UT Southwestern Electronic Theses and Dissertations

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1,3-Dipolar Cycloaddition of Electron-rich Alkynes and Optically Active Allenes in Asymmetric Catalysis
(2009-09-04) Qi, Xiangbing; Ready, Joseph M.
This dissertation includes two parts. The first part focuses on two 1,3-dipolar cycloadditions of electron-rich alkynes. Chapter one describes a copper-promoted cycloaddition reaction of acetylides with diazocarbonyl compounds. This novel cycloaddition offers a direct and efficient approach to the synthesis of pyrazoles. The method is a rare example of an inverse-electron-demand cycloaddition, it represents a conceptually novel approach to this important class of heterocycles. Chapter two investigates a cycloaddition reaction between donor-acceptor cyclopropanes and silyl ynol ethers. Lewis acid promoted ring-opening of donor-acceptor cyclopropanes generates a 1,3-zwitterion; cycloaddition with a silyl ynol ether leads to a general synthesis of cyclopentenones. Substitution is tolerated on the ynol and on all positions of the cyclopropane to give tri-, tetra-, and penta-substituted cyclopentenones in high yield. methyl(methoxyl)aluminumchloride, which is generated from dimethylaluminumchloride by oxidation, appears strong reactivity towards ring-opening of donor-acceptorcyclopropanes and cycloaddition with silyl ynol ethers. This infrequently used species might be extended to other classes of cycloadditions or Lewis acid promoted reactions. The second part describes a new asymmetric catalysis design using optically active allenes as backbone and the application in an asymmetric meso-epoxides opening reaction. Allenes are inherently chiral and can be prepared in optically pure form. They have not been incorporated into ligands or catalysts for asymmetric reactions. Since allenes project functionality differently than either tetrahedral carbon or chiral biaryls, they may create complementary chiral environments. Chapter three demonstrates that optically active, C2-symmetric allene-containing bisphosphine oxides can catalyze the addition of silicon tetrachloride to meso-epoxides with high enantioselectivity. The fact that high asymmetric induction is observed suggests that allenes may represent a new platform for the development other classes of organic catalysts or ligands for asymmetris reactions.
3 Tesla Magnetic Resonance Imaging of Hippocampal Asymmetry: Results from the Dallas Heart Study
(2013-06-01) Lucarelli, Richard T.; Peshock, Ronald M.
BACKGROUND: Asymmetry of the hippocampus is regarded as an important clinical finding but limited data on hippocampal asymmetry is available for the general population. Here we present hippocampal asymmetry data from the Dallas Heart Study determined by automated methods and its relationship to age, sex, and ethnicity. METHODS: 3D-MPRAGE MRI were obtained in 2082 DHS-2 participants. The MR images were analyzed using two standard automated brain segmentation programs, FSL-FIRST and Freesurfer. Individuals with imaging error, self-reported stroke, or major structural abnormalities were excluded. Statistical analyses were performed to determine significance of the findings across age, sex, and ethnicity. RESULTS: At the 90th percentile FSL-FIRST demonstrated hippocampal asymmetry of 9.8% (95% CI 9.3 to 10.5%). The 90th percentile of hippocampal asymmetry measured by the difference between hippocampii over the larger hippocampus was 17.9% (95% CI 17.0 to 19.1%). Hippocampal asymmetry increases with age (P=0.0216) and men have greater asymmetry than women as shown by FSL-FIRST (P=0.0036), but ethnicity is not significantly correlated with asymmetry. To confirm these findings Freesurfer was used. Freesurfer showed asymmetry of 4.4% (95% CI 4.3 to 4.7%) normalized to total volume, and 8.5% (95% CI 8.3 to 9.0%) when normalized by difference/larger hippocampus. Freesurfer also showed that hippocampal asymmetry increases with age (P=0.0024), and that men had greater asymmetry than women (P=0.03). CONCLUSION: There is a significant degree of hippocampal asymmetry in the population. The data provided will aid in the research, diagnosis, and treatment of temporal lobe epilepsy and other neurological disease.
[3+2]-Cycloadditions of Azomethine Imines and Ynolates and Progress Towards a Radical Three-Component Cross-Coupling Reaction
(2018-07-16) Winterton, Sarah Elizabeth; Chen, Chuo; Tambar, Uttam; De Brabander, Jef K.; Ready, Joseph M.
This manuscript consists of three chapters. The first chapter describes a novel [3+2]-cycloaddition of azomethine imines and ynolates to construct bicyclic pyrazolidinones in high yields and diastereoselectivities. This methodology is the first example in which the azomethine imine acts as a chiral auxiliary to control the cycloaddition. Optically active azomethine imines yield bicyclic pyrazolidinones in high yields, and conditions for removal of the azomethine imine chiral auxiliary have been defined to yield optically active monocyclic pyrazolidinones in high yields and enantioselectivities. The second chapter consists of progress towards a novel [2+2]-annulation/fragmentation to construct cyclohexanones. Among all the methods to synthesize highly substituted cyclohexanols/cyclohexanones, there is no general, one-step enantioselective, intermolecular method to synthesize a highly substituted cyclohexanol/cyclohexanone with a quaternary center at C4. Therefore, we developed a synthesis of cyclohexanones via a formal [2+2]-annulation/fragmentation between a cyclobutanone containing an electron-withdrawing group and an electrophile. The second chapter describes attempts to render the reaction asymmetric, which proved to be difficult. Future ideas to improve yields and to render the reaction asymmetric are also described. The last chapter involves progress towards a novel three-component, enantioselective radical cross-coupling reaction to synthesize complex products in a single step. Current literature contains only scattered examples of three-component radical cross-coupling reactions, all of which have a severely limited substrate scope (e.g., only alkynes, one nucleophile, contains fluorine). Therefore, we envisioned a three-component radical cross-coupling reaction with a broad substrate scope encompassing multiple radical initiators and olefin acceptors. This method could provide access to products that are currently challenging to synthesize. Progress towards this three-component enantioselective reaction is presented in chapter three, as well as future ideas to improve enantioselectivity and yields.
3D Animated Video Series for Patient Education of Pelvic Organ Prolapse and Sacrocolpopexy Surgery
(2012-12-20) Sumner, Elizabeth; Calver, Lewis E.
The objective of this video series was to present a comprehensive three-dimensional patient education video series that described basic female pelvic anatomy, types of pelvic organ prolapse, basic procedural steps for a sacrocolpopexy, and complications of the procedure. A survey of currently available patient education materials was conducted to assess possible areas for improvement. A storyboard and script were then developed and presented to several members of the Female Pelvic Medicine and Reconstructive Surgery Division and the Biomedical Communications Graduate School Program. Three-dimensional computer animations were created from CT data and compiled into video segments. The videos were embedded into a web platform and tested on a group of current and former patients. The result was that the test group correctly answered content questions and had an increased confidence with discussing the material. This may mean that patients have a better understanding of prolapse and sacrocolpopexy procedure after viewing this video series.
4D Study of Thoracic Cancer Radiation Treatment
(2007-05-22) Huang, Tzung-Chi; Zhang, Geoffrey
Respiratory motion causes an added uncertainty in the radiation treatment of thoracic malignancies due to an increase in the normal tissue irradiated and an uncertainty in the radiation coverage of the tumor. This results in a potential increase in complications from treatment and may be insufficient to ensure coverage of the tumor. Reduction of the volume of normal tissue irradiation while maintaining tumor coverage is used to accomplish this goal. The application of 4D CT imaging to radiotherapy treatment planning is an active area of research with the goal to reduce the required normal tissue irradiation and improve the tumor coverage. Deformable image registration holds the key to link the information of two images at various phases. The major purpose of this study is to develop and validate the optical flow method (OFM), a method of deformable image registration by which the image content properties are utilized to generate a displacement vector between each voxel in the reference image to the target image for registration. With OFM, we were able to develop and validate an automated method for intrathoracic motion estimation from breath-hold and 4-D computed tomography imaging; demonstrate the path integration of a four-dimensional dose distribution onto the 3-D anatomy; develop an automated target delineation technique; and to develop and implement a method to quantify tumor response and normal tissue damage by comparison of pre- and post-treatment and 18F-FDG-PET scans, all of which constitute meaningful applications and represent substantial progress in radiation treatment.
Abbreviated and Expanded Forms of the Montreal Cognitive Assessment for Dementia Screening
(2015-07-15) Horton, Daniel Kevin; Cullum, C. Munro; Hynan, Linda S.; Lacritz, Laura H.; Rossetti, Heidi; Weiner, Myron F.
Cognitive screening is becoming increasingly important as the general population ages and the prevalence of dementia rises. However, popular cognitive screening tools have been criticized for their insensitivity to subtle cognitive impairment, poor specificity, excessive administration time, and/or questionable methods of test development. The Montreal Cognitive Assessment (MoCA) is a cognitive screening instrument growing in popularity which has demonstrated increased sensitivity to mild cognitive impairment (MCI), but takes roughly 10-15 minutes to administer and was developed without an empirically-driven item selection process. We devised two studies to address common limitations of cognitive screening tools using the MoCA. The aim of Study 1 was to create a short form of the MoCA (SF-MoCA) including only the items found to be most sensitive to MCI and Alzheimer disease (AD) and compare the diagnostic classification accuracy of the SF-MoCA to the Mini-Mental State Examination (MMSE) and standard MoCA. Results revealed delayed recall, orientation, and serial subtraction items to be most useful in differentiating the diagnostic groups. Overall, diagnostic accuracy of the SF-MoCA was superior to the MMSE and comparable to the standard MoCA, suggesting that some MoCA items do not add to the sensitivity of the instrument in these populations. Given the brevity and sensitivity of the SF-MoCA, we suggested this measure may be useful for early detection of cognitive impairment in primary care and other settings where evaluation time is limited. Despite the advantages of the SF-MoCA, this tool only assesses three cognitive domains and may not be appropriate in settings where clinicians may want to efficiently assess additional domains affected in AD and MCI to gain a clearer picture of global functioning and assist in differential diagnosis. Therefore, we conducted a second study to determine if diagnostic accuracy of the SF-MoCA might be enhanced through the addition of several brief and well-validated neuropsychological measures shown to be sensitive to cognitive impairment. Results revealed that the addition of measures of processing speed, category fluency, and verbal recall resulted in an Expanded SF-MoCA with diagnostic classification accuracy superior to both the standard MoCA and SF-MoCA. Findings of these studies have implications for current cognitive screening procedures and techniques used to develop these tools.
Aberrant DNA Methylation and Cancer: A Global Analysis of Promoter Hypermethylation in Human Lung Cancers
(2006-12-20) Shames, David S.; Minna, John D.
Tumor-acquired alterations in DNA methylation include both genome-wide hypomethylation and locus specific hypermethylation. Global loss of DNA methylation destabilizes chromatin architecture, augments genomic instability, and may reactivate repetitive element expression. Promoter hypermethylation often coincides with loss of heterozygosity at the same loci, and together these events can result in loss of function of the gene in tumor cells. The "rules" governing which genes are methylated during the pathogenesis of individual cancers are unknown; however, it is known that certain genes are methylated with high frequency in selected tumors, whereas others are methylated across most types of tumors. The objective of the work described below was to use global profiling platforms (RNA and DNA) to identify epigenetically modulated genes that may be involved in cancer pathogenesis and bring these to the point where they could be developed as targets for diagnostic and treatment strategies. Using a global expression profiling approach and pharmacological inhibition of the DNA methyltransferases, 132 genes were identified that have 5' CpG islands, are induced from undetectable levels by 5-aza-2'-deoxycytidine (5-aza) in multiple non-small cell lung cancer cell lines, and are expressed in untreated immortalized human bronchial epithelial cells. Methylation analysis of a subset (45/132) of these promoter regions in primary lung cancer (N=20) and adjacent non-malignant tissue showed that 31 genes had acquired methylation in the tumors, but did not show methylation in normal lung or peripheral blood cells. Promoter methylation of eight of these genes were studied in breast cancers (N=37), colon cancers (N=24), and prostate cancers (N=24) along with counterpart non-malignant tissues. We found that seven loci were frequently methylated in both breast and lung cancers, with four showing extensive methylation in all four epithelial tumors. The data presented below suggest that while tumors differ in their molecular genetic phenotypes and pathogenesis, there may be underlying similarities in the pathways they follow toward malignancy. Some of these similarities may be reflected in the methylation programs tumor cells engage, which in turn, provides an opportunity to exploit for therapeutic applications and diagnosis. The approaches described herein entail a systematic and reproducible method to identify novel methylation markers in a variety of cancers, and the results of these studies provide a basis for developing a generic set of methylation markers for early detection screening across common epithelial cancers.
Ability of the Pain Disability Questionnaire in Predicting Healthcare Utilization
(2014-09-01) Rankin, McKenna Blair; Gatchel, Robert J.; Noe, Carl; Robinson, Richard C.
BACKGROUND: Chronic pain is one of the most expensive and prevalent healthcare problems in the United States. It affects not only the individual but also society as a whole. The Biopsychosocial Model of chronic pain is the standard model for understanding and treating the pain experience. As such, it is important to have accurate ways of predicting healthcare outcomes for the chronic pain population. Prior research has evaluated the predictive abilities of health outcome measures and healthcare utilization. However, only one study to our knowledge has evaluated the Pain Disability Questionnaire’s (PDQ) ability to predict healthcare utilization. SUBJECTS: The current study consisted of 50 [Mage = 54.76 (12.41) years] adult patients diagnosed with chronic pain who completed an interdisciplinary treatment program at the Eugene McDermott Center for Pain Management. METHOD: The PDQ was administered at baseline and total health care cost was obtained at three-month follow-up. Participants were placed in either the high or low health care cost group depending on how they compared to a national average estimate for chronic pain patients. Logistic and linear regression modeling, as well as receiver operator characteristic analyses were used to evaluate the predictive ability of the PDQ in determining healthcare utilization cost at a three-month follow-up time point. RESULTS: The logistic regression analysis indicated the PDQ accurately placed participants in the respective high or low healthcare cost group [X2 (1) = 10.67, p < .001]. Receiver operator characteristic analysis yielded an area under the curve of .76. A PDQ cutoff score of 96 produced the optimal sensitivity (.70) and specificity (.67) for determining whether patients fall in the high or low healthcare cost group. The linear regression established that PDQ scores at baseline statistically significantly predicted total healthcare utilization at three months following the end of their treatment program, where higher PDQ scores were related to higher healthcare cost [F (1, 48) = 11.41, p = .001]. DISCUSSION: These findings offer support for the use of the PDQ in predicting healthcare cost. Use of the PDQ in this context may help clinicians, caregivers, and patients in planning the cost of the chronic pain sufferers’ treatment. Knowing what to expect financially could help increase the patient’s quality of life.
Acanthamoeba spp. Secrete a Mannose-Induced Protein that Correlates with Ability to Cause Acanthamoeba Keratitis
(2004-01-14) Hurt, Michael Allen; Niederkorn, Jerry Y.
Acanthamoeba spp. are ubiquitously distributed in the environment. The trophozoite form can infect the cornea and cause sight-threatening corneal inflammation known as Acanthamoeba keratitis. The pathogenic cascade of Acanthamoeba keratitis begins when Acanthamoebae bind to mannose expressed on traumatized corneas. Published reports indicate that mannose is upregulated on the corneal surface during wound healing. Experiments in laboratory animals have shown that corneal abrasion prior to infection is essential for generating Acanthamoeba keratitis. Furthermore, supernatants from Acanthamoeba
Access to Care Factors Connected to Age at Evaluation for Children Assessed for Autism Spectrum Disorders
(2012-07-20) Castrejana, Jessica; Stewart, Sunita M.
The early identification of children with an autism spectrum disorder (ASD) is important for intervention programs to take place. The goal of this study was to examine the combination of several sociodemographic factors that research has indicated influence the age at which children are evaluated for ASDs. Primary variables of interest included sex, race/ethnicity, urbanicity, maternal education and severity of symptoms. This study analyzed 71 children ranging from 22 to 94 months as part of a database collected by the Autism Clinic at Children's Medical Center Dallas. We found that male children with higher Childhood Autism Rating scores were evaluated at an earlier age. For children diagnosed with ASD, maternal education was the strongest predictor of when a child would be evaluated. There were no differences between boys and girls in symptom severity at the time of evaluation. Implications for early identification of ASD are discussed.
Accuracy in Diagnosing Pediatric Appendicitis: Discordance in Diagnosis and Masqueraders
(2016-04-04) Farzal, Zehra; Fischer, Anne C.; Piper, Hannah; Schindel, David
BACKGROUND: The classification of perforated versus nonperforated appendicitis remains poorly defined despite being clinically significant and requiring dramatically different therapeutic approaches. We hypothesized that there is substantial variability in the determination of perforated and nonperforated appendicitis across specialties due to the lack of standardized criteria. To identify and quantify the degree of variability in the diagnosis of acute versus perforated appendicitis, a comparative analysis of radiologic results, operative findings, and pathologic review was undertaken to identify the degree of discordance across specialties as the primary objective. Given the new suggested paradigm of non-operative management of acute appendicitis, a secondary objective was to identify the incidence of atypical diagnoses detected among appendectomy specimens to better elucidate those potentially missed in non-operative management. METHODS: An IRB-approved retrospective review of 1311 appendectomies over a 16-month period at an independent children's hospital allowed a comparison of the diagnostic classification of appendicitis as perforated (PA) or nonperforated (NPA) based on radiology (R), operative (O), and pathology (P) reports. Three groups, P+O (N=1241), P+R (N=516), O+R (N=512), were compared to identify inter-specialty discordance in classification. The length of stay (LOS) was analyzed as a proxy for clinical behavior to confirm that the diagnostic classification was consistent with the clinical behavior of being perforated or nonperforated (PA with LOS >48hours and NPA with LOS ≤48 hours). Given the numerous pathologic descriptives of appendicitis, a more extensive pathologic review of all appendectomies was done to identify an intra-specialty variance in diagnostic classification and to identify any other diagnosis in the setting of presumed appendicitis. This arm of the study involved an expanded IRB-approved retrospective review of pediatric patients (n=6816) who underwent appendectomies at an independent children's hospital over an 11-year period from January 2000 to December 2010. Inclusion criteria were age <17 and surgery for presumed appendicitis, thus excluding incidental appendectomies (n=269) with a final review of 6547 specimens. RESULTS: In the assessment of the degree of discordance, the subsets P+O, P+R, O+R revealed a discordance of 11%, 15.7% and 16.6% within the classification of appendicitis respectively. In the O+R group, 35% of the cases that were operatively designated as 'perforated' contradicted the radiologic diagnosis of being 'nonperforated' appendicitis. Cases designated as PA in all subsets (P+O, P+R, O+R) clinically behaved as perforated with a mean LOS>48 hours (97, 95, 95, respectively), whereas the cases designated as nonperforated appendicitis (NPA) exhibited greater variation from the expected LOS≤48 hours, with means 35, 83, and 62, respectively. The more exhaustive pathologic review identified 7 classifications for appendicitis in 91.6% patients, 5% false positives, and ~3% coincident other diagnoses. 5998 (91.6%) subjects showed true appendicitis including acute non-perforated, perforated, chronic, suppurative, gangrenous, necrotizing, and catarrhal appendicitis. In 224 subjects (3.4%), diagnoses other than appendicitis were identified: non-inflammatory obstruction (n=71), other infectious etiologies (n=58), non-specific inflammatory changes (n=58), extra-appendiceal pathology (n=31), tumors (n=10), and foreign body (n=2). Of the tumors, 6 patients with true appendicitis had co-existing carcinoid tumors. 325 specimens (5.0%) were documented as negative appendicitis. CONCLUSION: Variability in the classification of appendicitis between specialties suggests an error rate inherent in diagnosis. This error rate may explain why identifying best clinical guidelines for length of antibiotic therapy and treatment has been elusive. Standardizing classification criteria across specialties may help identify best practices for optimal use of hospital resources and improve diagnostic accuracy for meaningful clinical trials. This study represents the largest analysis of the incidence of pathologies that mimic appendicitis in the pediatric population, conveying a broad overlap of diagnoses that present in a similar fashion. Given how commonly appendicitis is diagnosed, follow-up for routine appendectomies has been streamlined and expedited in such a way that the review of pathology by the practitioner may be overlooked. The number of infectious etiologies and tumors detected reinforces the increasing importance of pathology review in post-operative follow-up to appropriately diagnose uncommon conditions that may necessitate further work-up and treatment. Additionally, the possibility of missing an alternative or co-incidental diagnosis such as a carcinoid tumor in the non-operative management of appendicitis merits some reflection in planning operative versus non-operative management. Given the recent popularity of non-operative therapy for appendicitis, this research gives the clinician a reliable incidence of other potential diagnoses if symptoms recur. Having percentages for conditions that present similarly also allows for proper counseling of patients who opt for non-operative management.
Acetaminophen Dose Does Not Predict Outcome in Acetaminophen-Induced Acute Liver Failure
(2010-04-09) Gregory, Blake Elizabeth; Lee, William M.
Acetaminophen is a dose-dependent toxin. Prognosis in severe acute liver injury is related presumably in part to the dose ingested. We sought to assess the value of acetaminophen dosing information in patients with acute liver failure due to acetaminophen toxicity to determine the role of dose as a prognostic indicator. Methods: Prospective data from 113 acute liver failure patients having single time point ingestions of acetaminophen were analyzed. Multivariate and chi-square tests were used to determine the relationship of dose to clinical outcome. We also used the Mann-Whitney U test to compare prognosis and survival in ALF with acetaminophen dose ingested. Results: Multivariate and chi-square analysis failed to show any relationship between acetaminophen dose and spontaneous survival. A separate analysis showed no correlation between acetaminophen dose and clinical prognostic indicators. Conclusions: Dose of acetaminophen ingested did not seem to play a role in prognosis. The most important prognostic factor was coma grade on admission to study. Acetaminophen dosing information is not always obtainable. When it is, it adds little to the clinical assessment. Severity of encephalopathy is a more reliable indicator of prognosis in these critically ill patients.
Acres of Diamonds: Rediscovering Community Assets in Dallas Backyards
(2016-04-01) Chavda, Avinash Suresh; Rhee, Chanhaeng; Reed, W. Gary
BACKGROUND: Diabetes directly affects 8% of the US population and poses a growing burden to healthcare providers, to the health system, and to society. Cultivating self-efficacy enhances personal agency and enables patients to take an active role in the management of their disease. Effectively cultivating self-efficacy among patients from diverse backgrounds requires a patient-tailored assessment of existing gaps in patients' diabetes self-management skills. Tying patients to existing community health resources through ZIP code-based mapping is an effective patient-tailored intervention that creates long lasting change in diabetes self-management. To this end, a two-armed approach to community resource mapping was taken: In the first arm, existing resources in the categories of exercise, nutrition, and diabetes management were catalogued and mapped across Dallas County ZIP codes; in the second arm, patient awareness of existing resources across the same three categories was mapped across Dallas County ZIP codes. A comparison of each study arm across overlapping ZIP codes showed that resources abound in Dallas, but awareness of these resources is the limiting factor in fostering increased patient self-efficacy. To increase self-efficacy and empower patients to improve self-management of their diabetes, patients should be connected with community resources to tackle the issue of underutilization of community resources and thereby gain "mastery experiences." OBJECTIVE: This project hypothesizes that community resources are plentiful in Dallas and that there is a relative lack of awareness of community resources, limiting patient self-management of diabetes. METHODS: This study mapped community assets in the categories of exercise, nutrition, and diabetes management across the Dallas ZIP codes with the heaviest burden of diabetes. This study then mapped awareness of these same resources and compared both maps to identify a disconnect between community resources and the communities they serve. RESULTS: Sample maps of community assets in several of 14 target ZIP codes were produced with the aid of Google maps, showcasing the abundance of community resources throughout the ZIP codes of Dallas County, including those with poor clinical and socioeconomic measures. Though results may not be representative of individual ZIP codes, awareness of resources appears to vary more directly with these clinical and socioeconomic measures than does true resource distribution. CONCLUSION: This study applies quality improvement and process analysis tools to empirically advance theoretical population health frameworks. Asset cataloguing and geospatial mapping demonstrate an abundance of community resources evenly distributed throughout Dallas but a dearth of resource awareness that loosely correlates with negative community measures. Such measures include median household income, BMI, HbA1C, and crime index. Continued indexing of Dallas County resources with more sophisticated mapping software will yield asset catalogues more consistent in their value to society; and continued awareness surveying is necessary to develop representative ZIP code awareness maps for deep comparison of these objective resource and subjective awareness measures.
Actin Regulatory Dynamics Required for T Cell Activation: A Quantitative and Systems-Level Perspective
(2013-02-04) Roybal, Kole Thomas 1982-; Ward, E. Sally; Wülfing, Christoph; Rosen, Michael K.; Pasare, Chandrashekhar
T cell activation occurs through interaction with an antigen-presenting cell (APC). Upon activation, signaling ensues with the coordination of dozens of diverse signaling molecules in space and time, a feature of cell signaling we call ‘spatiotemporal patterning’. We performed a systems-scale analysis of the spatiotemporal patterning of T cell signaling and have found that it is highly diverse. Over 50 signaling sensors were imaged in live primary T cells activated with APCs under various physiological stimulation conditions, and no two signaling intermediates showed the same dynamic localization. The activation environment controlled spatiotemporal features of T cell signaling and specific spatiotemporal features correlated with efficient T cell activation. To identify underlying cell biological mechanisms controlling spatiotemporal organization of signaling, we complimented our live cell imaging with microscopy across multiple scales and identified a dense transient F-actin network that extends from a highly interdigitated T cell:APC interface several micrometers deep into the T cell lamellum. Systems-scale imaging revealed a large network of proximal T cell signaling intermediates that localized to the lamellal actin network and shared the spatial, temporal, and mobility features of F-actin. Interference with lamellal actin dynamics modulated the activity of the associated proteins and impaired IL-2 production. These data strongly suggest that the transient deep F-actin network by controlling lamellal localization modulates the activity of a substantial part of the T cell signal transduction system. As a next step in understanding how spatiotemporal dynamics of signaling controls T cell activation, we have developed a quantitative 4D analysis approach for signaling networks and coupled it with traditional cell biological techniques to uncover higher order mechanisms of the control of actin dynamics by CD28 co-stimulation during T cell activation. A group of nine actin regulatory proteins that mediate actin polymerization, capping, and severing were assessed and CD28 co-stimulation was required for their sustained activity at the T cell:APC interface. WAVE2 and Cofilin were especially sensitive to blockade of CD28 signaling. Functional relevance of the loss of WAVE2 and Cofilin enrichment was shown by the treatment of T cells with constitutively active Rac1 and Cofilin, which bypassed the requirement of co-stimulation for normal actin dynamics and AKT activation. This study highlights how a systems analysis of actin regulation could identify mechanisms that are inaccessible to more traditional single protein/gene approaches.
Activated RhoA Positively Regulates Exchange Activity of PDZ-RhoGEF
(2012-12-06) Medina, Frank J., III 1979-; Taussig, Ronald; Sternweis, Paul C.; Albanesi, Joseph P.; Cobb, Melanie H.
RhoA plays a key role in regulation of the actin cytoskeleton, cell migration and cell shape. Rho GTPases cycle between an inactive GDP-bound state and an active GTP-bound state. This cycle is mediated by guanine nucleotide exchange factors (GEFs) that increase the rate of dissociation of GDP by stabilizing the nucleotide-free state of the GTPases via their DH/PH domains; this facilitates binding of GTP and activation of the protein. The RGS subfamily of RhoGEFs (RGS-RhoGEFs) act as direct mediators of RhoA activation in response to stimulation of the heterotrimeric G12 and G13 proteins by hormone receptors. RhoGEFs usually bind most tightly to the nucleotide free form of RhoA, which represents the intermediate state for exchange of guanine nucleotides. Recently, our lab discovered that PDZ-RhoGEF (PRG), a member of the RGS-RhoGEFs, bound tightly to both nucleotide-free and activated RhoA (RhoA•GTP). Using deletion analysis and pulldown assays, I was able to show that this interaction occurs between the PH domain of PRG and activated RhoA. James Chen was able to define the molecular determinants of this interaction by solving the crystal structure of the PRG-DH•PHRhoA(GTPγS) complex. This structure revealed that the interface is comprised of the switch regions in RhoA and a conserved hydrophobic patch in the PH domain of PRG. Interestingly, activated RhoA does not regulate the exchange activity of PRG in solution. Here, I use reconstitution of the signaling pathway with phospholipid vesicles and recombinant proteins, to show that this interaction serves as a mechanism for spatially regulating PRG exchange activity, a feed-forward mechanism. We hypothesize that this feed-forward mechanism is also applicable in vivo and potentially may serve as a mechanism utilized by a larger group of RhoGEFs known as the Lbc subfamily of RhoGEFs.
Activation of Early Neural Progenitors Is Required for Traumatic Brain Injury-Induced Hippocampal Neurogenesis
(2008-09-19) Yu, Tzong-Shiue; Kernie, Steven G.
Traumatic brain injury (TBI) is the most common form of acquired brain injury in both children and adults in the United States. TBI causes neuronal loss and results in a variety of neurological impairments and deficits in hippocampus-dependent functions. However, cognitive recovery commonly occurs though the mechanism is unknown. Exploration of post-natal neurogenesis in the hippocampus raises the possibility that adult-born neurons may contribute to cognitive recovery from TBI. Several studies in animal models that mimic TBI demonstrate there is enhanced generation of adult-born neurons in the dentate gyrus and those adult-born neurons may correlate with cognitive recovery. Due to the limits of current methodology in studying neurogenesis, it remains unclear what relevance injury-induced neurogenesis may have in the recovery process following TBI. In order to explore the relevance of injury-induced neurogenesis, I have characterized a previously generated transgenic mouse line that has rtTA-IRES-eGFP expression under the control of a nestin promoter and also contains a neural progenitor-specific regulatory element. By using this line, I have demonstrated that eGFP-expressing cells represent early neural progenitors in the adult dentate gyrus. Performing unilateral controlled cortical injury (CCI) demonstrates that this injury depletes doublecoritn (Dcx)-expressing late neural progenitors while activating eGFP-expressing early neural progenitors. To address whether the subsequent recovery of Dcx-expressing late progenitors was derived from activation of early neural progenitors, I generated a transgenic line that expresses modified herpes simplex viral thymidine kinase (delta-HSV-TK) under the control of the neural progenitor-specific regulatory element of the nestin gene. This allows for temporally regulated ablation of dividing neural progenitors by exposing the animal to ganciclovir. Using this line, I demonstrate that ablation of dividing GFP-expressing early neural progenitors in neurogenic areas occurs only in the presence of ganciclovir. CCI on these mice, reveals that no newly born Dcx-expressing late neural progenitors are observed seven days after injury when exposed to ganciclovir. However, the repopulation of Dcx-expressing cells is apparent when ganciclovir was removed one day before injury. Four weeks after injury, those newly born Dcx-expressing cells became mature NeuN-expressing neurons. This suggests that injury-induced activation of early neural progenitors is required for the recovery of injured hippocampal neurons.
Activity-Dependent Regulation of Inhibition from Different Inhibitory Subtypes
(2007-08-08) Bartley, Aundrea Frances; Gibson, Jay R.
Neuronal activity, in the form of action potential firing, is critical in the maturation and maintenance of neocortical circuitry. A negative feedback mechanism by which neuronal circuits adapt to changing levels of average activity on a time scale of hours to days is known as homeostatic plasticity. At the simplest level, homeostatic adaptations occur to maintain firing rate of neurons at a particular set-point. To better understand homeostatic plasticity at the network level, one must understand the activity-dependent adaptations that occur in the different neocortical cells types. To this end, I examined the regulation of inhibitory neurons and their synapses. I used chronic pharmacological block of activity in a neocortical slice cultures to examine the role activity plays in regulating feedback inhibition defined by two biochemical inhibitory neuron subtypes - parvalbumin-positive (Parv+) and somatostatin-positive (Som+). The cellular and synaptic components of local feedback inhibition were examined. I found that chronic activity blockade caused the following: 1) an increase in the intrinsic excitability of Som+ neurons through the downregulation of 2 substhreshold currents. While not thoroughly examined in Parv+ neurons, a similar, but weaker, increase in excitability may occur in these neurons as well. These< changes are consistent with a homeostatic maintenance of firing rate in these neurons. 2) a differential regulation of monosynaptic inhibition based on subtype that was frequency dependent. At low frequency action potential firing, Parv+ mediated inhibitory drive was downregulated while Som+ was unchanged. Both subtypes were likely downregulated at high frequency firing. 3) an upregulation of excitatory drive onto both Parv+ and Som+ neurons. This was most dramatic at low frequency firing where both subtypes displayed an almost 3-fold increase. This is also consistent with homeostatic maintenance of firing rate in inhibitory neurons. 4) based on the above, a clear change in recurrent inhibition occurred at low frequency firing. First, net recurrent inhibition was increased for both subtypes, but the relative influence of the two changed, such that Som+ recurrent inhibition contributed more relative to that of Parv+ circuitry. At high frequency firing, a slight, but less resolvable, increase in net recurrent inhibition may have occurred in both subtypes without any change in relative contribution. 5) all of the synaptic changes were likely due to increases in presynaptic release probability and/or decreases in synapse number.
Acute Treatment Outcomes and Family Functioning of Children and Adolescents Diagnosed with Anorexia Nervosa
(2006-08-11) Hetrick, Maryann O.; Kennard, Beth D.
Recent studies have suggested that there is a relationship between treatment outcomes and baseline factors related to family functioning and specific eating disorder symptoms. However, these relationships have not been studied extensively within a pediatric population hospitalized for treatment. Therefore, it is unknown whether these relationships exist within an acutely ill population and whether these baseline characteristics improve significantly immediately following acute hospitalization. Given these limitations, the aims of the present study were to identify aspects of family functioning and eating cognitions and attitudes at admission that predict outcome at discharge, and evaluate what aspects of family functioning and eating cognitions and attitudes improve during an acute treatment period. The sample consisted of 41 patients diagnosed with anorexia nervosa or eating disorder not otherwise specified between the ages of 10 and 17 years. At admission, all patients were administered a structured clinical interview to obtain valid psychiatric diagnoses. Additionally, patients completed self-report measures of eating cognitions, eating attitudes, and family functioning; while parents completed a self-report measure of family functioning. Families also participated in a standardized clinician-rated observational measure of family functioning. All measures were re-administered at discharge, and the patient's body mass index (BMI) at admission and discharge were obtained from the medical record. The attrition rate from intake to discharge for this study was 26.8%. Overall, it appeared that parents and patients perceived their families to be healthy at intake, with little improvements noted over the course of treatment. However, standardized observations characterized these families as being affectively avoidant. Additionally, parental perception of adaptive family functioning at intake was predictive of outcome based upon the unit psychiatrist's assessment, and patient perception of healthy familial Expressiveness at intake was predictive of outcome based upon pathological eating attitudes. BMI and eating attitudes based upon eating behavior during treatment improved significantly over the course of treatment. However, patients continued to endorse unhealthy eating cognitions at discharge. These results suggest that weight restoration and pathological eating behavior are the first symptoms to improve during an aggressive treatment period, and psychological symptoms may require a longer period of treatment to remit.
Acyloxyacyl Hydrolase: Studies on Its Regulation and Function in Mus Musculus
(2004-01-14) Lu, Mingfang; Munford, Robert S.
Acyloxyacyl hydrolase (AOAH) is an enzyme that detoxifies Gram-negative bacterial lipopolysaccharides (LPS) by selectively removing secondary acyl chains from the lipid A moiety. Originally found in neutrophils, it is also produced by monocyte-macrophages and renal proximal tubule cells. In the studies described here, I found that both immature dendritic cells (DCs) of the XS52 cell line and bone marrow-derived DCs produce AOAH. AOAH expression decreased when DCs were incubated with IL-4, IL-1ᬠTNFa and an agonistic CD40 antibody (maturation cocktail), and increased following treatment with microbial agonists that engage 3 distinct Toll-like receptors (LPS, TLR4; CpG oligodeoxynucleotides, TLR9; and a Gram-positive bacterium (Micrococcus luteus), TLR2). Maturation cocktail treatment also diminished, while LPS treatment enhanced or maintained, the cells' ability to kill E. coli, deacylate LPS, and degrade bacterial proteins. Enzymatic deacylation of LPS is thus an intrinsic, regulated mechanism by which DCs may modulate host responses to this potent bacterial agonist. To study the biological functions of AOAH, AOAH-deficient mice were generated by targeted gene disruption. AOAH did not protect mice from lethal doses of LPS or Gram-negative bacterial challenge. In response to subcutaneous injections of LPS, however, AOAH-deficient mice produced significantly higher levels of non-specific (polyclonal) IgM and IgG3 than did wild type mice. Anti-double-stranded DNA and anti-nucleosome IgM and IgG antibody levels were also higher in LPS-immunized AOAH-deficient mice than in wild type control mice. In addition, the partially-deacylated LPS product (dLPS) induced lower polyclonal antibody responses in vivo than did mock-treated LPS, yet the anti-LPS specific responses to dLPS and LPS were equivalent. These results suggest that AOAH may diminish potentially harmful polyclonal antibody responses to Gram-negative infection but maintain the protective anti-LPS specific response. Since B cells do not produce the enzyme, my results also point to an important role for macrophages and DCs in modulating B-cell responses to LPS antigens. In addition, the absence of AOAH did not alter the ability of LPS to function as an adjuvant, indicating that this activity is mechanistically distinct from stimulation of polyclonal antibody production. Finally, the ability of a bacterial lipopeptide to stimulate polyclonal antibody production only in AOAH -/- mice suggests that the enzyme may also regulate immune responses to non-LPS bacterial agonists.
The Addicted Phenotype: Protein Phosphorylation Status and Dopamine Receptor Responsiveness
(2007-05-22) Edwards, Scott; Self, David W.
Up-regulation of cAMP/PKA signaling by drugs of abuse may contribute to escalation and relapse, possibly by differentially altering dopamine receptor-responsiveness in the mesolimbic dopamine system. To investigate this hypothesis, our initial studies measured alterations in cAMP-dependent and -independent protein phosphorylation in vivo produced by chronic cocaine and heroin self-administration, changes in mesolimbic protein phosphorylation compared to individual differences in the propensity for escalating cocaine self-adminstration, and, ultimately, dopamine receptor-mediated regulation of relapse to cocaine seeking in withdrawal. Chronic cocaine self-administration can produce either tolerance or sensitization to certain cocaine-regulated behaviors, but whether differential alterations develop in the biochemical response to cocaine is less clear. In Chapter 2, we studied cocaine-induced phosphorylation of multiple cAMP-dependent and -independent protein substrates in mesolimbic dopamine terminal regions following chronic self-administration. Changes in self-administering rats were compared to changes produced by passive yoked injection to identify regulation related to the context of behavioral reinforcement, whereas acute and chronic yoked groups were compared to identify the development tolerance or sensitization in the biochemical response to cocaine. Microwave-fixed brain tissue was collected immediately following 4 hrs of intravenous cocaine administration, and subjected to western blot analysis of phosphorylated and total protein substrates. Chronic cocaine produced region- and substrate-specific tolerance to cAMP-dependent protein phosphorylation, including phosphorylation of the AMPA GluR1 receptor subunit at serine 845 in striatal and amygdala subregions, and the NMDA NR1 receptor subunit at serine 897 in the CA1 subregion of hippocampus. Tolerance also developed to cAMP-independent GluR1S831 phosphorylation in the prefrontal cortex. In contrast, sensitization to cocaine-induced phosphorylation of the pre-synaptic vesicle protein synapsin I at serine 9 developed in amygdala and hippocampal subregions, while cAMP-dependent phosphorylation of the dopamine-synthesizing enzyme tyrosine hydroxylase at serine 40 decreased in pre-synaptic striatal dopamine terminals in striatal subregions. Cocaine-induced phosphorylation of extracellular signal-regulated kinase (ERK) was dissociated from downstream phosphorylation of the transcription factor cAMP-response element binding protein (CREB) in many brain regions, and failed to develop either tolerance or sensitization with chronic administration, and failed to develop either tolerance or sensitization with chronic administration. Positive reinforcement-related correlations between cocaine intake and protein phosphorylation were found only in self-administering animals, while negative dose-related correlations were found primarily with passive yoked administration. These regional- and substrate-specific adaptations in cocaine-induced protein phosphorylation are discussed in lieu of their potential impact on the development of cocaine addiction. In Chapter 3, we studied alterations in protein kinase A (PKA)-dependent and PKA-independent phosphorylation in multiple brain regions in rats undergoing either spontaneous or naltrexone-precipitated withdrawal (WD) from chronic intravenous heroin self-administration. Spontaneous WD from heroin self-administration produced region-specific increases in PKA-dependent GluR1S845 phosphorylation in the nucleus accumbens shell, basolateral amygdala, hippocampal CA1 and CA3 regions, and premotor cortex after 24 but not 12 hrs, and there were no changes in prefrontal cortex, nucleus accumbens core or caudate-putamen. Increased GluR1S845 phosphorylation occurred earlier (12 hrs) in the central amygdala, ventral tegmental area, and substantia nigra. In contrast, prominent ERK phosphorylation was found in both prefrontal and premotor cortex, CA1 and CA3, caudate-putamen, and basolateral amygdala, but not in nucleus accumbens, or central amygdala in spontaneous WD. Phosphorylation of striatal CREB increased in caudate-putamen but not in nucleus accumbens, paralleling ERK rather than PKA activity in heroin WD. Naltrexone administration potentiated GluR1S845 and ERK phosphorylation in the central amygdala, and ERK phosphorylation in nucleus accumbens core and shell. Thus, spontaneous WD from heroin self-administration produces region- and time-dependent changes in PKA and ERK activity that could contribute to the behavioral manifestation of opiate dependence. In Chapter 4 we studied PKA-dependent GluR1S845 phosphorylation and ERK phosphorylation mediated by ERK kinase in striatal subregions in an animal model of cocaine craving. Here, animals with chronic cocaine self-administration experience were re-exposed to the self-administration test chambers for 1 hr in the absence of cocaine to measure phosphorylation induced by the environmental context paired with cocaine reinforcement. After 1 day WD, GluR1S845 levels were elevated in both self-administering and yoked groups in the nucleus accumbens shell, but this effect persisted only in self-administering animals after 3 weeks WD. In the nucleus accumbens core, context-induced phosphorylation of both GluR1S845 and ERK increased from early to late WD from chronic cocaine self-administration, implicating this region in mediating the intensification of cocaine craving with longer periods of abstinence. These differential region- and substrate-specific adaptations to withdrawal- and context-induced protein phosphorylation could underlie the maintenance of cocaine addiction by exacerbating the potential for drug relapse in withdrawal. Finally, as a behavioral correlate, studies in Chapter 5 sought to compare these changes in protein phosphorylation status with alterations in dopamine-receptor mediated regulation of relapse to cocaine seeking in withdrawal. Here, the cocaine-addicted phenotype was modeled in rats based on individual differences in preferred levels of cocaine intake and a propensity for relapse in withdrawal. Since these cocaine-taking and -seeking behaviors are strongly but differentially regulated by postsynaptic dopamine D1 and D2 receptors in the mesolimbic system, we determined whether the development of cocaine addiction would be related to differential sensitivity in functional D1 and D2 receptor responses. Using a population of 40 outbred Sprague-Dawley rats trained to self-administer cocaine for 3 weeks, we found that animals with higher preferred levels of cocaine intake exhibited a vertical and rightward shift in the self-administration dose-response function, and were more resistant to extinction from cocaine self-administration, similar to phenotypic changes reported in other models of cocaine addiction. After 3 weeks of withdrawal from cocaine self-administration, high intake rats were subsensitive to the ability of the D1 agonist SKF 81297 to inhibit cocaine-seeking behavior, but supersensitive to cocaine seeking triggered by the D2 agonist quinpirole, when compared to low intake rats. Additionally, high intake rats developed profound increases in locomotor responses to D2 receptor challenge from early to late withdrawal times, whereas low intake rats developed increased responsiveness to D1 receptor challenge. In a second experiment, responses to the mixed D1/D2 agonist apomorphine and the NMDA glutamate receptor antagonist MK-801 failed to differ between low and high intake rats. These findings suggest that cocaine addiction is specifically related to differential alterations in functional D1 and D2 receptors that mediate opposing influences on cocaine-seeking behavior. Alterations in dopamine receptor signaling may issue from an enduring up-regulation of cAMP/PKA signaling in drug withdrawal. The potential for reduced D1 receptor (Gs/Golf-coupled) sensitivity to account for tolerance to cocaine-regulated PKA-dependent protein phosphorylation during self-administration (Chapter 2) is discussed in Chapter 6. Conversely, it is possible that PKA up-regulation in cocaine WD (Chapter 4) could account for the paradoxical sensitization of subsequent Gi/Go-coupled D2 receptor responses. Thus, addiction-related alterations in D1 and D2 dopamine receptor responses may ultimately involve complex reciprocal interactions between adaptations in PKA signaling pathways that differentially influence D1 and D2 receptor signaling.

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