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Genetic and Biochemical Analyses Reveal LitR\u27s Role in Vibrio fischeri Biofilm Formation
No full textQuorum sensing controls numerous processes ranging from production of virulence factors to biofilm formation. Biofilms, communities of bacteria that are attached to one another and/or a surface, are common in nature and, when they form, can produce a quorum of bacteria. One model system to study biofilms is the bacterium Vibrio fischeri, which forms a biofilm that promotes colonization of its symbiotic host. Many factors promote V. fischeri biofilm formation in vitro, including the symbiosis polysaccharide (SYP), cellulose, and the LapV adhesin but, so far, only a handful of studies have probed connections between biofilm formation and quorum sensing. In this work, I was able to further our understanding of quorum sensing’s contribution to biofilm formation, primarily through studying the quorum sensing-dependent transcription factor, LitR, and its downstream biofilm inhibition pathway. I first showed that LitR negatively influences V. fischeri biofilm formation in the context of a biofilm-overproducing strain. To better understand the importance of LitR, I then identified conditions in which the impact of LitR on biofilm formation could be observed in an otherwise wild-type strain. With these conditions, I investigated LitR’s role and the roles of upstream quorum regulators in biofilm phenotypes. In static conditions, LitR and its upstream quorum regulators, including autoinducer synthases LuxS and AinS, contributed to control over biofilms that were SYP-, cellulose-, and LapV-dependent. In shaking liquid conditions, LitR and AinS contributed to control over biofilms that were primarily cellulose- and LapV-dependent. LitR’s contribution to static pellicle formation also held true in an alternate isolate of V. fischeri, KB2B1, indicating that quorum sensing’s control over biofilm formation may be conserved. To understand how LitR affected the production of these biofilm components, I assessed the dependence of the ∆litR mutant biofilm on their known regulators and if LitR controlled the transcription of their genes. Of the syp regulators, LitR was dependent on the histidine kinase RscS and the Hpt domain of SypF for its pellicle phenotype. However, LitR did not substantially affect rscS or sypA transcription. LitR modestly inhibited cellulose transcription in a manner that depended on the transcription factor, VpsR. LitR induced transcription of the c-di-GMP degrading enzyme, PdeV, which is responsible for inhibiting the presence of surface-associated LapV. The gene upstream of pdeV, rpoQ, shares an intergenic region with VF_A1016, a histidine kinase with similarities to a known negative regulator of biofilm production. I showed that VF_A1016 also inhibited biofilm formation and that VF_A1016 was transcriptionally upregulated by LitR. Based on phenotypic assays, LitR appeared to control VF_A1016 and pdeV transcription to inhibit cellulose- and LapV-dependent biofilms, respectively. Chromatin immunoprecipitation sequencing revealed that LitR directly interacts with the rpoQ/VF_A1016 intergenic region to control their expression. Additionally, peaks were identified within the coding regions of other biofilm-relevant genes such as sypO, bcsA, and lapV, suggesting that LitR may bind within these genes to impact their transcription, and, thus, protein production. These findings expand our understanding of LitR and the quorum sensing pathway in the physiology of V. fischeri and illuminate negative control mechanisms that prevent robust biofilm formation by wild-type V. fischeri under laboratory conditions
A Novel Approach to Treat Trastuzumab Resistant HER2+ Breast Cancer: Activation PKCδ
Get PDFIn the United States, approximately one in eight women will be diagnosed with invasive breast cancer in her lifetime and one in forty will die from breast cancer. From among all the different subtypes of breast cancer, HER2+ can be found in approximately 20% of breast cancer tumors. HER2 overexpression has been associated with an aggressive form of breast cancer, increasing the risk of metastasis or recurrent tumors in 50% of patients. The standard of care for HER2 positive breast cancer, trastuzumab, has been effective for some patients; however, others develop resistance over time, leading to disease progression and metastasis. Drug resistance and tumor recurrence remain significant clinical challenges, limiting treatment efficacy. To address these issues, it is crucial to identify new mechanisms of action and develop novel therapeutic approaches to prevent or treat resistant disease. Previous work in the Osipo lab has shown a correlation between levels of PKCδ activity and resistance to trastuzumab therapy. It was hypothesized that by a TPA-mediated activation of PKCδ, trastuzumab resistant HER2+ breast cancer could be re-sensitized to trastuzumab. Surprisingly, TPA-mediated activation of PKCδ was sufficient to inhibit both proliferation and cancer stem cells survival of trastuzumab resistant cells lines. The addition of trastuzumab had little effect. Due to the nature of TPA to potentially cause cancer in other tissues, safer alternatives are needed to activate PKCδ. The hypothesis drawn to describe these findings is that: Activation of PKCδ is a novel therapeutic approach to treat trastuzumab resistant breast cancer using new compounds that mimic TPA. Two aims will be employed in order to test this hypothesis. Specific Aim 1: Test other PKC agonists as alternatives to TPA for inhibition of cell proliferation and cancer stem cell survival. After screening seven compounds raging between cyclic lactams, DAG lipids and Byostratin-1; a non-phorbol ester known as TPPB was the most potent inhibitor of cell proliferation and cancer stem cell survival using HCC1954 and BT474 resistant cell lines. Results showed that TPPB inhibited proliferation and cancer stem cell survival similarly to TPA. PKCδ was knocked down using an RNAi approach to show that the TPPB was on target for PKCδ. Furthermore, a pilot animal study using HCC1954 cells yielded information about potential toxicity. The rate of tumor growth with 1mg/Kg dose of TPPB was not significantly changed compared to the vehicle control group, suggesting that the dose may have been too low to elicit a measurable effect. Currently, a maximal tolerated dose (MTD) study is being performed using 5mg/Kg and 10 mg/Kg TPPB in HCC1954-harboring mice. The MTD study will provide enough information for larger scale in vivo studies in regard to toxicity and tumor growth. Specific Aim 2: Determine the mechanism by which activation of PKCδ inhibits growth of trastuzumab resistant HER2+ breast cancer. RNA sequencing was performed to determine the mechanism by which TPA-mediated activation of PKCδ inhibits proliferation and cancer stem cell survival of trastuzumab resistant breast cancer cell lines. The most significant pathway activated by TPA was the TNFα- NFKβ pathway. However, through several experimental approaches, it was determined that neither the canonical nor non-canonical NF-κB pathways were not required for cell proliferation and CSC survival inhibition. Alternative mechanisms, like JNK signaling and cellular senescence were also explored, but none of them were able to rescue the proliferative phenotype. After reinterrogating the RNA sequencing data, other signaling pathways such as p53 or cell differentiation emerged as possible mechanisms to explore. These results suggest that even though TPA has been shown to be an effective agent to activate PKCδ, TPPB could possibly be a safer alternative. Future studies will determine mechanisms by which activating PKCδ selectively inhibits growth of trastuzumab resistant breast cancer in vivo and if PKCδ is a novel vulnerability for trastuzumab resistant disease
Medical Students Trained in Emotional Intelligence Seem to be More Apprehensive of Artificial Intelligence in Healthcare
Get PDFThe use of artificial intelligence (AI) in healthcare is becoming increasingly widespread. While AI has been touted to reduce burnout and improve outcomes, its potential to undermine the doctor-patient relationship has been recognized. It is not known what future physicians believe regarding AI in healthcare and whether formal training on emotional intelligence and resilience (EIR) affects their opinion. Our medical students were asked to anonymously and voluntarily complete a 12-item survey regarding the potential impact of AI on the doctor-patient relationship (IRB #219038). Results were summarized as means and standard deviations, stratified by whether students did (EIR+) or did not (EIR-) take our institution’s EIR elective during their M2 year. Questions were measured via Likert-type scale with 1 = Strongly Disagree and 5 = Strongly Agree. Mean item responses were compared via two-sided Mann-Whitney U test (α \u3c 0.05) using Stata, v.18 (StataCorp, LLC., College Station, TX). 50 EIR+ students and 35 EIR- students replied. EIR+ students indicated greater disagreement vs. EIR- students to the item: “I believe AI technology will improve the doctor-patient relationship” (2.98 [±1.19] vs. 3.51 [±0.98]; p = 0.0327). While not statistically significant, EIR+ students reported similar disagreement vs. counterparts to the item: “Overall, I think adopting the use of AI in healthcare will be beneficial” (3.58 [±1.16] vs. 4.03 [±1.10]; p = 0.0521). These data suggest medical students who receive EIR training are less likely to believe AI will improve the doctor-patient relationship, which may reflect the enhanced understanding of EIR acquired by elective participants
“Mentors Need Mentors”: The Perspectives of Adolescent Cross-age Mentors of Color about their Instructors’ Supports
No full textThis study investigates instructor activities that 158 adolescent mentors of community children found meaningful in the context of their under-resourced urban communities. Theoretical frameworks draw from trauma theory, self-psychology, and community cultural wealth. Data include field notes, peer-to-peer program evaluation interviews, exit interviews, focus groups, and notes from clinical seminars for instructors. Mentors found instructor support vital for developing their empathy, especially with mentees behaving counter to expectations. Mentors also valued instructors’ authentic, supportive responses to stressors and feedback about the mentors’ positive impacts with mentees. As constructive social networks were formed, the program deepened the social capital form of community cultural wealth. From a self-psychology perspective, deepening mentor’s empathy occurred through iterative processes of instructor support and advice, experiences interacting with mentees and evaluating mentees’ responsiveness, and instructors’ affirmation of mentors’ competence and importance to mentees. Adolescents’ perspectives offer rich insights for optimizing social service provision, especially for marginalized youth
Post-Translational Modifications of PSD-95 in Glutamatergic Synapses
No full textPSD-95 is a pivotal postsynaptic scaffolding protein that regulates synaptic maturation, receptor localization, and signaling proteins, including mediating NMDA receptor clustering and function in synaptic plasticity. NMDA receptor-dependent LTD is dependent on multiple post-translational modifications. One of these modifications, phosphorylation of PSD-95, destabilizes the protein which can prompt the exit of associated AMPARs from postsynaptic spines. Our investigations highlight a potential factor in PSD-95 destabilization, isomerization of phospho-T19 by Pin1. We hypothesize that Pin1 mediated phosphorylation of PSD-95 may either protect or target PSD-95 for ubiquitination, playing a critical role in NMDAR-LTD
The Development and Validation of the Measure of Critical Reflection
No full textCritical reflection, a component of critical consciousness, is defined as a metacognitive ability that is engaged when identifying, critiquing, and transforming social oppression. This study developed and validated the Measure of Critical Reflection, with items that assess participants’ cognitive ability to critically reflect and are framed to be generalizable across time and social contexts. Items were generated through literature review and interviews with critical consciousness educators and data was collected across two samples for factor structure analysis (N = 300) and replication and validation (N = 286). Results confirmed that a unidimensional model was most appropriate to represent the data across samples. Evidence for reliability and validity were established. Implications for clinical interventions, psychology training, and research are discussed
Evaluating the Active Learning Experience of Prelicensure Nursing Students by Role and Level in Simulation
No full textAs the complexity of healthcare continues to grow, it is essential that nursing graduates enter the workforce with strong clinical judgment, confidence, and practice readiness. Simulation is an active learning strategy that creates a realistic, hands-on experiences for learners. Despite its widespread use, the impact of simulation roles within a simulated experience (participant versus observer) on student engagement remains unclear, particularly across different levels of nursing education. This study investigated differences in active learning engagement among sophomore and senior prelicensure nursing students (N = 161) based on their assigned role in a high-fidelity simulation scenario. Grounded in Kolb’s Experiential Learning Theory, a non-experimental pretest/posttest design was employed, using the Assessing Student Perspective of Engagement in the Classroom Tool (ASPECT) to measure engagement before and after debriefing. The ASPECT evaluates three engagement constructs: value of the activity, personal effort, and instructor contribution. From the study it was found that both a student\u27s simulation role (participant or observer) and level of study (sophomore or senior) influence their value of activity and personal effort invested during high-fidelity simulations. These insights can help guide simulation design and teaching strategies to better support learning and improve outcomes across the nursing curriculum
The Forced Sterilization of Quechua-Speaking Women in Peru: The Role of Epistemic injustice in The Perpetration of Genocide
No full textThe forced sterilization of Quechua-speaking women in Peru from 1996 to 2000 represents a case of genocide in which the state as an epistemic agent has a fundamental role. Although the systematic sterilization was carried out under the guise of a family planning program, the targeting of these women provides strong circumstantial evidence of genocidal intent. According to the testimonies of these women, medical staff, acting on behalf of the government, performed tubal ligation procedures through coercion, threats, lies, bribery, manipulation, and other tactics where the state intentionally withheld information, misinformed women, and failed to obtain proper consent. This research examines how epistemic injustice played a central role in the facilitation of this genocide, specifically focusing on how hermeneutical and testimonial injustice contributed to rendering Quechua women invisible and unheard. First, it deprived them of the necessary conceptual tools to understand the sterilization process, which went beyond a mere language barrier and resulted in a lack of transparency. Second, it discredited their voices, depriving them of consent and ultimately compromising their ability to gain public recognition of what occurred. The ongoing denial of this genocide is a clear reflection of the persistent epistemic injustice suffered by these women, which obstructs the path to meaningful reparations. Recognizing the genocide requires, first and foremost, confronting and eradicating epistemic injustice by adopting epistemic virtues and rejecting epistemic gaslighting. By restoring the epistemic conditions for these women to be heard, believed, and recognized as knowers, this work contributes to the broader conversation on epistemic justice
Forgive as We are Forgiven: Reframing a Trinitarian Model of Forgiveness in a Post-Genocide Rwanda
No full textThis dissertation seeks to develop a theological model of forgiveness rooted in a relational conception of the Trinity. The central argument of this dissertation is that the model of forgiveness practiced in post-genocide Rwanda and the forgiveness modeled by Jesus, and testified to in the New Testament and developed in the Christian tradition, are good, but not sufficient to address the kind of trauma and human disrepair, as well as the devastation of communities, inflicted by genocide. Rather, a theology of forgiveness modeled on a relational conception of the Trinity and its practical implications for a Trinitarian ecclesiology and anthropology of personhood, communion, and otherness can lead to the healing and restoration of peaceful relationships among individuals and communities. Since forgiveness is relational, I argue that a Trinitarian theology of forgiveness is embedded in a dynamic conception of the Triune God, which requires a shift from a substance-ontological thinking to a relational-ontological conception of the Trinity. This dissertation relies on the works of the Cappadocian Fathers and that of Catherine LaCugna and John Zizioulas, in their effort to develop a Trinitarian ontology of personhood and communion. The gift of divine forgiveness is not only God the Father forgiving us in Christ by the Holy Spirit, but it is also humanity receiving the grace to forgive humanity. A Trinitarian theology of forgiveness has a twofold dimension: Its verticality consists in the relationship with God and participation in the Trinitarian life as the source of human forgiveness. Such relationship, in turn, horizontally generates an ethical and spiritual configuration to God by living the commandment of love and by forgiving the enemy. Forgiveness is ultimately God giving us the capacity to forgive others. Therefore, forgiveness is an act of undeserved and unconditional love for the offender, by which the post-genocide Rwanda victim becomes Christ-like, an ambassador of God\u27s forgiveness and reconciliation. Unlike other studies that only present the Trinity as a prototype of community of forgiveness, this thesis will connect the foundation of faith in the Triune God to the act of forgiveness at the personal and community level, which renders forgiveness an act of faith, a form of spirituality and Christian discipleship. The last section emphasizes that the Christian community, especially the Church in Rwanda that is called to exist as the sacrament of Trinitarian communion and forgiveness, that gathers around the word of God and the Eucharist, can contribute to the healing and peaceful coexistence of individuals and communities. In the end, healing occurs when forgiveness becomes a way of connecting our faith in the Trinity with our daily life and a form a spirituality by connecting the lex orandi to the lex credendi through devotional and pastoral practice
Effectiveness of a Classroom-Based Stress Awareness and Coping Skills Program Among Adolescents from Diverse Backgrounds
No full textThe ongoing youth mental health crisis in the United States, worsened by the COVID-19 pandemic, has highlighted the urgent need for accessible and effective school-based interventions. This dissertation evaluates the effectiveness of Stress Less: Resilience for Success™, a single-session, classroom-based stress awareness and coping skills program implemented by NAMI Metro Suburban in diverse middle and high schools in the western suburbs of Chicago from 2018 to 2024. Using the CDC’s Framework for Program Evaluation, this study analyzes secondary data from 7,225 pre-test and post-test surveys and is complemented by qualitative informational interviews with current and former NAMI staff and volunteers to assess the program’s implementation and fidelity. The research explored changes in students’ knowledge of stress and coping skills, their intention to use coping strategies, and their willingness to speak with a trusted adult. Variations were analyzed based on race, gender, grade level, school type, and delivery method (in-person vs. virtual). The findings demonstrate statistically significant increases in students’ knowledge and coping strategy intentions post-intervention, with notable patterns based on demographic factors and modality. The study emphasizes the importance of culturally responsive, universally accessible interventions in promoting adolescent mental health and early help-seeking behaviors within school settings. Implications for social work practice, school-based mental health, and community-based program implementation are discussed