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Posttraumatic stress disorder (PTSD) affects 6-17% of college students in the United States, which can negatively impact academic achievement and is associated with other emotional difficulties. Exposure therapy, including written exposure therapy as been found to be efficacious for treating. However, concerns of treatment dropout and low symptom improvement are barriers to treatment success. Experiential avoidance in treatment has been problematic and may influence outcomes. The current study sought to reduce the use of experiential avoidance in exposure sessions through the promotion of self-compassion, an extension of kindness, humanity and mindfulness to the self. Furthermore, the current study also examined the impact of self-compassion induction on self-reported PTSD symptoms. The study recruited introductory psychology students who had PTSD symptoms following exposure to a traumatic event and randomly assigned these participants to two groups: an exposure-with-self-compassion component group and an exposure-only group. Using PROCESS mediation analysis, the main findings indicated that the self-compassion intervention was not effective in increasing state self-compassion and was not associated with any notable changes in state experiential avoidance. However, both conditions showed improvement in PTSD symptoms, providing support for written exposure treatment. [The dissertation citations contained here are published with the permission of ProQuest LLC. Further reproduction is prohibited without permission. Copies of dissertations may be obtained by Telephone (800) 1-800-521-0600. Web page: http://www.proquest.com/en-US/products/dissertations/individuals.shtml.]

The purpose of this qualitative study was to discover how communication professors at four-year private universities help students who exhibit public speaking apprehension (PSA) learn to cope with their anxiety. The research was framed in the narrative inquiry paradigm, interviewing eight college communication professors about their experiences working with public speaking students. Each of the eight professors worked at private universities in a large southwest metropolitan city. The researcher attempted to gather critical incidents that shed light on valuable learning experiences that could be useful to professors looking for ways to help reduce their students' PSA. The researcher found three common themes embedded in the interviews: (a) meeting with students individually and establishing relationships with them; (b) building community and trust in the classroom; and (c) placing less emphasis on grades. Each of the common themes contributed in one way or another to the critical incident that shaped the professor's teaching perspective. Although the purposeful sample was limited in size it revealed that Communication Orientation Motivation (COM) Therapy and systematic desensitization can both be useful treatment techniques for students willing to improve their public speaking ability. Findings revealed that professors were able to change an apprehensive student's perspective by focusing on public speaking as a communicative effort rather than a performance. The study offers recommendations for professors engaged with students at all levels of education in addressing this issue. [The dissertation citations contained here are published with the permission of ProQuest LLC. Further reproduction is prohibited without permission. Copies of dissertations may be obtained by Telephone (800) 1-800-521-0600. Web page: http://www.proquest.com/en-US/products/dissertations/individuals.shtml.]

In order to determine whether or not systematic desensitization or hypnosis have any effect in alleviating the test anxiety of community college students, 30 Massasoit Community College student volunteers were matched for GPA and assigned to one of three treatment groups: (1) a systematic desensitization group, in which subjects listened to tape recorded training sessions in relaxation, and were asked to imagine the anxiety provoking scenes described on tape; (2) a hypnosis group, in which subjects listened to hypnotic inductions, and were asked to imagine the same anxiety provoking scenes; or (3) a control group. At the outset, each subject had a two-semester GPA of 3.0 or below and each had scored in the upper quartile on the Mandler-Sarason Test Anxiety Scale. Pre- and post-treatment examinations of GPA, scores on the Mandler-Sarason Scale, and two other self-report measures of anxiety, showed no significant effects of the treatment. It is concluded that these behavioral change techniques, found successful with other populations, must be critically examined for their suitability for two-year college students. Also included are a review of the evolution of community colleges in the United States, a discussion of the unique characteristics of community college students, and a survey of recent approaches to behavior change. Results are tabulated and the testing instrument is appended. (NHM)

A study examined the relative effectiveness and synergistic effect of two treatments for reducing speech anxiety--Self-Statement Modification (SSM), a therapy focused on modification of cognitive behavior; and Modified Desensitization (MD), a therapy focused on physiological variables, utilizing relaxation training, group hierarchy construction, and reciprocal inhibition of anxiety through the pairing of imaginal scenes with relaxation. Four treatments--SSM, MD, Cognitive Modification (a combined version of SSM and MD), and an attention-placebo treatment--were administered to four different groups (a total of 53 university students with high self-ratings of speech anxiety) in four two-hour periods, preceded and followed by assessment sessions. A five-week follow-up assessment was performed, which included self-report measures of speech and social anxiety, as well as stress-condition measures (self-report and behavioral) of acute speech anxiety. Results indicated that SSM was the most effective strategy for reducing speech anxiety, and that students experiencing high social anxiety benefited more from treatments than those experiencing low social anxiety. The synergy hypothesis, designed to examine an additional unique effect resulting from the interplay of procedures, was not supported. (Fourteen tables and three figures are included, and appendixes providing the rationale and guidelines for treatment, speech anxiety hierarchy, handout material on Self-Statement Modification and Speaking Skills, assessment instruments, confidential evaluation, and 187 references are appended.) (MM)

A study involving 97 students (79 females and 18 males) at New York City Technical College was undertaken to determine the effectiveness of desensitization in reducing test anxiety and improving grade point averages (GPAs). The study compared the GPAs of students who completed workshops using the desensitization hierarchy developed by R. Strieby (1974) with those of control students. The study used J. Wine's (1971) theory that systematic desensitization should be effective in reducing test anxiety. In all, 42 students did and 55 students did not participate in the workshops. Test anxiety levels of all subjects were assessed by administration of the debilitating and facilitating scales of the Achievement Anxiety Test before and after a treatment period of 6 weeks. The achievement level was assessed for all subjects through the comparison of GPAs at the end of the semester. In addition, a Mann Whitney u test was used to evaluate the significance of the results. Results indicate that students participating in the workshops improved their academic averages more than did control students. The experimental group also reported less debilitating anxiety than did the control group, although there was no comparable finding for facilitating anxiety. Early screening of students to identify test anxiety and the use of desensitization workshops using counselors in college learning centers are recommended. Study instruments and instructions for subjects and a 37-item list of references are included. (TJH)

Anti-human leukocyte antigen antibodies (aHLA Ab) can form during a humoral immune response to non-self HLA encountered during pregnancy, blood transfusion, or transplantation. Due to shared epitopes across HLA alleles, highly sensitized patients can have aHLA Ab that recognize and would destroy an allograft from >99% of deceased donors; thus, their pool of available organs is severely limited compared to non-sensitized patients. To increase organ availability for highly sensitized patients, aHLA Ab must be sustainably reduced which requires depleting their source: plasma cells (PCs). Our group has focused on PC depletion using proteasome inhibitors as PCs are exquisitely dependent on the proteasome to maintain proteostasis due to their high rate of antibody synthesis. Despite significant PC depletion, proteasome inhibitors alone are insufficient to sustainably reduce aHLA Ab. We hypothesized this was in part due to PC residence in protective bone marrow (BM) microenvironments. Because the CXCR4:CXCL12 axis is instrumental in recruiting and maintaining PCs in the BM, we conducted a proof-of-concept clinical trial to determine if CXCR4 antagonism with the small molecule inhibitor, plerixafor, would mobilize PCs out of the BM. We evaluated the effects of plerixafor on PC residence in blood and BM, its safety profile (alone and in combination with the proteasome inhibitor, bortezomib), and the transcriptional impact on BMPCs in HLA-sensitized kidney transplant candidates. We found that plerixafor indeed mobilized PCs to peripheral blood and had varying effects on BM residence depending on the dose of plerixafor and bortezomib used. Single cell RNA-sequencing on BMPCs pre- and post-treatment revealed multiple populations of PCs, with post-treatment enrichment of oxidative phosphorylation, proteasome assembly, cytoplasmic translation, and autophagy-related genes. Follow up in vitro studies using mouse BMPCs demonstrated that dually inhibiting the proteasome and autophagy resulted in greater BMPC death than did monotherapies. Taken together, our findings suggest the potential utility of plerixafor-based PC mobilization and autophagy- and proteasome-inhibitor-based PC depletion in desensitization regimens.

This study uses design to create a children’s medical picture book that is presented in first-person perspective through illustrations and text that teach children coping strategies for going to a medical visit. Many children fear what they do not understand. Children’s medical picture books are a useful tool to teach children coping strategies as well as what to expect from a visit to a doctor.

This thesis focuses on the signaling and trafficking functions of beta-arrestins, which are flexible regulators and transducers for the large G protein-coupled receptor (GPCR) family. The introductory chapter will briefly cover GPCR biology, how the β-arrestins were discovered, the findings that led the canonical understanding of β-arrestin signaling and trafficking functions, recent structural and biophysical advances, as well as the approaches used to obtain the data presented in this thesis. The second chapter describes a new cellular mechanism for β-arrestin function termed ‘catalytic activation’ in which β-arrestins can transiently bind to and be activated by GPCRs. The third chapter provides evidence that lipid engagement by β-arrestins is a key regulator of β-arrestin conformation and function. The fourth chapter will discuss new approaches to understanding the conformational states of β-arrestins and how they may explain the functional flexibility of β-arrestins. The fifth chapter describes a new mechanism of β-arrestin–mediated trafficking and provides a framework for understanding how β-arrestins transduce receptor-specific functions. The final chapter summarizes the major advances from the previous chapters, discusses how these fit with the established findings, and suggests future directions.

Pediatric headache is a global health concern, with up to 60% of youth experiencing persistent headache pain for at least three months (Abu-Arafeh et al., 2010). Often accompanying the headache are symptoms such as sensitivity to light and sound (Hayne & Martin, 2019; Neut et al., 2012). Headache pain and its associated sensitivities often result in a significant reduction in daily function and avoidance of school, leisure, and social activities (Langdon & DiSabella, 2017). Sensitivities to light and sound are important yet understudied symptoms of pediatric headache disorders. The lack of empirical evidence to guide occupational therapists in the treatment of headache-related sensitivities in children and adolescents presents a major gap in the practice area of pediatric chronic pain. Pediatric Auditory and Visual Exposure (PAVE) was developed to address this gap and to provide occupational therapists with an effective, standardized approach to treat headache-related sensitivities in youth. PAVE is a theoretically driven, exposure-based occupational therapy intervention for youth (ages 8–17) presenting with a diagnosed pediatric headache disorder and experiencing light and/or sound sensitivities that interfere with their daily functioning. The intervention consists of six treatment modules employed over the course of several weeks. The primary goal of PAVE is to improve participant daily functioning through reduction of avoidance behavior, sensory sensitivity, and pain intensity, all of which contribute to functional disability.

Mammalian cells are surrounded by fluctuations of various cytokines, small proteins that control a broad range of cellular signaling. In order to survive, cells must respond appropriately to these different dynamics of these signaling molecules. This thesis work presents partial answers to how human epithelial cells decode information encoded in the dynamics of the external stimuli and successfully execute proper responses. We focus on interferon (IFN) pathway which is important for viral infection as well as tumorigenesis. We employed CRISPR/Cas9 technology to generate reporter cell lines, time-lapse microscopy, microfluidics, single cell analysis and computational modeling to dissect the molecular mechanism governing the cytokine responses. In chapter 1, we studied how IFN-a pretreatment could lead to two contradictive effects: priming and desensitization. We discovered that short pretreatment duration produced priming effect while prolonged IFN treatment caused activation of delayed negative feedback, USP18, responsible for desensitization. Intriguingly, USP18 induction was duration and cell-cycle dependent. Understanding this regulation paves ways to improve the usage of IFN in virus infection such as SARS-CoV-2 and cancer therapy. In chapter 2, we systemically studied the cellular response to different type of IFN. We found that three types of IFN process distinct dynamic responses. Type I IFN (IFN-I) was a potent IRF9 inducer than type III but succumb to desensitization. Single cells analysis showed heterogenous response to IFN-III stimulation. Type II was strong STAT1 activator but was unable to induce IRF9. Interestingly, combination of IFN-II and -III generated a synergy effect mimicking IFN-I response but without USP18 induction and therefore no desensitization. Short pulses of low IFN-II dose were sufficient to maintain the synergy. We believe that this will provide alternative methods to deliver IFN to patients especially against virus infection.

Pentameric ligand-gated ion channels (pLGICs) are transmembrane proteins which mediate fast excitatory or inhibitory neurotransmission. The composition of the plasma membrane regulates the function of these channels. Bioactive lipids and cannabinoids have been shown to allosterically modulate the function of pLGICs and the mechanisms by which they affect receptor activity continue to be actively investigated. Here, we examine how essential omega-3 and omega-6 fatty acids, and endogenous/ plant cannabinoids modulate pLGICs by functional and structural studies. This work also addresses the structural basis for inhibition in a cationic pLGIC by a clinically approved antagonist. We show that docosahexaenoic acid (DHA) enhances desensitization in two cationic pLGICs, GLIC and 5-HT3AR, immediately upon application. DHA has no effect on a non-desensitizing GLIC double mutant confirming that DHA enhances agonist induced desensitization. We crystallized GLIC with DHA and found that DHA binds at the channel periphery near transmembrane helix 4 (M4) and is stabilized by an Arg118 residue from the Cys-loop. The effect of DHA was reduced when we mutated the Arg to Ala thus validating the DHA binding site. Notably, the upper half of the channel pore was wide open and the lower half was constricted resulting in loss of water and ions, thus stabilizing a lipid-induced potential desensitized state. Conversely, cannabinoids mediate their effect after pre-application suggesting that they likely diffuse into the membrane before affecting channel activity. A 3-D reconstruction of the 5-HT3AR in complex with ∆-9-Tetrahydrocannabinol (THC) and serotonin (5-HT) shows the extracellular and transmembrane domains in a potentially activated conformation and a partially occluded intracellular domain at the lateral portals. This preliminary structure provides a glimpse into the potential mechanism underlying cannabinoid modulation of the 5-HT3AR. To better understand orthosteric inhibition we show the cryo-electron microscopy structure of the 5-HT3AR receptor in complex with granisetron displaying the precise binding orientation of the ligand, the interacting residues and the conformational changes that stabilize a non-conducting conformation. We highlight the differences between the resting non-conducting and inhibited non-conducting conformations. Collectively, these results begin to shed light into the structural basis for the modulation of pLGICs by lipids, cannabinoids and antagonists.

Many clients with developmental and/or intellectual disabilities (ID/DD) do not tolerate routine medical or dental procedures and may require intrusive interventions, including restraint of various types (i.e. chemical, mechanical, physical, etc.) during appointments. Graduated exposure, or stimulus fading, along with reinforcement for compliance, have been shown to increase cooperation and tolerance in some clients; however, many do not respond to these types of interventions. Nine participants diagnosed with ID/DD recieved compliance/tolerance training for routine medical or dental procedures. Results of these interventions were evaluated in the context of several potential indices of readiness, such as medical diagnoses, level of disability, and presence of challenging behavior, among others. Several of the variables appeared to be correlated with program responsiveness; however, a larger sample will be necessary to draw definitive conclusions. Client characteristics and past assessments (anecdotals, preference assessments, terminal probes, and survey data) were evaluated. The analytical framework developed for this analysis may be useful to future researchers and clinicians as a model for assessing readiness for tolerance training programs.

Adverse effects of video game violence have been well documented in psychology research for decades. As technology advances, violence depicted in games becomes more realistic and features more diverse types of aggression. A new trend in violent games is the depiction of women as sexualized targets of violence. Research suggests that exposure to sexual content and violence in games can be harmful to both male and female players, but no studies to date have looked at specific differences in outcomes based on the victim’s gender in a violent game. The present study examines how opponents’ gendered appearance in a violent video game affects players’ implicit attitudes related to gender and gender violence. By having 235 undergraduates play a game fighting either male or female opponents, I found that women who fought female opponents expressed more negative implicit attitudes toward women.

Food allergy is defined as an abnormal immune response following ingestion of food-based allergens. Ingestion of a food allergen, by a sensitized individual, can induce shock and cause the development of different localized responses such as urticaria, wheezing, cramping, vomiting, or diarrhea. Food allergy is a type I hypersensitivity that is mast cell- and basophil-dependent; an absorbed allergen binds to IgE associated with the high-affinity IgE receptor, Fc?RI, on the cell surface of mast cells and basophils leading to the secretion of compounds causing signs and symptoms. However, the role of mast cells, along with the mediators released, is not well understood. Two mouse models were utilized to investigate the clinical response of food allergy as determined by measuring hypothermia and the development of diarrhea. One model, the active model, is sensitization through injection of a food antigen with an adjuvant. Sensitized mice were then orally challenged two times a week until both hypothermia and diarrhea were observed. The second model, the passive model, requires an intravenous injection of antigen-specific IgE monoclonal antibody (mAb) twenty-four hours prior to an oral challenge with the specific antigen. Previous studies in our lab indicated that an allergic response, modeled in mice utilizing the active immunization method, induces both hypothermia and diarrhea in response to an oral antigen challenge. However, the passive immunization model induces hypothermia without diarrhea. This led to the hypothesis that changes within the gastrointestinal tract induced by active sensitization were required for the development of diarrhea. Our results indicated that mice sensitized to egg white protein (EW), using the active model of sensitization, developed diarrhea in response to a challenge with TNP-BSA after sensitization with IgE anti-TNP mAb. This contrasted with mice that were not actively immunized and only developed a temperature drop to a similar IgE anti-TNP/TNP-BSA challenge. These observations provide support for the idea that organ specific changes, induced by prior sensitization with an antigen can facilitate the development of an organ specific local response such as diarrhea to a separate and systemic allergen.Treating allergy mainly focused on avoidance of the allergen but another method, oral immunotherapy, has been suggested. Oral immunotherapy, is carried out by increasing doses of an allergen until a large dose does not elicit a response and the patient has been desensitized. However, this process can take weeks to month, may not work in some individuals, and is temporary. Efforts to understand desensitization have discovered mechanisms associated with an establishment of a non-responsive state however, the effect of desensitization on mast cells has not been understood. We hypothesized that mast cell degranulation and/or sensitivity to mast cell mediators, as measured by a temperature drop, might be decreased. Our studies show degranulation of mast cells increased and decreased sensitivity to histamine was a short-lived phenomenon. Additionally, treatment with propranolol one day after desensitization re-established a temperature response. These observations suggested novel mechanisms, such as decreased sensitivity to vasoactive mediators and an induction of an adrenergic response in desensitization.

A number of studies have indicated that violent video gameplay is associated with higher levels of aggression, and desensitization to violent content contributes to this association. Utilizing a rapid serial visual presentation (RSVP) task, the current study used event-related potentials (ERPs) to investigate selective attention (N1 activation), cognitive control (N2 activation), and desensitization (P3 activation) as neurocognitive mechanisms potentially underlying the association between gameplay and subtypes of aggression. Results showed video game players and non-players differed significantly in brain activation when engaged with violent imagery. N1 and P3 amplitude moderated the association between gameplay and pleasure-oriented aggression. Follow-up analyses further revealed that individuals who play games for many hours and show large N1 activation (high selective attention) in the face of violence have small P3 activation (heightened desensitization). Thus, our results suggest that selective attention to violent content and subsequent desensitization effects moderate the association between video gameplay and aggression.

The yeast G-protein-coupled receptor (GPCR) Ste2 is an integral plasma membrane protein that initiates response to an extracellular stimulus (the peptide mating pheromone α-factor) by mediating ligand-dependent activation of a cognate heterotrimeric G-protein. Prolonged pathway stimulation is detrimental, and feedback mechanisms have evolved that act at the receptor level to limit the duration of signaling and stimulate recovery from pheromone-induced G1 arrest. In the research described in this dissertation, I found that three α-arrestins — Rod1/Art4, Rog3/Art7 and Ldb19/Art1 — serve as adaptors to promote the ubiquitinylation-dependent internalization of Ste2 and block its ability to signal, thereby desensitizing the cells to continued stimulation. Deleting the genes encoding these three α-arrestins increases the sensitivity of a MATa haploid cell to mating pheromone and results in an increase in Ste2 abundance at the plasma membrane. Conversely, overexpression of either Rod1 or Rog3 enhances the rate of adaptation. To contribute to negative regulation of the mating pheromone response pathway, Ldb19 requires binding of a HECT domain class of ubiquitin ligase, Rsp5, and most likely clears misfolded Ste2 from the PM. I found that Rod1 and Rog3 contribute to clearance of the pheromone-bound state of Ste2 and negatively regulate the mating pheromone response pathway by both Rsp5-dependent and Rsp5-independent mechanisms. In addition, I identified two classes of protein kinases (Snf1/AMPK and Ypk1/SGK) that phosphorylate and inactivate Rod1. Conversely, I showed that the phosphoprotein phosphatase responsible for dephosphorylating and re-activating Rod1 is calcineurin / PP2B. Because the S. cerevisiae genome does not encode any β-arrestins, the findings I made and present in this dissertation are the first to show that α-arrestins alone are capable of negatively regulating a GPCR.

No longer are village elders and literature the only sources of stories that compel consumers to become emotionally and cognitively involved with fictional characters. Rather, technologies such as television, Netflix, and digital video recording allow media consumers to not only become involved with mediated characters, but even feel empathy with them. Coupled with a media-heavy culture, submersing oneself in emotion-inducing narratives may bode well for consumers’ entertainment. However, constant connections to characters may inhibit viewers’ empathic capabilities toward other human beings by way of desensitization. This study examined television consumption as a predictor of empathy in 491 college students at a midsized Midwestern university. Findings suggested that television use does not directly impact empathy. However, increased consumption of crime dramas, situational comedies, and overall television predicted higher desensitization to violent and emotional portrayals, and increased desensitization predicted lower empathy. In addition, men were more desensitized and less empathic than women. By exploring desensitization to nonviolent content, these findings expand upon prior desensitization research which only focused on violence. Limitations to the study are acknowledged, and implications are discussed in terms of attempting to better understand how the increasing time viewers spend immersed in their mediated worlds may impact their real worlds.

Community violence exposure (CVE) is a major environmental stressor that threatens the psychological well-being of many youth living in American cities. Although research to date has documented the many psychosocial correlates of CVE among youth of all ages, issues regarding the definition and measurement of CVE limit our complete understanding of precisely how exposure to various forms of community violence relates to youths’ well-being. This study uses longitudinal data from the Project on Human Development in Chicago Neighborhoods (PHDCN) to investigate relations between CVE and both internalizing and externalizing symptoms among 4,149 youth ages 3 to 15 at baseline. In particular, this study examines patterns of emotional desensitization to CVE, the differential impact of chronic versus isolated CVE, the moderating roles of age and gender in these patterns, and the impact of CVE severity on youths’ internalizing and externalizing symptoms. Overwhelmingly, results provided support for the hypothesis that youths become emotionally desensitized to violence, as evidenced by quadratic associations between CVE and internalizing symptoms both over youths’ lifetimes and a one-year period. Quadratic associations between CVE and externalizing symptoms also emerged in some cases, but tended to be weaker than those for internalizing symptoms. Whereas linear associations were greater among older youth than younger youth, neither cohort nor gender moderated the quadratic associations between CVE and well-being. Isolated CVE was not distinctly associated with internalizing or externalizing symptoms compared with more chronic exposure, and less severe CVE was more strongly associated with well-being than more severe CVE. These findings are integrated with evidence for desensitization. Further, both theoretical and practical implications of the findings are considered, and next steps for research are offered.

Background: Desensitization has been used for some decades to treat patients with the allergenic drug when an alternative drug with similar effi cacy and safety is not available. We present the results from a series of oncology patients desensitized at our hospital during the last 2 years. Objective: To assess the effi cacy of a new desensitization protocol in patients allergic to chemotherapy drugs. Methods: We performed an observational retrospective study of 11 women (6 breast cancer and 5 ovarian cancer) who underwent our desensitization protocol. Four patients had immediate reactions to carboplatin, 3 to docetaxel, 3 to paclitaxel, and 1 to both docetaxel and paclitaxel. Premedication was administered in all cases. A 5-step protocol based on 5 different dilutions of the drugs was used. Results: We performed 39 desensitization procedures: 14 to carboplatin, 3 to oxaliplatin, 16 to docetaxel, and 6 to paclitaxel. Eight patients tolerated the full dose in 36 procedures. One patient suffered an anaphylactic reaction to carboplatin that reverted with treatment. One patient had dyspnea after a paclitaxel cycle. One patient experienced dyspnea due to chronic pulmonary thromboembolism related to her disease. Conclusion: Desensitization is a useful procedure in patients who are allergic to their chemotherapy agents.

Desensitization of the μ-opioid receptor (MOR) has been implicated as an important regulatory process in the development of tolerance to opiates. Desensitization of G-protein coupled receptor (GPCR) is thought to involve receptor phosphorylation and subsequent recruitment of βArrestins (βArrs). However, the roles of receptor phosphorylation and βArr in morphine-induced MOR desensitization remain to be demonstrated; this may result from the insensitivity of the methods used to study receptor function. Using MOR-induced intracellular Ca2+ ([Ca2+]i) release to monitor receptor activation, [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO) induced MOR desensitization in a receptor phosphorylation- and βArr-dependent manner. DAMGOinduced desensitization was blunted in HEK293 cells expressing the MORS375A mutant and was eliminated in MEF cells isolated from βArr2 knockout mice expressing the wild type MOR. However, although morphine induced a more rapid desensitization of [Ca2+]i release than DAMGO did and could induce the phosphorylation of the Ser375 residue of MOR, morphine-induced desensitization was not influenced by mutating MOR phosphorylation sites or in MEF cells lacking βArr1 and 2. In contrast, morphine induced MOR desensitization via protein kinase C (PKC). By using subtype-specific inhibitors, PKCε was shown to be the PKC subtype activated by morphine and the subtype responsible for morphine-induced desensitization. Meanwhile, DAMGO did not increase PKCε activity and DAMGO-induced MOR desensitization was not affected by a PKCε inhibitor. Among the various proteins within the receptor signaling complex, Gαi2 was phosphorylated by morphine-activated PKCε. Moreover, mutating v three putative PKC phosphorylation sites, Ser44, Ser144 and Ser302 on Gαi2 to Ala attenuated morphine-induced, but not DAMGO-induced desensitization. In addition, pretreatment with morphine desensitized cannabinoid receptor CB1 agonist WIN 55212-2-induced [Ca2+]i release, and this desensitization could be reversed by pretreating with a PKCε inhibitor or overexpressing of Gαi2 with the putative PKC phosphorylation sites mutated. Thus, depending on the agonist, activation of MOR could lead to heterologous desensitization and probable crosstalk between MOR and other Gαi-coupled receptors such as the CB1 receptor.

The current study attempted to discern the extent to which a gender bias influences the adult ratings of observed childhood pain. While gender differences in pain sensation are well documented in physiologically mature individuals, there seems to be no such difference in children. The effect of manipulating gender on the procedural pain ratings of 201 university undergraduate and nursing students was examined via a deceptive pain observation task. Results demonstrated no significant difference between gender conditions; however a strong link was established between prior exposure to painful pediatric medical procedures and lower pain ratings. The results suggest that, while a gender bias failed to alter pain ratings, desensitization to viewing painful procedures could alter how much pain healthcare professionals believe a patient is experiencing.

GABAA receptors are chloride selective ligand-gated ion channels that mediate inhibitory neuronal signaling in the adult central nervous system. Native receptors are pentameric combinations of various subunits with expression patterns that are highly regulated with respect to anatomical location as well as developmental period. Although the physiological significance of such molecular diversity remains poorly understood, the striking subunit-dependence of kinetic properties and pharmacological modulation may provide a broad range of inhibition 'strategies' to the brain. The primary aim of this thesis was to determine the relevance of various GABAA receptor properties for synaptic (non-equilibrium) and extra-synaptic (steady-state) forms of neuronal inhibition. A fibroblast expression system allowed molecular manipulations (mutations, chimeras) of receptor subunits, as well as precise kinetic measurements (concentration jump technique) of GABAA receptor function to be achieved. This combined approach revealed several important receptor properties that may regulate the time course, efficacy, and pharmacological modulation of GABAA receptor-mediated inhibition. Specifically, we developed a novel functional assay to demonstrate a fundamental constraint on the relationship between GABA binding and channel gating: all kinetic states from which channel opening occurs are agonist-bound; GABA unbinding is unlikely or not possible from open and desensitized states. This finding provided a conceptual framework for subsequent hypothesis testing of the specific impact of gating efficacy, desensitization and microscopic affinity on GABAA deactivation, the major determinant of inhibitory post-synaptic current (IPSC) duration. Experimental data, supported by computer simulation results, indicated independent contributions of these three receptor properties to GABAA receptor deactivation. These insights laid the foundation for functional evaluation of the first GABAA receptor mutations linked to human epilepsy and sleep syndromes. Additional work on the properties and regulation of putative extra-synaptic GABAA receptor isoforms, that are thought to mediate tonic inhibition, revealed clear subunit-dependent biophysical and pharmacological properties. Analysis of modulation by an endogenous neurosteroid indicated that these isoforms are subject to a surprisingly dynamic range of regulation. Moreover, detailed functional analysis led us to propose a novel mechanism for isoform-specific allosteric modulation that distinguishes GABAA receptors based on differences in gating efficacy rather than primary protein structure.

The opioid receptor-like receptor ORL1 was previously cloned as an orphan receptor. We expressed ORL1 in Xenopus oocytes and in mammalian cells to study its activation by endogenous opioid peptides. Dynorphins were found to activate the coexpressed G protein-activated K + channel (GIRK1) by stimulating ORL1 at nanomolar concentrations. The dynorphin effect was antagonized by the non-selective opioid antagonist naloxone, but only at high concentrations. Dynorphins also activated the negative coupling of ORL1 and adenylyl cyclase in transfected mammalian cells. These results suggest that ORL1 is a member of the opioid receptor gene family, and that dynorphins may be endogenous ligands for this receptor. Dynorphins were considered kappa opioid receptor (KOR) agonists with high affinity and potency. Previous brain tissue binding and animal behavioral studies showed that dynorphins interact with receptors other than KOR. To better understand the kinetics of dynorphin binding to ORL1 and opioid receptors, we performed radioligand binding studies using mammalian cells transfected with each of the cloned receptors ORL1,μ (MOR), δ (DOR) and KOR. Dynorphin bound to all the receptors at nanomolar concentrations, with higher selectivity to KOR. When each of these receptors was coexpressed with GIRK1 in Xenopus oocytes, dynorphin activated potassium currents at physiological concentrations. These results suggest that, as potential endogenous agonists, dynorphins can bind to and functionally activate all four members of the opioid receptor gene family. Animal studies demonstrated that ORL1 functions as an "anti-opioid" receptor to modulate morphine effects. To understand the cellular mechanisms underlying the interaction of ORL1 and MOR, we studied the calcium channel inhibition by both receptors in human neuroblastoma SH-SY5Y cells expressing both receptors endogenously. The endogenous agonist for ORL1, ociceptin/orphanin FQ (N/OFQ), and morphine both inhibited whole-cell N-type calcium channel currents in a dose-dependent manner. ORL1 underwent homologous and heterologous desensitization induced by N/OFQ and morphine, respectively. However, MOR was only desensitized after long-term morphine treatment, and was completely resistant to N/OFQ treatment. These results indicate a nonreciprocal nature of heterologous desensitization between ORL1 and MOR. Further studies suggest that the crosstalk between these receptors is mediated by a mechanism other than receptor internalization.

Mechanistic models of G-protein coupled receptor (GPCR) signaling are used to gain insight into how changes in drug properties affect cellular response. Broadly, this work is divided in to three areas focusing on drug screening, desensitization, and receptor dimerization. First, ordinary differential equation models are used to examine biases in drug screening assays such as those used in drug discovery. It is shown that some screens should be innately biased against detecting inverse agonists and as such may miss pharmaceutically valuable drug leads. However, the results also suggest ways in which the screening assay can be modified to correct this bias. Second, Monte Carlo simulations of protein diffusion and reaction are used to determine the effects of drug properties on GPCR activation and desensitization. For most GPCRs, drugs cause an initial burst of activity (activation) followed by an attenuation of the signal over long times (desensitization). Simulations of this activation and desensitization process show that the mean drug-receptor lifetime can affect desensitization in a way that allows receptor activation and desensitization to be partially decoupled. Third, Monte Carlo simulations of receptor dimerization and diffusion are used to show how dimerization can affect membrane organization. Many membrane bound proteins, including GPCRs, form transient dimers, but the physiological reason for dimerization is not clear. The simulations show that dimerization under diffusion limited conditions can lead to the formation of extended clusters. These clusters, in turn, can alter the receptor internalization rate and the degree of cross-talk among receptors, in agreement with experimental findings. Overall, this work has a variety of implications. Pharmacologically, this work presents a new way of making drug discovery a more rational process by focusing assays toward drugs with desirable efficacies and improved desensitization profiles. Similarly, receptor dimerization could also provide a novel mechanism for affecting drug signaling. For basic biology, the modeling work presented here suggests that dimerization could provide a new way to control protein organization within the cell membrane. Together this work helps us to provide us with a more mechanistic understanding of how cells communicate via GPCRs.

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Since the cloning of GABA$\sb{\rm A}$ receptor (GABAR) subunit families, whole-cell patch-clamp studies have provided extensive information about the biophysical and pharmacological properties of recombinant GABARs over a time scale of hundreds of milliseconds to several seconds. Yet, at GABAergic synapses, events that shape GABAR currents take place on a scale of $\mu$secs to tens of milliseconds. To understand the factors that shape synaptic GABAR currents, the biophysical and pharmacological properties of recombinant GABARs need to be examined on the time scale of synaptic events. To examine the time course of recombinant GABAR currents on such a rapid time scale, we have utilized a technique for rapidly applying GABA to outside-out membrane patches. First, we examined the contributions of the $\delta$ and $\gamma$ GABAR subunit families in shaping the rapid kinetics of recombinant GABAR currents, finding that $\delta$-containing GABARs activate more slowly, desensitize more slowly and less completely, and deactivate more rapidly than $\gamma$2L-containing GABARs. These macroscopic currents are shaped by the underlying pattern of activity of many single channels. Therefore, we examined GABA steady-state single-channel currents at a predicted synaptic GABA concentration. We utilized these rapid kinetic and single-channel data to develop more comprehensive kinetic models that predict the behavior of recombinant GABARs containing $\delta$ or $\gamma$2L GABAR subtypes. These models reconcile microscopic and macroscopic current kinetics and predict that differences in against unbinding rates, channel opening rates, and desensitization are responsible for the differences in $\delta$-containing and $\gamma$2L-containing GABAR kinetics. We used $\delta$-$\gamma$2L subunit subtype chimeras and site-specific mutagenesis to explore the structural bases behind these kinetic differences, localizing the determinants of gating and desensitization to a region that included the M1, the M1-M2 linker, and M2 domains. We examined the roles of different GABAR agonists and of the anticonvulsant agent, pentobarbital, in shaping the rapid kinetics of GABAR currents. These experiments, in conjunction with the insights gained from developing the kinetic models, enhance our understanding of the factors that are likely to be important in shaping the time course of GABAergic IPSCs.

To survive, cells must be able to sense and respond to their environment. They accomplish this through a process known as signal transduction, and the most common mechanism involves the binding of extracellular ligands to cell surface receptors that are coupled to guanine nucleotide binding proteins (G-proteins). In this thesis the importance of several parameters involved in G-protein coupled receptor signaling was assessed. A mathematical model including the dynamic events of G-protein activation and receptor desensitization was developed and used along with experimental data to identify the most important parameters in determining a ligand's efficacy. It was found that the conformational selectivity (the influence of ligand binding on conversion of the receptor from an inactive to an active state) was the predominant factor in determining the activation and desensitization elicited by a particular ligand. The ligand/receptor complex lifetime, as controlled by the ligand dissociation rate constant, was postulated to be an additional ligand-specific determinant of efficacy, and experimental techniques for further examining its role were described. The generality of the mathematical model was tested by taking activation and desensitization data for the human neutrophil N-formyl peptide receptor. Because responses were measured far downstream of G-protein, the model was extended to include several hyperbolic functions in series that represented a signal transduction cascade made up of many saturable steps. With this addition, two N-formyl peptides with different binding kinetics were predicted to have different values of conformational selectivity but the same desensitization rate constant. Response behavior of neutrophils was further examined at the single cell level using flow cytometry. Pertussis toxin treatment was used to vary the number of G-proteins, and it was shown that actin polymerization and oxidant production exhibit threshold behavior in terms of G-protein number. In contrast, as ligand concentration was varied actin polymerization underwent a graded change in response, while oxidant production showed both graded and threshold behavior. These results were interpreted with a mathematical model to show the importance of the G-protein threshold in determining response behavior and that a G-protein independent event was necessary to account for neutrophil responses.

ATP has been established as a neurotransmitter or cotransmitter in both the central and the peripheral nervous systems. Fast excitatory responses to ATP are mediated by P2X receptors, which are ATP-gated receptor/channel complexes. Seven P2X subunits have been cloned so far and named P2X$\sb1$ to P2X$\sb7$ respectively. When expressed in heterologous expression systems, all seven receptors form ATP-gated channels that are selectively permeable to cations, such as Na$\sp+$, K$\sp+$ and Ca$\sp{2+}.$ One physiological property that characterizes some, but not all, P2X receptor channels is that they pass inward current more easily than outward current, a characteristic referred to as inward rectification. Among the seven subunits, P2X$\sb2$ shows the most profound inward rectification. In this thesis, I used two-electrode voltage clamp and patch clamp recording to investigate the underlying mechanism of inward rectification of P2X$\sb2$ expressed in Xenopus oocytes and a mammalian cell line (HEK 293 cells). Inward rectification of P2X$\sb2$ could be accounted for by a combination of voltage-dependent gating and voltage-dependence of the single channel conductance. To investigate the molecular determinants of inward rectification, I examined the effects of altering charged amino acids in the membrane on rectification. The positions altered by site-directed mutagenesis were R34, R313, D315, K324 and D349. R34 in the first putative alpha helical transmembrane domain and R313 and D315 in the putative pore lining loop are important for inward rectification. Mutations of these amino acids changed the voltage dependence of gating of the channel by shifting the midpoint of the steady state P-V relation (probability of channel being open vs. voltage). However, these amino acids are unlikely to be part of the voltage sensor, since mutations did not change the slope of the P-V relation. None of these mutations changed the instantaneous current-voltage relation or the channel selectivity, which suggests that they probably had no effect on single channel conductance. I also tested the effects of these mutations on P2X$\sb2$ desensitization and found that mutations at D349 accelerated the rate of desensitization more than 8-fold, while neutralization of the other four charged amino acids produced receptors that had desensitization rates as slow as wild type or even slower.

This investigation focused on an empirical evaluation of the efficacy of preventative systematic desensitization, a recently developed psychotherapeutic technique intended to preclude the formation of a phobic or adverse autonomic reaction. While earlier research suggested the therapeutic viability of this technique, various methodological difficulties inherent in the experimental designs negated any clear an unequivocal statements regarding therapeutic efficacy. The design of the experiment central to this dissertation was developed expressly to avoid the methodological problems which characterized prior research, expand upon the hypothesis of earlier investigators, and evaluate the effectiveness of prophylactic desensitization. The following results were obtained: (a) the experimental group did not show an increase in fear on the dependent measures following conditioning, (b) the placebo control group did show a significant increase in fear on the dependent measures following conditioning, (c) the group that received traditional systematic desensitization showed a significant fear increase after conditioning which was reduced to baseline following systematic desensitization, and (d) the measurements control group confirmed the above result and showed no significant differences in fear at retests. The major conclusion of this experiment is that within the context of the laboratory, it would appear that preventive systematic desensitization is an efficient psychotherapeutic technique. Further research designs and therapeutic strategies are discussed with regard to clients who may be expected to develop significant clinical phobias.

The current study investigated the efficacy of Wolpe's original (1954) paradigm of anxiety-relief conditioning. The procedure consisted of administering a mildly aversive electric shock to a subject for several seconds until the subject said the word "Relax," and the shock was terminated. Repeated pairings were claimed by Wolpe to condition physiological relief to the cue word, "Relax," which could then be employed in order to reduce anxiety in various anxiety provoking situations. Since there does not appear to be a generally accepted theoretical rationale to account for the reported efficacy of anxiety-relief conditioning, several theoretical rationales were discussed. In addition, a distinction was made between the anxiety-relief paradigm described by Wolpe (1954) and the aversion-relief paradigm employed by subsequent investigators (Gaupp, Stern, & Galbraith, 1972; Solyom, McClure, Heseltine, Ledwidge, & Solyom, 1972; Thorpe, Schmidt, Brown, & Castell, 1964). It was suggested that this distinction might be used to account for the failure of the current investigation to support the efficacy of anxiety-relief conditioning, as a review of the major study supporting its efficacy (Turnage & Wenrich, 1974) indicated that aversion-relief, rather than anxiety-relief, may have been employed. In the absence of strong supportive evidence for the efficacy of Wolpe's anxiety-relief paradigm, the need for further research elucidating the relevant parameters was discussed, and specific areas requiring more intensive study were delineated.

The purpose of this study was to test the therapeutic usefulness of a completely self-administered, behavior-therapy technique: systematic desensitization. The method involved the comparison of behavior change following the treatment of two groups of subjects. One group received the actual systematic-desensitization procedure, while the other underwent a similar but theoretically ineffective treatment termed attention-placebo.

The present research was undertaken to investigate the effects of two independent variables considered potentially important to the reduction of fear through systematic desensitization. The first independent variable investigated was the importance of making covert motor responses when instructions were given to imagine motor behavior. Electromyographic measures were obtained on subjects' covert muscular activity as they imagined themselves raising their arms. The subjects were then classified, on the basis of their average electromyographic responsiveness, as high-responders (those showing relatively high levels when imaging movement) and low-responders. A 2 X 2 analysis of covariance showed a significant difference in the posttreatment scores of the high- and low responders on performance measures, the high-responders performing better. The difference between the two instruction groups was not statistically significant. Additional analyses indicate the importance of the instruction variable is mitigated by the extent to which subjects actually follow the particular instructions given. These data imply electromyography may be used in clinical practice as an assessment tool to determine which subjects are likely to respond to systematic desensitization. They also suggest the possibility of using response measures to train self-monitoring of imagery. Concerning the imagery construct, the present study offers an empirical alternative to the traditional conceptualization. The data obtained support the utility of the analysis of imagery advanced in this paper.

Basic data are lacking in the literature concerning the reactivities of desensitizing dyes to Iodine, and concerning their redox potentials while undergoing such reactions. Such information may be useful in deciding on the mechanism of desensitization, since there is good evidence that the halogen liberated from an Iodobromide emulsion during photolysis is actually Iodine. Therefore, experiments were undertaken to measure the polarographic anodic potential of the first addition product of a series of thiacyanine dyes, if such a species existed. Also, preliminary experiments were undertaken to determine the relative reactivities of the dyes to molecular Iodine and Bromine while they were adsorbed to a dilute Silver Bromide sol. Both experiments proved unfeasable because, first, it was not possible to achieve a stable sol for the purpose of spectrophotometry, and second, the dyes did not show a stable, reactive intermediate. However; basic data concerning the relative reactivities of the dyes to Iodine and Bromine were obtained. The dyes prove to be at least as reactive to Iodine as they are to Bromine, and the reactivities to both halogens increased with increasing chain length by orders of magnitude. There was no evidence of dye regeneration nor of equilibrium with the halogen.

The problem of this study was to investigate the effectiveness of systematic desensitization on test anxiety, general anxiety, and attitude toward school among fifth-grade elementary-school children.

This investigation was concerned with the potential effectiveness of systematic desensitization as a technique in the prevention of pervasive anxiety. It was hypothesized for investigatory purposes, that if two specific, potentially anxiety-evoking stimuli could be pre-desensitized, this would be strongly suggestive that pre-desensitization programs might also prove successful in the prevention of pervasive anxiety.

Since fear of public speaking has been considered a good example of anxiety and an example that is correlated with behavioral and cognitive measures of anxiety levels, a study of Rogerian therapy, desensitization, and modeling techniques in reducing this anxiety seemed appropriate.

The present study was an attempt to show that the behavioral technique of desensitization is superior to insight-oriented psychotherapy in terms of not only behavior change for individuals undergoing desensitization but in terms of case of acquisition to novice therapists who have virtually no clinical experience.