Sympathetic stimulation occurs in the heart after injuries such as ischemia/reperfusion (I/R) and myocardial infarction and affects myocardial remodeling. Prolonged sympathetic stimulation can result in myocardial dysfunction through its effects on cardiac myocyte apoptosis and myocardial fibrosis. Ubiquitin (UB) is well known for its role of tagging old or damaged proteins for degradation via the UB-proteosome pathway. The role of exogenous UB however, is not fully understood. Previously, our lab showed that β-adrenergic receptor (β-AR) stimulation increased levels of extracellular UB in the conditioned media of adult rat ventricular myocytes and that UB inhibits β-AR-stimulated apoptosis. This study investigates the role of extracellular UB after myocardial I/R injury in terms of infarct size, function, inflammation and proteomic changes in vivo as well as the effects of extracellular UB on cardiac fibroblast function in vitro. First, we validated a method of consistently measuring real-time myocardial ischemia and reperfusion in vivo. Second, cardiac function was studied 3 days post I/R injury in the presence or absence of UB infusion. Echocardiographic analysis determined UB infusion increased cardiac function after I/R injury in terms of ejection fraction and fractional shortening. UB decreased infarct size and infiltration of inflammatory cells including neutrophils and macrophages as well as reduced activity of neutrophils. UB increased protein levels of matrix metalloproteinase (MMP)-2 and transforming growth factor-β1 and increased activity of MMP-9. Third, in adult rat primary cardiac fibroblasts, we demonstrate that extracellular UB interacts with CXCR-4. UB treatment decreased serum-mediated increases in fibroblast proliferation and enhanced the contraction of fibroblast-populated collagen gels. Thus, extracellular UB likely interacts with CXCR-4 to influence fibroblast function and proliferation. Additionally, UB influences cardiac remodeling in terms of heart function, infarct size, inflammatory response and proteomic profile.

Adult obesity is associated with increased morbidity and mortality. Increasing success in weight loss maintenance will decrease the prevalence of overweight and obesity, and therefore help control the adverse health effects of excess weight. Much is known about the behavioral characteristics of successful long-term weight loss maintenance, but less is known about the cognitive processes behind weight loss maintenance. The purposes of this study were to (1) identify differences in visual attention to high-energy dense foods between individuals who are normal weight, weight loss maintainers, and overweight/obese in a high-risk (food-buffet) situation; (2) to evaluate differences in food choices from a food buffet between weight status groups; (3) to analyze correlations between food attention and food choice across weight status groups. No significant differences were found between groups with respect to food attention or food choice. Overall, findings from this study may have been limited by methodology, technology, and sample size. Future research is needed to better understand the interaction of cognitive processes and weight loss maintenance.

<p>Cytochrome c (Cyt c) is a heme-containing protein that is a component of the electron transport chain as well as the mitochondrial apoptotic pathway. It is released from the mitochondrial intermembrane space to the cytosol during apoptosis and is also thought to be a biomarker for cancer and liver disease. Therefore, an efficient Cyt c biosensor would be a very useful tool for studying apoptosis. Here we show the process of development of Cyt c-dependent aptazymes, derived by <em>in vitro </em>selection. These aptazymes consist of 3 components: 1) a substrate with a cleavage site that consists of a single ribonucleotide flanked by a fluorophore and quencher; 2) a DNAzyme (catalytic DNA) motif capable of cleaving the substrate; 3) an aptamer, a short piece of single- stranded DNA that can specifically bind Cyt c. When Cyt c is absent, the aptamer occludes the catalytic core of the DNAzyme and the fluorophore of the intact substrate is quenched. However, when Cyt c is present, the aptamer binds Cyt c, allowing the DNAzyme to cleave the embedded ribonucleotide, separating the fluorophore and quencher, resulting in a fluorescent signal. Simulations of <em>in vitro </em>selection of Cyt c- dependent aptazymes were also performed. The simulations revealed several methods that can improve the success rate of future <em>in vitro </em>selections of aptazymes.</p> <p>Further analysis of the previously derived DNAzyme DEC22-18 was also performed. A detailed understanding of this DNAzyme will allow it to be developed into a biosensor.</p> / Master of Science (MSc)

<p>Application of previously learned knowledge to new problems or contexts is a cognitive process known as transfer. Undergraduate medical education is optimized when learners are able to transfer basic science knowledge to clinical learning. A long history of transfer research suggests that spontaneous transfer of conceptual knowledge is not easy for learners, thus creating an educational challenge during undergraduate training. However, not all transfer tasks are equally difficult. When conceptual problems are presented in familiar contexts (e.g. similar surface details or semantic content in word problems), this <em>near </em>transfer is facilitated for learners. But when contextual familiarity does not exist, the problem is one of <em>far </em>transfer and becomes more difficult. Previous research suggests that using contextual information and focusing on conceptual teaching can improve transfer performance for novices.</p> <p>This thesis investigates how emphasizing contextual information versus conceptual information can impact transfer of principles of physics relevant to physiology (the concepts) to different organ systems (the contexts). Across three experimental studies, students were assigned to different learning and practice conditions where conceptual and contextual teaching were manipulated. The results showed 1) while emphasizing conceptual information can improve transfer, contextual alignment (near transfer) between learning and problem solving had the highest performance for all students. 2) Novices use contextual information as</p> <p>recognition cues for new problems but can be shifted to examine deep conceptual structure when provided with in-depth conceptual teaching as well as varying the number of contexts used to practice concepts. This shifts novices to equal success at near and far transfer. 3) Novices can revert to relying on contextual information if teaching interventions do not provide contextual variation and instead promote a close association between contextual details and conceptual information.</p> <p>This research suggests that shifting novices to examine conceptual problems at the deep structure level should be a key goal for teaching basic science for transfer. Novices default to using surface details to encode and retrieve conceptual information. While in some near transfer problems this can be an effective strategy, for far transfer it can lead to errors. Basic science teaching during undergraduate training must emphasize transferability of concepts by providing more relatable ways to understand conceptual information and showing the variation of a concept’s presentation.</p> / Doctor of Philosophy (PhD)

INTRODUCTION: The detection of a second inorganic phosphate (Pi) resonance, a possible marker of mitochondrial content in vivo, using phosphorus magnetic resonance spectroscopy (31P- MRS) at 3T is technically challenging, which may prevent its reproducible quantification. PURPOSE: To determine the reproducibility of resting alkaline inorganic phosphate (Pialk) measurement using 31P-MRS in human skeletal muscle at 3 tesla (T). METHODS: Resting 31P- MRS of the quadriceps muscles was acquired on two separate visits, within seven days, in 13 healthy, sedentary to moderately active young adults using a whole-body 3T MR system. Measurement variability related to coil position, shimming procedure, and spectral analysis were also quantified. 31P-MRS data were acquired with a 31P/1H dual-tuned surface coil positioned on the quadriceps using a pulse-acquire sequence. Test-retest absolute and relative reproducibility were analyzed using coefficient of variation (CV) and intra-class correlation coefficients (ICC), respectively. RESULTS: Pialk demonstrated a within-subject reproducibility marginally greater then the 10% cutoff (CV: 10.6 ± 5.4%; ICC: 0.80), but still appropriate given its small concentration in relation to other 31P metabolites. Proximo-distal change in coil positioning along the length of the quadriceps induced large variability in Pialk quantification (CV: 21.1%). In contrast, measurement variability due to repeated shims on consecutive scans from the same muscle sample (CV: 6.6%), and the automated spectral processing procedure, were minor (CV: 2.3%). Both metrics of absolute and relative reproducibility of Pialk were of similar magnitude to other well-resolved metabolites (e.g., phosphocreatine, Pi, and phosphodiester). CONCLUSION: Using multiple metrics, the present study established the high reproducibility of Pialk quantification by 31P-MRS using a surface coil on the quadriceps muscle at 3T. However, large variability in Pialk quantification can originate from positioning the coil on the most distal part of the quadriceps, which should be avoided due to shimming inhomogeneity.

Obesity is strongly associated with hypertension and cardiovascular diseases. An activated renin-angiotensin system (RAS) has long been suggested as a critical contributor to elevated blood pressure with obesity. Angiotensin II (AngII), the main effector of an activated RAS, can be catabolized by angiotensin-converting enzyme 2 (ACE2) to form angiotensin-(1-7) (Ang-(1-7)), which, acting through the mas receptor (MasR), has been shown to oppose the effects of an activated RAS. Therefore, further understanding of the mechanisms of this counter-regulatory arm, called the ACE2/Ang-(1-7)/MasR axis, may lead to new therapies for obesity-induced hypertension. Previously, we demonstrated that differences in the regulation of ACE2 in a tissue-specific manner contribute to sexual dimorphism of diet-induced obesity-hypertension in mice. Whereas male mice fed a high fat (HF) diet developed hypertension, HF-fed female mice were protected from obesity-hypertension, and this was associated with increased activity of ACE2 in adipose tissue of females. Both upregulation of adipose ACE2 and protection against obesity-hypertension were lost when females were ovariectomized (OVX). We hypothesized that estrogen-mediated increases in adipose ACE2 reduce the AngII/Ang-(1-7) peptide balance and protect females from obesity-hypertension. To test this hypothesis, we first determined if estrogen restores protection of Ovx female mice from obesity-hypertension, and therapeutically protects male mice from obesity-hypertension. We demonstrated that estrogen administration to Ovx HF-fed females activates adipose ACE2, reduces plasma Ang II concentrations, and decreases blood pressure in wildtype, but not of ACE2-deficient obese females. In contrast, estrogen administration to HF-fed male mice had no on the development of obesity-hypertension, regardless of genotype. These results demonstrate that estrogen protects female mice from obesity-hypertension through an ACE2-dependent mechanism. Next we defined the role of MasR deficiency on the development of obesity-hypertension in male and female mice. In HF-fed MasR-deficient female mice, diastolic blood pressure (DBP) was significantly elevated compared to LF-fed controls, suggesting that protection from obesity-hypertension was abolished by MasR deficiency. In contrast, HF-fed male mice with MasR deficiency exhibited reduced blood pressure compared to wildtype controls which was associated with reduced cardiac function. Overall, these studies indicate that the ACE2/Ang-(1-7)/MasR axis plays an important role in sexual dimorphism of obesity-hypertension, and in the regulation of cardiac function. Moreover, these studies suggest that the effects of this counter-regulatory arm of the RAS may be sex-specific.

Background: Vitamin D is crucial for skeletal muscle function. 25-hidroxyvitamin D (25(OH)D) has been correlated with skeletal muscle mass and intramyocellular lipid (IMCL) content. The purpose of this study was to understand how calcitriol, the active vitamin D metabolite, directly affects myocellular size and lipid partitioning. Methods: C2C12 myotubes were treated with calcitriol (100nM) or vehicle control for 24 or 96 h. Myotube diameter and protein synthesis rate were measured to determine effects of calcitriol on myocellular size. Intramyocellular triacylglycerol (IMTG), diacylglycerol (DAG), and ceramide content were measured by LC/MS. Expression of genes involved in lipid packaging and lipolysis were measured by RT-PCR. Insulin-stimulated phosphorylated Akt (Thr 308) was determined by western blot. Results: Calcitriol did not affect myocellular size or protein synthesis rate. Calcitriol increased total DAG and ceramides in a sub-species specific manner. Calcitriol increased IMTG area, but did not affect total IMTG content. Calcitriol reduced mRNA content of diglyceride acyltransferase and increased mRNA content of lipid packaging genes. Calcitriol did not negatively affect insulin-stimulated pAkt. Conclusions: These results suggest calcitriol directly alters lipid content and packaging in skeletal muscle cells. Altering the expression of lipid packaging genes and increasing IMCL subspecies content may be mechanisms by which vitamin D improves skeletal muscle function in vivo.

The inhibitor of DNA binding protein 4 (ID4) is a dominant negative regulator of basic helix loop helix (bHLH) family of transcription factors.1 Recently, Patel et al., demonstrated that inhibitor of differentiation 4 (ID4) acts as a tumor suppressor and its loss, frequently observed in prostate cancer, promotes castration-resistant prostate cancer (CRPC) through constitutive androgen receptor (AR) activation.2 However, the mechanism by which loss of ID4 promotes constitutively active AR signaling in the CRPC conditions is unknown. The rationale of the present study was to unravel the underlying molecular mechanisms through which loss of ID4 potentiates AR signaling in this setting. Initially, chromatin immunoprecipitation (ChIP) assay results demonstrated a significant increase in binding of AR to its respective response elements on PSA, FKBP51, TMPRSS2, and ETV1 promoters in L(-)ID4 cells, further implicating constitutive AR activity. Among the notable findings, proteomic profiling between prostate cancer cell line LNCaP (L+ns) and LNCaP lacking ID4 (L(-)ID4) revealed elevated protein levels of Heat shock protein 27 (Hsp27) and the 52-kDa FK506-binding protein (FKBP52), suggesting a role for these AR-associated co-chaperones in promoting constitutively active AR signaling in L(-)ID4 cells. Interestingly, protein interaction studies further confirmed a direct interaction between ID4 and FKBP52 in vitro but not with AR. Recent evidences suggest that FKBP52 is a positive regulator of AR signaling in cellular and whole animal models.3-6 Thus, we hypothesized that ID4 acts as a tumor suppressor by selectively regulating AR activity through interaction with FKBP52. To address the underlying mechanism, we blocked the FKBP52-AR signaling using a specific inhibitory compound known as MJC13.4, 6-7 The results demonstrated that MJC13 effectively inhibited AR-dependent expression and activity in a dose-dependent manner. In addition, xenograft studies further confirmed that inhibiting FKBP52-regulated AR activity via MJC13 significantly attenuated the growth of subcutaneous L(-)ID4 xenografts in vivo. Collectively, our results suggested that ID4 selectively regulates AR activity through direct interaction with FKBP52 in vitro, and, its loss promotes CRPC through FKBP52-mediated AR signaling. Increased AR signaling along with a subsequent decrease in ID4 expression levels in prostate cancer strongly supports this model.

Endogenous bioelectric fields guide morphogenesis during embryonic development and regeneration by directly regulating the cellular functions responsible for these phenomena. Although this role has been extensively explored in many peripheral tissues, the ability of electric fields to regulate wound repair and stimulate regeneration in the mammalian central nervous system (CNS) has not been convincingly established. This dissertation explores the role of electric fields in regulating the injury response and controlling the regenerative potential of the mammalian CNS. We place particular emphasis on their influence on astrocytes, as specific differences in their injury-induced behaviors have been associated with differences in the regenerative potential demonstrated between mammalian and non-mammalian vertebrates. For example, astrocytes in both mammalian and non- mammalian vertebrates begin migrating towards the lesion within hours and begin to proliferate after an initial delay of two days; subsequently, astrocytes in non-mammalian vertebrates support neurogenesis and assume a bipolar radial glia-like morphology that guides regenerating axons, whereas astrocytes in mammals do not demonstrate robust neurogenesis and undergo a hypertrophic response that inhibits axon sprouting. To test whether injury-induced electric fields drive the astrocytic response to injury, we exposed separate populations of purified astrocytes from the rat cortex and cerebellum to electric field intensities associated with intact and injured mammalian tissues, as well as to those electric field intensities measured in regenerating non-mammalian vertebrate tissues. Upon exposure to electric field intensities associated with uninjured tissue, astrocytes showed little change in their cellular behavior. However, cortical astrocytes responded to electric field intensities associated with injured mammalian tissues by demonstrating dramatic increases in migration and proliferation, behaviors that are associated with their formation of a glial scar in vivo; in contrast, cerebellar astrocytes, which do not organize into a demarcated glial scar, did not respond to these electric fields. At electric field intensities associated with regenerating tissues, both cerebellar and cortical astrocytes demonstrated robust and sustained responses that included morphological changes consistent with a regenerative phenotype. These results support the hypothesis that physiologic electric fields drive the astrocytic response to injury, and that elevated electric fields may induce a more regenerative response among mammalian astrocytes.

Hyperhomocysteinemia (hHcys) is a widely known pathogenic factor in the progression of end-stage renal disease (ESRD) and it is also associated with an increased risk for injurious cardiovascular pathologies during ESRD. HHcys is linked to the formation and activation of the NOD-like receptor protein 3 (NLRP3) inflammasome, characterized as a critical early mechanism initiating the inflammatory response. NADPH oxidase (NOX)-derived reactive oxygen species (ROS) mediate the activation of the NLRP3 inflammasome in podocytes in response to elevated levels of homocysteine (Hcys) in vitro and in vivo. However, it remains unknown how NLRP3 inflammasome activation is triggered by NOX. The aim of the present study sought to determine the signaling cascade that triggers glomerular injury and sclerosis during hHcys mediated by Vav2, a guanine nucleotide exchange factor (GNEF). Using both genetic and pharmacological interventions of Vav2, we first tested whether this GNEF is involved in hHcys-induced NLRP3 inflammasome activation in podocytes by its role in activation of the Rac-1-NOX complex. Further, we explored whether pharmacological targeting of Vav2 activation may regulate NLRP3 inflammasome signaling pathway during hHcys-induced glomerular injury. We found that mice with hHcys (on the FF diet) or oncoVav2 (a constitutively active form of Vav2) transfection in the kidney exhibited increased colocalization of NLRP3 with apoptosis-associated speck-like protein (ASC) or caspase-1 and elevated IL-1β levels in glomeruli, indicating the formation and activation of the NLRP3 inflammasome. This glomerular NLRP3 inflammasome activation was accompanied by podocyte dysfunction and glomerular injury, even sclerosis. Local transfection of Vav2 shRNA plasmids significantly attenuated hHcys-induced NLRP3 inflammasome activation, podocyte injury, and glomerular sclerosis. In cultured podocytes, Hcys treatment and oncoVav2 transfection increased NLRP3 inflammasome formation and activation. This NLRP3 activation was inhibited by Vav2 shRNA, associated with reduction of Rac-1 activity and ROS production. Administration of NSC23766, a Rac-1 inhibitor substantially attenuated inflammasome formation, desmin expression and decreased podocin expression in glomeruli of hHcys mice. These results suggest that elevated Hcys levels activate Vav2 and thereby increase NOX activity, leading to ROS production. ROS trigger NLRP3 inflammasome activation, podocyte dysfunction and glomerular injury. Therefore, the present study defines a novel mechanism underlying hHcys-induced NLRP3 inflammasome activation and its progression to ESRD.