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Cholinergic Neurotransmission During Performance of a Sustained Attention Task after Traumatic Brain Injury
No full textAttentional deficits are prevalent following traumatic brain injury (TBI), and treatment options are limited due to an inadequate understanding of their etiology. Attentional functioning relies on an intact cholinergic network originating in the nucleus basalis of Meynert (nbM) and projecting to the medial prefrontal cortex (mPFC). In vivo microdialysis studies in non-TBI rats show task-evoked increases in acetylcholine (ACh) in the mPFC correlating with attentional performance, such studies are lacking in preclinical TBI models. It was hypothesized that TBI would decrease in vivo task-evoked release of ACh in the mPFC, correlating with impaired real-time performance on the 3-Choice Serial Reaction Time Test (3-CSRT) and with cholinergic neuron morphology reflective of a neurodegenerative phenotype in the nbM. Adult male rats (3-4 months old) trained in the 3-CSRT to the 2-s cue duration received either a moderate right controlled cortical impact or a Sham injury (n=10/group). On post-injury day (PID) 14, a guide cannula was surgically implanted in the right mPFC. At PID 21, dialysate samples were collected before and during 3-CSRT testing. Samples underwent analysis via high performance liquid chromatograph. After testing, rats underwent subsequent daily 3-CSRT testing for 5 days. At PID 26, cresyl violet staining was used to quantify cortical lesion volume and verify probe placement. Morphological assessments of cholinergic neurons in the contralateral and ipsilateral nbM were reconstructed and analyzed via Interactive Microscopy Image Analysis software (IMARIS). TBI rats exhibited significant deficits in sustained attention compared to their baseline performance and the Sham group. There were no differences in basal ACh efflux between Sham groups (p>0.05). After 3-CSRT onset, Sham rats showed a significant increase in task-related ACh release in the mPFC compared to both their baseline (p<0.05) and the TBI group (p<0.05). The TBI group did not exhibit a comparable increase in ACh release during the task. Morphological assessments revealed following TBI a significant reduction in soma area and volume in the ipsilateral nbM. In vivo sampling techniques such as microdialysis, provide increased temporal resolution and when combined with real-time behavioral performance can elucidate the correlative relationship between behaviorally-driven chemical dynamics
Application of transcriptomics, computational modeling, and evolutionary analysis to investigate the regulation of transcription elongation factors
No full textBetween transcription initiation and termination, RNA Polymerase II (RNAPII) associates with a variety of transcription elongation factors. These proteins modulate RNAPII elongation rate, RNAPII processivity, and the activity of enzymes that co-transcriptionally process RNA. The evolutionarily conserved Paf1 complex (Paf1C), consisting of Paf1, Ctr9, Rtf1, Cdc73, and Leo1, associates with elongating RNAPII and has been implicated in a variety of co-transcriptional processes. Defects in Paf1C have been associated with disease states and misregulation of co-transcriptional events. Therefore, an understanding of how Paf1C functions in transcription elongation is paramount to understanding how these functions impact human health. Thus, for my dissertation, I investigated Paf1C and other transcription elongation factors to advance understanding of the fundamental processes underlying gene expression. Herein I describe my progress in characterizing the roles of Paf1C and other transcription elongation factors through multiple approaches. Through one investigation, I elucidate the direct and indirect contributions of Paf1C subunits to chromatin and transcription regulation using multi-omic and computational approaches. This work uncovers subunit-specific functions of Paf1C regulating transcription elongation processivity and an indirect role of Paf1C in modulating transcript processing through splicing. I have additionally developed a model of transcription elongation dynamics (TED) to predict how RNAPII elongation is altered in the short- and long-term absence of Paf1C. Furthermore, I interrogate the contributions of Paf1C-dependent histone modifications towards Paf1C-dependent transcriptional phenotypes. This work comprehensively analyzes both the immediate and extended roles of each Paf1C subunit in transcription elongation and transcript regulation. I also describe my contributions to another study characterizing the phylogenetics of core transcription elongation factors including Paf1C across the tree of life. In collaboration with Aakash Grover, we uncovered patterns of apparent reductive evolution which may render some transcription elongation factors disposable in select clades. This work reveals flexibility in the composition of the transcription elongation machinery across species. Together, this work advances our understanding of the fundamental biological processes regulating transcription in a model eukaryote and across all domains of life
Appreciated and motivated: Examining whether, when, and why receiving gratitude enhances instrumentality intentions in romantic relationships
No full textInstrumentality—the act of facilitating another person’s goal pursuit—is common and beneficial in romantic relationships. Indeed, instrumental individuals can promote their partner’s goal progress and efficiency (Cappuzzello & Gere, 2018), while also fostering feelings of relational closeness (Fitzsimons & Fishbach, 2010). Despite our understanding of instrumentality’s benefits, little is known about how to motivate instrumentality in romantic relationships. Drawing from evidence showing that receiving gratitude can increase people’s prosocial and helping behaviors (Grant & Gino, 2010; Kubacka et al., 2011), I sought to understand whether and how receiving expressions of gratitude from one’s romantic partner might predict one’s intentions to be instrumental to the partner’s goal(s) in the near future (future instrumentality intentions; FIIs). In Study 1 (N = 244), I examined the correlation between gratitude receipt and FIIs using a recall design, wherein participants described a time when they were instrumental to their romantic partner’s goal(s), reported the extent to which their partner expressed gratitude, and reported their FIIs. In Studies 2 (N = 348) and 3 (N = 439), I experimentally manipulated whether or not romantic partners expressed gratitude for participants’ instrumental support using hypothetical scenarios, and examined whether the presence of gratitude increased FIIs. In Study 3, I also manipulated whether one’s romantic partner achieved or failed to achieve a focal goal, allowing investigation into whether the effects of gratitude receipt on FIIs might depend on the outcome of the goal pursuit. Across all studies, I found evidence that receiving gratitude positively predicted FIIs, even when controlling for factors known to impact support provision motivation (e.g., attachment insecurity). Additionally, gratitude receipt increased FIIs regardless of whether romantic partners succeeded or failed at achieving their goal. I also found consistent evidence for a mechanism through which gratitude receipt increased FIIs: by enhancing individuals’ self-efficacy beliefs. To my knowledge, this work is the first to consider how expressions of gratitude between romantic partners might be leveraged to motivate future instrumentality. These findings serve as a foundation for future work examining additional antecedents to instrumental support provision, and longitudinal consequences of gratitude receipt for support processes in romantic relationships
The Therapeutic Application of Medium Chain Acylcarnitines for Genetic Disorders of Long Chain Fatty Acid Oxidation
No full textMedium chain fatty acids (MFA) are commonly used as oral therapeutics to correct energy deficits causes by genetic long-chain fatty acid oxidation disorders (LCFAOD); however, patients taking medium chain fatty acid therapies often continue to experience hospitalization due to rhabdomyolysis. Before fatty acids of any chain length can be shortened and utilized to produce cellular energy through ß-oxidation, the free fatty acid must be conjugated to coenzyme A (CoA) to allow acyl-CoA production. The fatty acid chain length specific enzyme family known as acyl-CoA synthetases catalyze this necessary reaction. We show the five known medium-chain acyl-CoA synthetases (ACSMs) are expressed in the liver and the kidney but not in the skeletal muscle or heart tissue. Two pre-clinical mouse models of LCFAODs were used to show that current medium chain fatty acid therapies are ineffective in tissues lacking ACSMs. We demonstrate in vivo and in vitro that without the expression of the ACSMs, medium chain fatty acids are inefficiently oxidized through the carnitine shuttle and long-chain fatty acid enzymatic pathway. In testing the oral bioavailability of medium-chain therapies, we found that most of the oral treatment is taken up by the liver and does not reach the blood stream for systemic therapeutic use.
Carnitine-O-acetyltransferase (CrAT) is a mitochondrial enzyme shown to reversibly react short and medium-chain carnitines to their CoA conjugate forms and vice versa to balance cellular pools of free-carnitine and free-CoA in support of the ß-oxidation pathway. We show that CrAT is robustly expressed in the skeletal muscle and heart. Liver expresses very low levels of CrAT, allowing oral doses of medium-chain carnitines to partially escape first-pass liver metabolism and reach systemic circulation. We exploited CrAT’s expression profile and activity, testing the therapeutic potential of medium-chain carnitines against skeletomuscular symptoms of LCFAOD using three preclinical LCFAOD mouse models in both in vivo and in vitro experiments. Additional studies were performed with non-human primate tissue, wild type mice, and human cell lines, demonstrating treatment efficacy in near-human models. Finally, we demonstrate the therapeutic effects of medium-chain carnitines are ablated in the absence of CrAT, using a CrAT deficient mouse model
Bioorthogonal and Photoswitchable Tools for Protein Control
No full textI have utilized key tools in chemical biology research, namely bioorthogonal chemistry, genetic code expansion, and targeted protein degradation to investigate new ways to impart specific and selective control over protein function. I have developed and optimized a new tool for in cellulo protein bioconjugation quantification using simple western blotting techniques (Chapter 1.0) and site-specifically recruited bioorthogonally modified E3 ligase ligands to proteins with an expanded genetic code for investigation of protein degradability (Chapter 2.0). Expanding on these methods, I have also imparted optical control of site-specific degradation via photocaged, bioorthogonal E3 ligase ligands (Chapter 3.0) and developed a tool for the investigation of hydrophobic degradation through bioconjugation of two hydrophobic ligands (Chapter 4.0).
In addition to my extensive work in conjugating various ligands to proteins with expanded genetic codes and investigating the resultant impact on protein expression, I have also utilized photoswitchable molecules to impart reversible optical control over proteins and peptides. We developed the best photoswitchable amino acid to date and utilized it to impart optical control over protein translation (Chapter 6.0). I have also developed a series of fluorinated azobenzene chromophores for rigid stapling and reversible isomerization of strategically designed peptides (Chapter 7.0), and fluorinated unnatural amino acids for photocontrol over protein structure and resultant function in biological systems (Chapter 8.0)
Stroke of genius: how TGFα promotes angiogenesis & post-stroke recovery
No full textStrokes are a major global health issue, causing approximately 5.5 million deaths annually and imposing a financial burden on survivors and caregivers. With the aging population, stroke incidences are expected to increase significantly, and by 2030, about 4% of adults are projected to have experienced a stroke. Strokes can be classified as ischemic or hemorrhagic, with ischemic strokes, characterized by blood clots blocking blood flow to the brain, making up 87% of cases. Current treatments for ischemic strokes, such as intravenous thrombolysis and endovascular thrombectomy, have strict time constraints, leaving a substantial need for therapies that improve tissue recovery.
Post-stroke recovery involves neurogenesis, synaptogenesis, and angiogenesis, all critical for restoring function in affected brain areas. Enhancing revascularization, especially through endothelial cell proliferation and migration, is essential for creating a supportive environment for brain repair. Growth factors play vital roles in promoting post stroke brain repair. Our previous study shows that transforming growth factor alpha (TGFα) is critical in promoting white matter integrity and long-term recovery after stroke, however its role in post-stroke angiogenesis remains underexplored.
This study used TGFα knockout (KO) mouse and wild-type controls to examine the role of TGFα in post-stroke angiogenesis and functional recovery. Immunofluorescence quantification revealed that KO mice showed significantly lower endothelial cell (EC) proliferation and angiogenesis, as revealed by the reduced numbers of BrdU+CD31+ cells and Ki67+CD31+ cells, respectively. Additionally, in vitro studies using mBMECs confirmed that TGFα directly enhances EC proliferation and migration. These molecular findings align with murine behavioral tests, which demonstrated increased cognitive deficits in the KO group, correlating with the decreased angiogenesis. Collectively, these results indicate that TGFα supports endothelial network formation and vascular recovery, highlighting its therapeutic potential to promote revascularization and improve functional outcomes after ischemic stroke
Olfactory Navigational Strategies in Aqueous Water Plumes and Deterministic Casting Algorithm Analysis
No full textThis dissertation explores olfactory navigational strategies in aqueous water plumes and analyzes deterministic casting algorithms, providing insights into the complex dynamics of odor-guided navigation. The study comprises two main parts: an investigation of simple olfactory algorithms in various odor landscapes, and an in-depth analysis of a deterministic casting algorithm. In the first part, two local algorithms, bilateral search and temporal comparison (”casting”), are compared for navigating to an odor source in various air and water plumes. Using planar laser-induced fluorescence (PLIF) datasets, these algorithms were simulated under different flow conditions and odor source configurations. The model parameters are optimized to maximize success rates and minimize path tortuosity, revealing the trade-offs between exploration and direct navigation. My findings demonstrate that both algorithms can be tuned to successfully locate odor sources in a wide range of odor landscapes, with performance varying based on plume characteristics and algorithm parameters. The second part focuses on a deterministic casting algorithm, examining its fixed points, stability, basin of attraction, and bifurcation behavior. Mathematically, I prove the existence of two fixed points and analyze their stability in relation to key parameters such as velocity, sensor length, and casting angle. Through bifurcation analysis, transitions from stable behavior to chaos are observed as the casting angle varies. I also apply the algorithm\ to real water plume data, optimizing parameters for success rate and efficiency. This research contributes to our understanding of olfactory navigation in complex environments and has implications for both biological systems and artificial olfactory navigation in robotics. The insights gained from this study could inform the development of more efficient and adaptive navigation strategies in various fields, from environmental monitoring to search and rescue operations
Exploiting ferroptosis as a therapeutic vulnerability in cyclin E1-high ovarian cancers
No full textAmong ovarian cancer subtypes, CCNE1-amplification is frequently associated with aggressive tumor growth and limited treatment options, resulting from homologous recombination (HR) proficiency and a subsequent de novo resistance to DNA damaging agents. This underscores a need for alternative, targeted therapies. Ferroptosis is an iron-dependent form of regulated cell death, characterized by generation of reactive oxygen species (ROS) and excessive membrane peroxidation leading to cellular death. Here, we present the metabolic consequences of cyclin E1 overexpression highlighting ferroptosis as a vulnerability of this ovarian cancer profile. We demonstrate that cyclin E1 overexpression promotes a proliferative phenotype that correlates with an upregulation of iron-containing proteins that are involved in DNA replication and repair. We also demonstrate that cyclin E1-high cells exhibit a marked increase in labile iron content, potentially to meet an increased iron demand, which in turn leads to elevated ROS production. This increase in labile iron and ROS sensitizes cyclin E1-high cells to ferroptosis, while treatment with ferroptosis inhibitors and antioxidants rescues cell viability. Our findings highlight the link between cyclin E-overexpression and dysregulated iron metabolism in ovarian cancer, offering a novel therapeutic opportunity. By targeting ferroptosis in cyclin E1-amplified tumors, these results suggest a novel strategy to treat this aggressive cancer subtype
Comparison of the Metabolic Capacity Between Human and Porcine Liver Microsomes
No full textOrgan transplantation is currently the treatment of choice for end stage organ diseases. However, organ shortages have greatly limited the number of patients who may receive organ transplantation. Even with the use of organs from living-donors, the number of patients on the waiting list continues to increase every day. It is critical to find a solution for the organ shortages. Xenotransplantation, a process of transplanting organs from one species to another has received much attention over the past several years. Pig has been identified as an ideal organ donor for xenotransplantation. Researchers have successfully produced pigs with gene editing and human gene insertion. Liver is the second most transplanted organ and the primary organ that metabolizes endogenous and exogenous chemicals. Given that transplant patients receive treatment with several drugs that are metabolized, it is essential to understand the metabolic capacity of pig livers. This study compares the metabolism of testosterone by human and porcine liver microsomes. The formation of five typical testosterone metabolites were evaluated, highlighting the metabolic differences between the two species. Comparison of enzyme activities and production rates of major and minor metabolites revealed that both human and pig microsomes primarily produced 6β-hydroxytestosterone (6β-OHT) as the major metabolite. Human microsomes generally produced more 6β-OHT and exhibited higher enzyme activity compared to pig microsomes. Among the minor metabolites, human microsomes produced 2⍺-hydroxytestosterone (2⍺-OHT) and 6⍺-hydroxytestosterone (6⍺-OHT), while pig microsomes did not produce any detectable amount of 2⍺-OHT.
The study observed a decline in the enzyme activity for 6β-OHT production over time for both human and pig microsomes, potentially due to NADPH depletion, autoinhibition, or enzyme denaturation. At 60 minutes, the concentrations of certain metabolites, including 6β-OHT and 16β-hydroxytestosterone (16β-OHT), were lower than at earlier time points, suggesting further transformation or breakdown of metabolites.
By providing detailed metabolic profiles and enzyme activity comparisons, this research contributes to the foundational knowledge necessary for understanding drug metabolism in human and porcine liver microsomes, which will be critical for advancing xenotransplantation of the liver
Doubly Robust Estimation of Causal Effects in Observational Data with Time-to-event Outcomes
No full textThere is a growing need for novel methods to estimate causal effects in observational data with time-to-event outcomes to provide reliable guidance for treatment strategies in life-threatening conditions. Bias in causal effect estimation can arise from treatment effect heterogeneity, model misspecification, and unmeasured confounders. This dissertation proposes novel approaches to address these challenges.
In the first part of the dissertation, I propose a framework for estimating conditional average treatment effects (CATE) in time-to-event data with competing risks. It accounts for treatment effect heterogeneity and protect against model misspecification. Using targeted maximum likelihood estimation (TMLE), I develop a substitution estimator based on cumulative incidence functions (CIF), derived from the efficient influence function (EIF). This estimator is doubly robust, and achieves asymptotic efficiency under mild conditions. Simulations demonstrate its favorable performance across various settings, confirm the double robustness, asymptotic normality and the flexibility of the framework incorporating different regression and machine learning models. Additionally, I construct variable importance measures to identify variables contributing to treatment effect heterogeneity and estimation, providing guidance for clinicians on the critical biomarkers or information to collect. The method is applied to electronic health record data to evaluate the treatment effect of steroids on ICU mortality among sepsis patients.
In the second part, I develop a novel instrumental variable (IV) method for estimating average treatment effects in data with unmeasured confounding. Derived from the EIF, this model-free estimator achieves double robustness and asymptotic efficiency under certain mild conditions. Defined by CIF, the method is adaptable to time-to-event data with competing risks. Our method also enables the incorporation of various models for outcome, treatment, and censoring. Extensive simulations demonstrate the double robustness, asymptotic normality, and the capability to analyze complex data. This proposed IV method is applied to investigate the effect of hydrocortisone on mortality among ICU patients with vasopressor-dependent septic shock.
Public health significance: The proposed methods address key challenges in estimating causal treatment effects in time-to-event data, including treatment effect heterogeneity, model misspecification, and unmeasured confounders. This dissertation provides powerful tools for optimizing treatment strategies, improving estimation reliability, and advancing healthcare research