• 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.
    • Development And Characterization Of Polymeric Formulations And Microneedles For Dermal Drug Delivery

      Kim, Yujin
      Skin provides an attractive route for drug delivery, as transdermal delivery systems can ensure noninvasive and sustained delivery of medication across the skin into systemic circulation. Although the skin is a convenient site for drug administration, it only permits passive penetration of permeants with certain physicochemical properties. As a result, different strategies or devices have been developed to enhance the drug permeation into and across skin. The first aim of this study was to evaluate the efficacy of multiple dosing of trolamine salicylate (TS) as a topical analgesic on in vitro skin permeation. Also, the effect of sonophoresis on topical delivery of analgesic was evaluated. Results suggested that the multiple dosing (17.4 ± 4.1 μg/sq.cm) delivered a significantly higher amount of drug than the single dose (6.5 ± 0.6 μg/sq.cm). Also, the use of ultrasound enhances topical absorption of TS into and across skin. In the second aim, we focused on developing a new formulation to reduce the frequency of application of topical analgesics. A Poly lactic-co-glycolic acid (PLGA) based bio-adhesive polymeric solutions were prepared successfully and formed a thin film upon application in situ. A significantly higher amount of TS was delivered from a formulation containing 20% PLGA (45 ± 4 µg/sq.cm) as compared to PLGA-free counterpart (0.6 ± 0.2 µg/sq.cm). In the third aim, hyaluronic acid (HA) microneedles (MNs) were fabricated with and without magnesium ascorbyl phosphate (MAP) to facilitate the delivery of the active agent into and across the skin. The results showed that successful fabrication of HA MN, and the application of the current MN technology enhanced the delivery of MAP into skin (96.8±3.9 µg/sq.cm) compared to passive delivery (44.9±16.3 µg/sq.cm). In the fourth aim, the feasibility of transdermal delivery of heparin, a hydrophilic macromolecule, through laser-microporated skin was investigated. There was no passive permeation of heparin across the skin. However, permeation of heparin from the laser-treated group delivered 13.4 ± 0.62 µg/sq.cm after 24 h. Furthermore, heparin was not delivered to either the epidermis or dermis passively, whereas the laser-treatment group enabled delivery of heparin to the epidermis as well as the dermis.
    • Development And Evaluation Of An Adolescent Chronic Restraint Stress (acrs) Protocol To Model Adult Depression In Female Rats / By Meghan Hibicke.

      Hibicke, Meghan
      Major depressive disorder (MDD), a mood disorder that disproportionately affects women, is often predicated by childhood or adolescent stress. However, females have traditionally been excluded from pharmacological research, and the influence of adolescent stress on depressive-like behaviors has not been widely reported. Additionally, reports correlating adolescent stress with adult depressive-like behaviors have largely neglected to evaluate the effect of common antidepressants. The purpose of this research was to develop and validate an adolescent chronic restraint stress (aCRS) protocol using female rats in order to address the impact of adolescent stress on female adult depressive-like characteristics. During the pilot study, rats were restrained 60 minutes daily for 12 consecutive days beginning in early adolescence, then allowed to mature to adulthood. Control and restrained animals were handled and weighed daily. Behavioral testing included sucrose preference testing (SPT), elevated plus maze (EPM), locomotor activity, novel object recognition (NOR) and object location (OLT), and forced swim tests (FST). Rats were sacrificed immediately following the FST, and trunk blood was collected for corticosterone (CORT) analysis. Four subsequent studies modified the aCRS protocol by increasing the restraint period from 12 to 14 days, using exact-age animals, and by including a treatment period during which antidepressants (fluoxetine and desipramine) or saline were given, and by changing the number and timing of the behavioral assays and sample collections. Additionally, brain-derived neurotrophic factor (BDNF) was assessed. In all studies, aCRS rats displayed significantly increased depressive-like behavior in the FST, but no differences in anxiety-like behaviors or locomotor activity vs control rats. Desipramine, but not fluoxetine, consistently decreased depressive-like behavior in restrained rats. Results of SPT, NOR, OLT, and CORT were inconsistent across experiments. Serum, but not HIP or FCX BDNF, positively correlated with immobility in the FST (a depressive-like behavior), which is not consistent with the literature. In the final experiment, we observed that uterine weights were not normally distributed, with aCRS rats trending low. In conclusion, aCRS elicits increased depressive-like behaviors in adult female rats, without altering anxiety-like behaviors or locomotor activity. Future investigations using aCRS rats should include further evaluation of reproductive hormones and BDNF.
    • Development and Evaluation of Micro/Nanoparticulate Vaccines for Human Papillomavirus and Influenza

      Vo, Trinh; College of Pharmacy
      Influenza and Human papillomavirus (HPV) are among the most common infectious deseases prevalent globally. The influenza viruses cause approximately 200,000 hospitalizations each year, resulting in a significant financial burden to the healthcare industry. Similarly, HPVs cause virulent infections that have multiple serotypes capable of producing genital warts and cervical cancer. In both cases the infection is spread through viral agents, The influenza virus is transmitted by aerosolized mist of coughs and sneezes with HPV is a sexually transmitted disease. Prevention of influenza and HPV is possible with the help of vaccines, and currently, there are several vaccines for both infectious diseases. However, in the case of influenza, development of a vaccine is challenging as the virus undergoes rapid mutations and hence, the vaccine needs to be modified every season. Along with the constant need for vaccination rates. In the case of HPV, the need for a novel vaccine delivery system is more crucial. HPV is directly linked to cervical cancer and developing a novel strategy to improve vaccination globally is a major priority. Commercial vaccines are not only expensive but in developing countries the financial cost of maintaining cold storage and vaccine administration is significantly challenging. The developing non-parenteral vaccines is essential to improve patient compliance without affecting safety and efficacy of the vaccine. This project has two main sections focusing on development of an oral microparticulate vaccine for influenza and HPV. In the first part of this project, we developed an oral microparticulate vaccine for influenza. The influenza vaccine consists of a M2e protein which is a viral protein common to all influenza viruses encapsulated into a microparticle. Adjuvants such as Alum, MPL. R848, MF59, Flagellin, CpG and P4 were encapsulated in microparticles separately. Microphages werestimulated with the vaccine-adjuvants combinations and subsequently., splenocytes were incubated with activated macrophages to investigate the T-cell activity. We observed significant enhancement of immune markers such as nitric oxide, CD80, CD86, and MHC II with several adjuvants. Furthermore, CD4 and CD8 T cell expression was also significantly enhanced in groups treated with particulate adjuvants. In the second part of this project, we formulated a novel oral and transdermal vaccine delivery system for HPV. Development of potent vaccines against HPV can prevent serious complications such as genital warts and cervical cancer. Therefore a novel non-parenteral vaccine against HPV may significantly reduce incidence of HPV worldwide. In this study, three different microparticualte vaccine formulations have been prepared and characterized: Bovine Albumin Serum (BSA) cross0linked with glutaradehyde; b-cyclodextrin, and cellulose acetate succinate matrices. We systematically studied activation of the immune markers such as nitirc oxide. CD80, CD86, CD40 and MHC-II. In vivo studies demonstrated elevated humoral and cell-mediated immune response in murine models. We observed elevated IgG titers in mice vaccinated with microparticulate HPV16 VLP vaccine, Furthermore, flow cytometry analysis revealed expansion of specific T-cell populations in vivo. This study proves the efficacy of the HPV-16 oral microparticulate vaccine as a promising alternative to conventional vaccines, In the alter part of the HPV vaccine project we investigated the potential of transdermal HPV vaccine as a novel strategy to improve vaccinations globally. In order to combat HPV infection, vaccines must be affordable, self-administered and efficacious. Transdermal vaccines can be self-administered. are almost painless and tap the immune cells present under the skin. Transdermal vaccines have gained immense attention due to their efficacy. In this project, our main aim was to develop a microparticulate HPV16 VLP vaccine for transdermal vaccination. HPV-16 VLP was encapsulated in microparticles and characterized for size, zeta potential, encapsulation efficiency and microparticle yield. Scanning electron microscope images confirmed the particles were irregular with surface indentations. Western blot analysis was performed on HPV16 VLP extracted from microparticles, which confirmed the retention of antigenicity of the epitope following microparticles, which confirmed the retention of antigenicity of the epitope following microparticle fromulation. Following in vitro characterization studies, we performed extensive in vivo studies to investigate the potential of the microparticulate vaccine to initiate a robust and sustained immune response. IgC titers for mice vaccinated with microparticulate vaccine were significantly elevated as compared to antigen solutions adminstered by the transdermal route. Flow cytometry was performed on vaccinated and control groups to investigate the expansion of specific T-cell populations. Vaccinated mice demonstrated significant increase in CD4, CD45R, CD27 and CD62L cell populations. Expansion of these specific T-cell subsets demonstrates the efficacy of the microparticulate vaccine when administered transdermally. Thus, our study proves the efficacy of the microparticulate vaccine over vaccine solutions and the novel approach of administering the vaccine through a minimally invasive, pain-free route that will help to improve acceptability of vaccination against HPV.