• Contributions of the N and C – Termini of Varicella – Zoster Virus Portal

      Nale Lovett, Dakota J; School of Medicine
      The VZV portal protein is a multimeric protein found at a single vertex of the viral capsid that is essential for encapsidation (packaging) of viral DNA. All viruses within the herpesvirus family contain structurally homologous portal proteins. PORT compounds have been shown to target herpesvirus portal proteins and show potential as broad-spectrum herpesvirus antivirals. We are specifically interested in the interaction of PORT compounds with the VZV portal since the activity of our compounds against VZV was shown to be in the single nanomolar range. Unfortunately, VZV portal structure has yet to be resolved by transmission electron microscopy (TEM) due to complications with size and aggregation. We aimed to observe the effects of pORF54 (VZV portal protein) terminal truncation on virus viability to determine the minimal portal protein that can be used for structural analysis. Six recombinant viruses containing N and C- terminal mutations were created using bacterial artificial chromosome (BAC) technology and targeted recombineering to create the mutant VZV strains. PCR was used to engineer stop codons at 29, 49, and 75 AAs from the C-terminal end. In addition, a second set of recombinants was constructed where the first start codon of the 769 AA pORF54 open reading frame was deleted creating a 40 AA N-terminal truncation. Sequencing of all mutant strains confirmed the expected changes for the ORF54 gene and also that no gross off-target mutations occurred. All constructs showed a similar restriction digestion pattern, upon gel electrophoresis, compared to the parent BAC, pOKA, suggesting the mutant genomes were stable. Mutagenized BACS were used to create infectious viral stocks after lipofectamine-based transfection into ARPE19 cells. Viruses with non-functional mutations in pORF54 were transfected into ARPE54 (complementing) cells, that constitutively express wild-type pORF54. The ∆29, ∆49, N40∆29, and N40∆49 viruses were replication competent in ARPE19 cells. Replication characteristics suggested that some of these viruses grew less efficiently in vitro. Overall, we confirmed that a protein of 679 AAs can form a functional viral portal. This portal multimer may represent a strong candidate for detailed structural studies.
    • Coping with Death and Grief: Mount Zion Baptist Church Widows' Stories

      Bright, Eddie Lee; McAfee School of Theology
      ABSTRACT EDDIE LEE BRIGHT COPING WITH DEATH AND GRIEF: MOUNT ZION BAPTIST CHURCH WIDOWS’ STORIES Under the direction of DENISE M. MASSEY, PH.D. Three widows were selected from Mount Zion Baptist Church to participate in my thesis project on Death and Grief. Their stories are individually told respectively as shared by them during the interview sessions. Although a semi-structured format was used and open-ended questions were asked, their stories are conveyed in this writing through a narrative as opposed to a verbatim style. Three widows were chosen and deemed sufficient to satisfy this project and to attain the data necessary to organize the Death and Grief Ministry. Two distinctive criteria existed among the chosen participants: (1) Widows had to have been married more than half their years of age when their husbands died. This criterion enabled the project to specifically examine the effect a loss of a husband has on a widow in a relationship that lasted more than half of the existence of her life. (2) Widows must have had the same husband the entire length of their marriage, without separation and/or divorce, regardless of whether the remarriage was to the former husband. This project empowers widows to share their stories, including their thoughts and feelings concerning how their spouses’ death has affected them. Because the narration comes directly from the widows, the uniqueness of each woman’s plight has been demonstrated. These widows’ stories will also be used to educate the congregation and community on how to cope with death and grief based on their shared experiences and stories. The long-term intention of this project is to institute a Death and Grief Ministry at Mt. Zion.
    • Counseling Self-efficacy And Counselor-in-training Anxiety: The Moderating Role Of Mindfulness

      Koth, Kristen Heather
      Mindfulness has been suggested as a predictor in counselor self-efficacy and has been shown to have a negative correlation with anxiety.� However, the relationship between anxiety and counselor self-efficacy with the possibility of mindfulness playing a moderation role has not been examined.� This study examined the relationship between counseling self-efficacy and counselor-in-training anxiety and the potential moderating effect of mindfulness.� Master’s level counselors-in-training were surveyed using the Five Facet Mindfulness Questionnaire (FFMQ), the Trimodal Anxiety Questionnaire (TAQ), and the Counselor Self-Efficacy Scale (CSES) (N = 156). Levels of anxiety, counselor self-efficacy, and mindfulness were obtained and a Pearson product-moment correlation coefficients revealed significant pairwise relationships between the three variables.� A moderated path analysis supported the hypothesis that mindfulness is a significant predictor of anxiety and self-efficacy.� However, results indicated that mindfulness was not a moderator of the relationship between anxiety and counselor self-efficacy. Implications for the use of mindfulness as part of counseling program curriculum are discussed.
    • Critical Consciousness: A Measurement Tool For English To Speakers Of Other Languages (esol) Teachers In U.S. K-12 Education

      Simmons, Camelle L.
      Critical consciousness is an increasingly important component of pedagogy. However, the measurement of critical consciousness among teachers remains underdeveloped, partly because of the absence of psychometric testing of appropriate scales. In this study, the researcher adapted and tested Diemer et al.’s Critical Consciousness Scale, which was developed for students, with a sample of 178 ESOL teachers. The theoretical context for the study was pedagogical social justice theory, particularly as developed by Freire, which suggests that critical consciousness is likely to be highly developed in educators who, like ESOL teachers, are well-situated to understand the relationships between power, knowledge, and practice. Psychometric testing revealed that the Critical Consciousness Scale as adapted for teachers (a) possessed an internal reliability of .91; (b) consisted of three factors (each with an eigenvalue greater than 1), corresponding to critical reflection on perceived inequality, critical reflection on commitment to egalitarianism, and sociopolitical participation; and (c) was highly right-skewed, indicating that most ESOL teachers had a high level of critical consciousness. The findings were interpreted in light of pedagogical social justice theory, which predicts that the nature of ESOL teaching develops critical consciousness in educators. The study findings confirmed that the Critical Consciousness Scale can be utilized with teachers, thereby supporting future empirical research on critical consciousness.
    • Deciphering the Role of Sumoylation During EBV Replication

      Jenkins, Jessica L; School of Medicine
      Epstein Barr Virus, a gamma herpes virus, is the known causative agent in infectious mononucleosis and is highly ubiquitous in nature. Although primary infection typically yields no long term issues, viral latency is associated with lymphomas and epithelial cell carcinomas. We documented that the presence of LMP1, the principal EBV oncogene, dysregulates cellular sumoylation processes in lymphoma tissues, modulates innate immune response, and maintains viral latency. Sumoylation is a dynamic process were target proteins are modified with free small ubiquitin like modifier (SUMO) proteins. The SUMO modification is vital for cellular processes including: immune response, DNA damage repair sensing, cell cycle progression, resistance to apoptosis, and metastasis. Several cancers display dysregulation of the sumoylation process, making the SUMO machinery a sufficient target for anti-cancer therapies. Known sumoylation inhibitors include natural extracts and antibiotics. However, many of these agents are nonspecific and/or demonstrate adverse effects like allergic reactions with botanical extracts. This piqued our interest in investigating synthetically engineered compounds along with a well-known natural extract inhibitor, Ginkgolic Acid (GA). ML-792, 2-D08, and TAK-981 are synthetically derived small molecule inhibitors that were identified as selective SUMO-inhibitors, interfering at different stages of the sumoylation process. We hypothesize that the SUMO-inhibitors will have therapeutic effects for the treatment of EBV-associated malignancies by modulating the EBV life-cycle. Results showed that each of the tested inhibitors decreased global levels of sumoylated proteins, though ML-792 and TAK-981 showed greater inhibition when compared to GA and 2-D08. Additionally, the SUMO-inhibitors induced low levels of spontaneous reactivation in latently infected B cells. We also confirm that sumoylation is important for maintaining EBV latency and lytic replication in B cells. Lastly, we note anti-viral potential for each tested inhibitor, particularly GA and 2-D08 have a better affect than ML-792 and TAK-981 in this regard. Of the tested sumoylation inhibitors, we now propose 2-D08 as the best potential therapeutic drug to aid the treatment of EBV-associated malignancies due to its ability to significantly reduce viral DNA levels following induced reactivation and decrease the ability of produced virus to infect additional cells.
    • Design And Evaluation Of Novel Topical, Transdermal And Microneedle Formulations

      Sivaraman, Arunprasad
      Arunprasad Sivaraman Design and evaluation of novel topical, transdermal and microneedle formulations (Under the direction of AJAY K. BANGA, Ph.D) Purpose: The broad aim of this project was to formulate passive transdermal patches, topical gel and microneedle and evaluate their physical properties and delivery efficiency via skin. Methods: Polyvinyl alcohol polymer transdermal patch was prepared in combination with DURO-TAKTM 387-2516 acrylate adhesive using oxybutynin and characterized for coating efficiency, matrix structure, rheology, tack, shear, peel adhesion and tested for in vitro permeation using dermatomed human skin for 72 hours. A semi-synthetic opioid transdermal patch was prepared using a formulation blend of oleic acid, BIO-PSA 7 - 4301 silicone adhesive and an amphiphilic solvent and characterized for coating efficiency, matrix structure, thickness, tack, peel adhesion and tested for in vitro permeation using dermatomed human skin for 72 hours. In situ forming hydrogel microneedles were investigated using a non-ionic triblock thermosensitive copolymer with methotrexate. The microneedles were characterized for sol-gel transition, geometry, depth of micropores, histology, rheology, methylene blue staining and evaluated for transdermal delivery in full thickness porcine ear skin and dermatomed human skin. A topical diclofenac gel was prepared using polyvinyl alcohol polymer and characterized for matrix arrangement, pH, adhesion, spreading efficiency, crystallization, rheology, skin irritation and evaluated for in vitro drug distribution and permeation with dermatomed human skin. Results and Conclusions: The polyvinyl alcohol transdermal patch showed an emulsion matrix formation with an average in vitro cumulative permeation and flux of 343.80 ± 74.36 μg/cm2 and 4.79 ± 1.3 μg/cm2/h respectively with no skin irritation. The semi-synthetic opioid transdermal patch delivered an average drug cumulative permeation and flux of 25.98 ± 0.19 μg/cm2 and 0.28 ± 0.2 μg/cm2/h respectively with no drug crystallization. The in situ formed hydrogel microneedles delivered an average cumulative drug amount of 32.2 ± 15.76 μg/sq.cm and 114.54 ± 40.89 μg/sq.cm for porcine ear skin from 0.2% w/w and 0.4% w/w methotrexate formulations. The topical diclofenac gel delivered an average cumulative drug amount of 22.85 ± 9.41 μg/cm2 and distribution of 10.30 ± 9.09 μg/cm2 for 24 h with no skin irritation. In conclusion, transdermal patches, topical gel and microneedles were formulated and characterized for their physical properties and evaluated for skin delivery.
    • Developing Empathy From Storytelling In The Congregational Diversity Of Church For The Highlands / By John Henson.

      Henson, John Craig
      JOHN HENSON DEVELOPING EMPATHY FROM STORYTELLING IN THE CONGREGATIONAL DIVERSITY OF CHURCH FOR THE HIGHLANDS Under the direction of Robert N. Nash, Jr., Ph.D. The focus of this project thesis is on how members of Church for the Highlands, a congregation of pronounced diversity, can move from awareness and acceptance of one another to mutual understanding and a deeper level of relationships. The diversity, age and pace of missional activity create a challenge for members when it comes to deepening self-awareness of commonality. This project is concerned with how the practice of storytelling can produce empathy among members of Church for the Highlands and can become a sustainable practice to help the church experience commonality. The research involves a qualitative method and use of a focus group to determine how storytelling creates empathy in the focus group members who attended three storytelling events. An assistant moderator recorded notes and responses of focus group members on a matrix chart for both the pre and post-events session. The data was interpreted using Micro-interlocutor analysis. The conclusion of the project is that the focus group members who attended the storytelling events and heard the stories were able to empathize with the storytellers. Various methods of storytelling were utilized and all provided opportunity for listeners to develop empathy.
    • Development and Characterization of a Novel Immunotherapy for Treatment of Breast Cancer by Combining Particulate Cancer Vaccines with Immune-Modulators

      Mulla, Nihal S; College of Pharmacy
      In the last half of the century, advances in the field of cancer therapy including chemotherapy, hormonal therapy and targeted therapy have been responsible for improvements in breast cancer related mortality. Although such advances have benefited all cancer types, there are considerable challenges faced by researchers striving to realize the goal of complete tumor remission. Immunotherapy is a great alternative as it has minimum side effects and several advantages over traditional cancer therapies. The aim of this project is to develop a novel immunotherapy for treatment of cancer by combining cancer vaccines with various immune-modulators. We take advantage of micron-sized particles to deliver vaccine along with other immune modifiers to target immune cells and to initiate immune response against breast cancer antigens. These particles were evaluated for their size, charge, surface morphology, release profiles, cyto-toxicity and particle uptake by various in vitro studies. The efficacy of breast cancer vaccine microparticles was tested in a murine breast cancer model. The immunized animals showed significantly lower tumor growth compared to the naive animals that did not receive any treatment. The delay in tumor growth in vaccinated animals was due to a strong immune response generated against tumor- associated antigens encapsulated within the microparticles. We observed a significant increase in the CD4+ T cell population. The suppression mechanism employed by regulatory T cells are thought to contribute significantly to the failure of current therapies that rely on potentiation of anti-tumor responses. We evaluated the therapeutic efficacy of vaccine microparticles after depleting the immunosuppressive regulatory T cells. We observed a significant improvement in the efficacy of vaccine microparticles by depleting regulatory T cells. The tumor inhibitory effect of vaccine microparticles was due to depletion of regulatory T cells by cyclophosphamide. We observed a significant increase in the CD8+ T cell population. The final aim of my research project was to enhance the immunogenicity of cancer vaccines using adjuvants. Based on our findings we conclude that Alum, Addavax (like MF59) R848 and CpG significantly enhanced the immunogenicity of breast cancer associated antigens.