In the present study, I examined how the quality of relatedness (operationalized as Mutually Responsive Orientation) in the mother-child relationship in kindergarten students affects the association between the mother's values about school and the child's emotional engagement in school. Relatedness, as described by Self-Determination Theory, posits when a child feels a sense of relatedness—supported, respected, and connected with another individual—the child will be more likely to integrate that person's values into their own belief system. Sixty-six mother-child dyads were observed and videotaped doing four everyday activities (mother worked while child played independently, mother and child had a snack, mother and child played a game, mother and child cleaned up). In addition, the mothers filled out a questionnaire reporting their own valuing of school, and children participated in the Berkeley Puppet Interview, a semi-structured interview between researcher and child in which children reported their levels of emotional engagement in school to two dog puppets. Data were coded and then analyzed using multiple regression analysis. Relatedness between mother and child was found to have a moderating effect on the relation between mothers' values about school and children's school engagement. The strongest relation between mothers' values and children's school engagement was found when mother-child relatedness was low. When mother-child relatedness was high, the engagement of the child was not affected by the mother's valuing of school. The study findings offer implications for how children experiencing high levels of relatedness with their mothers will be able to be more successful in the school setting, regardless of the mothers' valuing of their own school experiences.

Skeletal muscle atrophy is a common, debilitating consequence of muscle disuse, malnutrition, critical illness, musculoskeletal conditions, neurological disease, cancer, and organ failure. Despite its prevalence, little is known about the molecular pathogenesis of this devastating condition due in large part to an incomplete understanding of the molecular mechanisms that drive the atrophy process. In previous studies, we identified the transcription factor ATF4 as a critical mediator of skeletal muscle atrophy. We found that ATF4 is necessary and sufficient for skeletal muscle atrophy during limb immobilization. However, ATF4 mKO mice were only partially protected from skeletal muscle atrophy during limb immobilization, indicating the existence of another pro-atrophy factor that acts independently of the ATF4 pathway. Using mouse models, we identify p53 as this ATF4-independent factor. We show that skeletal muscle atrophy increases p53 expression in skeletal muscle fibers. In addition, overexpression of p53 causes skeletal muscle atrophy. Further, p53 mKO mice are partially resistant to muscle atrophy during limb immobilization. Taken together, these data indicate that like ATF4, p53 is sufficient and required for skeletal muscle atrophy during limb immobilization. Importantly, overexpression of p53 induces muscle atrophy in the absence of ATF4, whereas ATF4-mediated muscle atrophy does not require p53. Furthermore, overexpression of p53 and ATF4 induces greater muscle atrophy than p53 or ATF4 alone. Moreover, skeletal muscle lacking both p53 and ATF4 is more resistant to skeletal muscle atrophy than muscle lacking either p53 or ATF4 alone. Taken together, these data indicate that p53 and ATF4 mediate distinct and additive mechanisms to skeletal muscle atrophy. However, the precise mechanism by which p53 and ATF4 cause skeletal muscle atrophy remained unclear. Using genome-wide expression arrays, we identify p21 as a skeletal muscle mRNA that is highly induced by p53 and ATF4 during limb immobilization. Further, overexpression of p21 causes skeletal muscle atrophy. In addition, p21 is required for muscle atrophy due to limb immobilization, p53, and ATF4. Collectively, these results identify p53 and ATF4 as critical and complementary mediators of skeletal muscle atrophy during limb immobilization, and discover p21 as an essential downstream mediator of the p53 and ATF4 pathways.

Preeclampsia is a hypertensive disorder of pregnancy that is diagnosed after the 20th week of gestation. It is defined by the American College of Obstetrics and Gynecology as de novo hypertension of at least 140/90 in a pregnant woman. Proteinuria with the hypertension is sufficient but not required for the diagnosis, especially if a woman displays severe symptoms such as headache, blurry vision, right upper quadrant pain, and low platelet count. Despite significant research, preeclampsia continues to kill 76,000 mothers and 500,000 babies per year worldwide. It causes short and long term consequences such as future metabolic and cardiovascular events for the mother and the child born during a pregnancy affected by preeclampsia. A delay in diagnosis and delayed access to appropriate care is a core cause of the preeclampsia related morbidity and severe mortality worldwide. Despite being in the medical literature since the time of the ancient Greeks, there is currently no significant predictive, preventative, therapeutic, and curative agent for preeclampsia except for an often preterm delivery of the fetus. The complex pathogenesis of preeclampsia has challenged the ability to effectively predict preeclampsia to decrease the delay in this diagnosis. Consequently, an early intervention or triage to higher level obstetric care is hindered. The lack of an early biomarker for preeclampsia also represents a major barrier to treat preeclampsia before major clinical symptoms emerge and the cycle of future cardiovascular risk for mom and baby begins. Novel, very early pregnancy predictive tests for preeclampsia may provide significant clinical utility. Furthermore, a biomarker that is linked with an early pathogenic mechanism in the first trimester development of preeclampsia would reveal a new avenue of early, first trimester intervention to treat and prevent this devastating disease. This work details the search for such a biomarker linked to an early initiator of the molecular pathogenesis of preeclampsia. These microRNA data highlight very important dysregulated mechanisms including immunologic, cell growth, and angiogenic mechanisms. T cells and the role of indoleamine 2,3 dioxygenase (IDO) is important in the early, maternal immune tolerance to the placenta and pregnancy. As poor placentation is a core cause of preeclampsia, a decreased immune tolerance to it is hypothesized to lead to preeclampsia. Furthermore, low IDO activity has been observed in the placentas of preeclamptic pregnancies which may make it a viable biomarker. These IDO-knock out mouse data, demonstrate that chronic IDO deficiency is sufficient to cause some of the core phenotypes of preeclampsia including renal dysfunction, vascular endothelial dysfunction, fetal growth restriction, and a slight increase in systolic blood pressure. This model does not completely phenocopy human preeclampsia. An investigation of early markers that are linked to vascular, immune, and renal abnormalities highlights the vasopressin pathway as a potential biomarker and early initiator of the pathogenesis of preeclampsia. These data demonstrate that copeptin, as a stable marker of vasopressin secretion, is robustly predictive of the development of late pregnancy human preeclampsia, as early as the 6th week of gestation. Furthermore, a mouse model with chronic infusion of vasopressin throughout mouse gestation phenocopies all the essential aspects of human preeclampsia: pregnancy specific hypertension, proteinuria, pathognomonic glomerular endotheliosis, fetal growth restriction, and increased fetal death. Further research must be done to elucidate the immunologic, vascular, and fetal programming phenotypes of this model. This work posits the possibility that the vasopressin pathway may provide new predictive, preventative, therapeutic, and potentially curative modalities for preeclampsia.

Parkinson's disease (PD) is a slow-progressive neurodegenerative disorder affecting 5-6 million people around the globe. The disease is manifested by the rapid deterioration of dopaminergic cells in the substantia nigra portion of the brain; however, the pathological mechanism of selective dopaminergic neuronal death is unknown. A reduction in levels of 3,4-dihydroxyphenylacetaldehyde (DOPAL) is biologically critical as this aldehyde has been shown to be toxic to dopaminergic cells and is a highly reactive electrophile. Investigating neuronal protein targets is essential in determining the cause of toxicity. An essential protein-GAPDH (e.g., glyceraldehyde-3-phosphate dehydrogenase) is an abundantly expressed enzyme known for its glycolytic activity, and recent research has implicated its role in oxidative stress-mediated neuronal death. This work positively shows GAPDH as a target for DOPAL modification, and, for the first time, DOPAL is identified as a potent inhibitor for GAPDH enzymatic activity. LC-MS and other chemical probes (ie. thiol and amine modifiers) show that DOPAL modifies specific –Lys, -Arg, and –Cys residues in the cofactor binding-domain of GAPDH. The enzyme inhibition is also time and DOPAL dose-dependent. DOPAL has a unique structure, containing two reactive functional groups: an aldehyde and catechol ring. In-house syntheses of DOPAL analogues, containing the catechol group and lacking the aldehyde, and vice versa have been tested on GAPDH and do not inhibit or modify GAPDH. Therefore, both the catechol and aldehyde groups of DOPAL are specific to binding with GAPDH and are necessary to achieve modification and enzyme inhibition. In addition to finding a novel enzyme inhibited and modified by DOPAL, this work has also confirmed linking DOPAL levels to a fungicide associated with PD risk. This benzimidazole fungicide, benomyl was shown to inhibit ALDH2 in the SH-SY5Y neuroblastoma cell line via an increase in DOPAL and a decrease in DOPAC. The ratios of DOPAL and DOPAC, the product of ALDH, were measured by HPLC-ECD, and found that benomyl does inhibit ALDH2 in this dopaminergic cell model. The cytotoxicity of benomyl, DA, DOPAL and the combination of DA or DOPAL with benomyl was assessed by MTT assay. Surprisingly, the only toxic combination was the combination of DA or DOPAL with benomyl. In fact, this toxicity appears to be synergistic, as none of the single treatments are significantly toxic to the cells. This synergistic effect also affects GAPDH aggregation. The cell morphology is also drastically different in the presence of the combined treatments, compared to individual treatment of DA, DOPAL or benomyl; cells start to ebb and show apoptotic-like features at just 2h. A second class of pesticides, named chlorpyrifos and chlorpyrifos-oxon were tested for toxicity in PC6-3These compounds were toxic to these cells due to DOPAL accumulation reaching high levels in the 100 µM range. Exposure to environmental toxins such as pesticides and fungicides has long been linked to PD risk, but only recently to DOPAL levels. This work provides a novel mechanism by which fungicide exposure may stimulate PD pathogenesis.

Pseudomonas aeruginosa is a Gram-negative bacterium that causes acute nosocomial infections as well as chronic infections in cystic fibrosis (CF) patients. P. aeruginosa utilizes a type III secretion system (T3SS) during acute infections to promote host cell cytotoxicity and inhibit phagocytosis. Regulation of T3SS expression can be classified into two distinct categories: intrinsic and extrinsic. T3SS intrinsic regulation involves the well-characterized ExsECDA cascade that controls T3SS gene transcription. Extrinsic regulation involves global regulatory systems that affect T3SS expression. Despite general knowledge of global regulation of T3SS expression, few specific mechanisms have been elucidated in detail. The overall goal of my thesis work was to provide clarity to global regulatory mechanisms controlling T3SS expression. One well-documented observation is that P. aeruginosa isolates from CF patients commonly have reduced T3SS expression. In chapter II, I describe how the MucA/AlgU/AlgZR system, commonly activated in CF isolates through mutation of the mucA gene, inhibits T3SS gene expression. My experiments demonstrate that the AlgZR two-component system inhibits ExsA expression through two separate global regulatory systems. First, as previously described, AlgZR inhibits ExsA expression by reducing activity of the cAMP/Vfr signaling pathway. Vfr, a homolog of Escherichia coli Crp, regulates T3SS gene expression through an unknown mechanism. Second, AlgZR alters the activity of the RsmAYZ system to specifically reduce ExsA expression. The RNA-binding protein RsmA, a homolog of E. coli CsrA, activates ExsA expression at a post-transcriptional level. Previous studies in our laboratory identified several transposon insertion mutants that appeared to be novel extrinsic regulators of T3SS gene expression. One of those candidates, named DeaD, is a putative ATP-dependent RNA helicase. My experiments in chapter III reveal that DeaD regulates T3SS expression by directly stimulating exsA translation. Mutants lacking deaD have reduced exsA translational reporter activity and ExsA expression in trans fails to complement a deaD exsA double mutant for T3SS gene expression. I demonstrate that purified DeaD stimulates ExsA expression in a coupled in vitro transcription/translation assay, confirming our in vivo findings. In chapter II, I observed that RsmA activates the transcription of RsmY and RsmZ, two small non-coding RNAs that act to sequester RsmA from target mRNAs. My experiments in chapter IV begin to dissect the RsmA-activation mechanism of RsmY/Z expression. I show that RsmA activation requires the previously described Gac/Lad/Ret system that controls RsmY/Z expression. RsmA, however, does not alter Gac/Lad/Ret gene transcription or translation. Interestingly, an RsmA variant deficient in RNA-binding, RsmA R44A, was able to complement an rsmA mutant for RsmY/Z expression. I hypothesized that RsmA interacts with an unknown protein to activate RsmY/Z expression and identified several potential interaction partners using co-purification assays. Together, my combined experiments elucidate novel global regulatory pathways controlling T3SS gene expression during acute and chronic P. aeruginosa infections, and provide a foundation towards the goal of developing future treatment options.

Exercise has numerous health benefits. Yet, exercise can exacerbate pain for individuals with chronic musculoskeletal pain conditions such as myofascial pain syndrome (MPS) and fibromyalgia (FM). The exacerbation is out of proportion to the activity performed and lasts for long periods of time even after the cessation of activity. This pain acts as a barrier to healthy exercise and physical rehabilitation, which, when applied consistently, are effective treatments for MPS and FM--two diseases that produce substantial suffering and disability. The goal of the proposed studies is to determine the underlying peripheral mechanisms that contribute to enhanced pain following exercise. A better understanding of these mechanisms will lead to better pain management and prevention for these diseases. Previous data show that two hours of running wheel activity lowers the threshold necessary to induce muscle pain by acidic saline injection, producing robust pain behaviors to normally innocuous stimuli. Muscle activity that produces fatigue is associated with extracellular increases in protons, lactate, and ATP. These fatigue metabolites can directly activate muscle nociceptors and, when combined, produce a potentiated effect. Acid sensing ion channels (ASICs) are non-selective cation channels that open in response to increased proton concentrations, a response that is enhanced when lactate binds at a separate location. Ionotropic purinergic receptors (P2X) similarly produce an inward current in response to elevated ATP. Evidence suggests certain ASIC and P2X subtypes are capable of a physical interaction that allows ASIC activation at lower proton concentrations in the presence of ATP. This suggests that ATP, lactate, and protons released during exercise could activate ASIC and P2X receptors on muscle nociceptors, exciting the nociceptors and sensitizing them to subsequent muscle insult. However, the limitations of these experiments leave several gaps. First, the running wheel task fails to produce measurable increases in fatigue metabolites, possibly due to the fact that there was minimal fatigue (10%) or that their levels quickly return to baseline. Further, the running wheel task depends on central nervous system (CNS) activity and volitional running, which may introduce confounding factors upstream of muscle activation and result in large variation in the rate and duration of running. Second, it is unclear whether ASICs are necessary for the development of mechanical hyperalgesia induced by muscle activity, nor is it understood which ASIC subtypes might be required for such an effect. Finally, the molecules necessary for the induction of mechanical hyperalgesia after exercise are not known. Protons, lactate, and ATP have been suggested, but it is not known if these compounds are themselves sufficient or if they interact in an additive or synergistic manner. We address these concerns by developing an electrically-stimulated muscle fatigue paradigm that reliably fatigues a single muscle independent of the CNS, allowing for metabolite measurement during muscle activity and in vivo study of molecular mechanisms of muscle pain in the peripheral tissue. We then use genetic and pharmacologic approaches to test the role of ASIC subtypes in the development of mechanical hyperalgesia after exercise. Finally, we test the effectiveness of by-products of muscle activity in recapitulating the effects of the exercise-enhanced pain model.

Neurons are specialized cells that communicate via electrical and chemical signaling. It is well-known that homeostatic mechanisms exist to potentiate neuronal output when activity falls. Likewise, while neurons rely on excitable states to function, these same excitable states must be kept in check for stable function. However, the identity of molecular factors and pathways regulating these pathways remain elusive. Chapter 2 of this thesis reports the findings from an RNA interference- and electrophysiology-based screen to identify factors necessary for the long-term maintenance of homeostatic synaptic potentiation. Data is reported to resolve a long-standing question as to the role of presynaptic Cav2-type channels in homeostatic synaptic potentiation at the Drosophila NMJ. It is shown that reduction in Cav2 channel expression and resultant activity is not sufficient to occlude homeostatic potentiation. Thus, the homeostatic block of a amino-acid substituted Cav2-type calcium channel (cacS) channel is presumed to be due to loss of a specific signaling or binding activity, but not due to overall diminishment in channel function. It is also reported that both Drosophila homologs of phospholipase Cβ (PLCβ) and its putative activator Gαq were found to be necessary for a scaling up of neurotransmitter release upon genetic ablation of glutamate receptors. These factors are canonically involved in the activation of intracellular calcium stores through the inositol trisphosphate receptor (IP3R) and the closely related ryanodine receptor (RyR). Likewise, the Drosophila homolog of Cysteine String Protein (Csp) is identified as important for long-term homeostatic potentiation. CSP has also been reported to be involved in regulation of intracellular calcium. PLCβ, Gαq, and CSP are also known to regulate Cav2-type channels directly, and this possibility, as well as others, are discussed as mechanisms underlying their roles in homeostatic potentiation. Chapter 3 of this thesis reports the extended findings from expression of a gain-of-function Cav2-type channel. The Cav2.1 channel in humans is known to cause a dominant, heritable form of migraine called familial hemiplegic migraine (FHM). Two amino-acid substitutions causative for migraine were cloned into their analogous residues of the Drosophila Cav2 homolog. Expression of these migraine-modeled channels gave rise to several forms of hyperexcitability. Hyperexcitability defects included abnormal evoked waveforms, generation of spontaneous action potential-like events, and multi-quantal release. It is shown that these forms of hyperexcitability can be mitigated through targeted down-regulation of the PLCβ-IP3R-RyR intracellular signaling pathway. Chapter 4 presents an extended discussion as to the roles for presynaptic calcium channels, PLCβ, and CSP in homeostatic synaptic potentiation, and the mechanism underlying hyperexcitability downstream of gain-of-function Cav2-type channels. The proposed model aims to bridge the involvement of the PLCβ pathway in both homeostatic potentiation and neuronal excitability. Last, the implications for these findings on human disease conditions are elucidated.

Traumatic injuries lead to articular cartilage lesion formation and result in the development of osteoarthritis. Recent research suggests that the early stage of mechanical injuries involve cell death (apoptosis and necrosis) and inflammation. In this thesis, we focus on building mathematical models to investigate the biological mechanism involving chondrocyte death and inflammatory responses in the process of cartilage degeneration. Chapter 1 describes the structure of articular cartilage, the process of carti- lage degeneration, and reviews of existing mathematical models. Chapter 2 presents a delay-diffusion-reaction model of cartilage lesion formation under cyclic loading. Computational methods were used to simulate the impact of varying loading stresses and erythropoietin levels. The model is parameterized with experimental results, and is therefore clinically relevant. Due to numerical limitations using delay differential equations, a new model is presented using tools for population dynamics. Chapter 3 presents an age and space-structured model of articular cartilage lesion formation un- der a single blunt impact. Age structure is introduced to represent the time delay in cytokine synthesis and cell transition. Numerical simulations produce similar tempo- ral and spatial patterns to our experimental data. In chapter 4, we extend our model under the cyclic loading setting. Chapter 5 builds a spatio-temporal model adapted from the former models, and investigates the distribution of model parameters using experimental data and statistical methods. Chapter 6 concludes.

The loss of physical activity after a spinal cord injury results in musculoskeletal deterioration and metabolic dysfunction. Rehabilitation often overlooks the importance of physical activity in the paralyzed limbs for systemic metabolic health. There is a need for safe, feasible exercise interventions to increase physical activity levels in the paralyzed limbs of people with chronic paralysis that have severe musculoskeletal loss. The goal of this work is to 1) develop a gene expression signature after a single dose and long term training using a high force exercise in people with an acute spinal cord injury; 2) develop a novel low force exercise intervention using electrical muscle stimulation to limit force production and increase routine physical activity for chronically paralyzed human skeletal muscle; 3) determine the gene expression signature after a single dose of this novel low force exercise in people with long term paralysis; 4) develop a dose estimate of this low force exercise needed to initiate a phenotype transformation of chronically paralyzed skeletal muscle. The major findings of this research are 1) a single dose of high force exercise increases the expression of key regulatory genes needed for the transformation of paralyzed skeletal muscle observed after long term training; 2) our novel low force exercise intervention challenges chronically paralyzed muscle but not non-paralyzed muscle; 3) a single dose of low force exercise increases the expression of key regulatory genes needed to improve skeletal muscle health; 4) a dose of at least 4 days per week of our low force exercise is needed to initiate a phenotype transformation of chronically paralyzed skeletal muscle. Together, this work supports the use of a low force exercise intervention for people with long term spinal cord injury and establish the need for future work assessing effects of our low force exercise on the systemic health and quality of life of people with long term spinal cord injury.

Motivated by the AdS/CFT correspondence, new supersymmetric solutions to Type IIB and Type IIA supergravity are presented. These solutions contain $AdS_5$ or $AdS_4$ factors and are generated using T-duality symmetries of supergravity. The technique used to generate these solutions consists of performing a series of non-Abelian and Abelian T-dualities, sometimes with coordinate shifts in-between, to Freund-Rubin type seed backgrounds. An added bonus of the gauge fixing procedure inherent in non-Abelian T-Duality is the freedom to generate backgrounds with extra free parameters, some examples of which are presented. Aspects of the dual field theories of these new solutions are analyzed using holography techniques. The supersymmetry of these new backgrounds is also discussed. In addition to supergravity backgrounds with AdS, the study of generalized Calabi-Yau manifolds in the context of flux compactifications is briefly reviewed. The particular case of the resolved cone over $Y^{p,q}$ and its admission of generalized SU(3) structure solutions is examined. Contrary to geometries with $AdS$ factors, whose field theory duals are conformal field theories, these types of geometries can be phenomenologically interesting to study, as their gauge theory duals are minimally supersymmetric and confining, thus they could someday help aid our understanding of strongly-coupled QCD (Quantum Chromodynamics).