In this dissertation, several new tools for neurotransmitter measurement and analysis will be discussed. A new method, alternating current-coupled voltammetry has been developed, making in vivo neurotransmitter measurements safe for the subject, and moving fast-scan cyclic-voltammetry (FSCV) towards FDA compliance. A measurement and software platform was developed that expands the capabilities of FSCV, and fast-scan controlled-adsorption voltammetry (FSCAV). This new system not only allows for FSCV and FSCAV measurements to occur simultaneously, allowing for the measurement of both tonic and phasic signaling but also expands the instrumentation to allow for more individually addressable electrodes for neurotransmitter measurement. The system can also measure multiple neurotransmitters with different waveforms on discrete electrodes which previously was limited. Data analysis for FSCV was also expanded by the application of machine learning in automated neurotransmitter identification. We built classification and regression models to interpret and quantify unknown raw voltammetric data without input from the operator. Lastly, commonly used carbon-fibers in microelectrodes were studied to determine the structure-function relationship between the chemical properties of each, for application selection. Together these new tools are an important advancement in neuroanalytical instrumentation by enabling multi-region analysis, and automated identification of chemical species.

Elucidating the chemical changes that underlie dysfunction of the central nervous system requires tools and techniques that enable rapid and accurate neurotransmitter quantification. The dopaminergic system is of major interest in many research groups due to its involvement in Parkinson’s disease, chronic pain, schizophrenia, and many more conditions. However, dopamine measurement in the CNS is challenging due to complex signaling dynamics. Phasic signals occur on the second to sub-second temporal regime while tonic changes arise over minutes to hours or longer. Presently, tonic and phasic dopaminergic signals cannot be measured simultaneously at a single probe. Using fast-scan cyclic voltammetry (FSCV) and fast-scan controlled-adsorption voltammetry (FSCAV) these studies investigated the response of the mesolimbic dopamine system to pain and pain relief as well as drug therapies. Opioids, which are known to interact with the mesolimbic reward pathway, were given to determine their influence on tonic and phasic dopamine signaling dynamics in various pain states. Using rodent models for pain, these investigations elucidated: (1) how ongoing (or sustained) pain alters both tonic and phasic dopamine release, (2) that acute thermal pain and pain relief cause a biphasic dopaminergic response in the mesolimbic pathway, and (3) a modulatory effect of mu opioids in the anterior cingulate cortex on mesolimbic dopamine release during acute pain. Thus, enhanced understanding of the the underlying biochemical mechanisms involved in pain perception and response will facilitate the development of more effective, less addictive pain therapeutics.