• A Comparison of SUMOylation in HK1 and BL41 Cell Lines

      Suarez, Persia; School of Medicine
      Nearly 96% of the population is infected with Epstein Barr virus (EBV), a gammaherpesvirus that results in a life-long infection. EBV lytically infects B lymphocytes and epithelial cells, and it establishes latency in B lymphocytes. Latent EBV infection often evades the host’s immune system; however, the presence of the EBV genome in certain cancers suggests that the virus is associated with approximately 200,000 new cases of cancer, specifically Burkitt’s lymphoma, Hodgkin’s lymphoma (HL), and nasopharyngeal carcinoma (NPC), each year. One cellular process commonly dysregulated in cancers, including EBV-positive lymphomas, is the post-translational modification of lysine residues by the Small Ubiquitin-like Modifier (SUMO), and SUMOylation inhibitors have been proposed to have potential anti-cancer properties. Our recent work focused on the small molecule inhibitor ML-792, which decreases global levels of SUMOylated proteins in EBV-positive and EBV-negative B lymphocytes. Similar experiments repeated with paired EBV-negative and EBV-positive nasopharyngeal cell line HK1 revealed that ML-792 only inhibited SUMOylation processes in the EBV-positive epithelial cells and not in their EBV-negative counterparts. We hypothesized that EBV may differentially modulate SUMOylation processes in epithelial cells when compared with B lymphocytes. This study aims to elaborate on the role of EBV on SUMOylation in epithelial cells. Paired primary B lymphocytes and epithelial cells were examined to determine the expression of the SUMO machinery. Results showed that EBV infection coincided with increased levels of SUMO-modified proteins and the SUMO-activating enzyme (SAE1 and SAE2), but not the SUMO-conjugating enzyme (Ubc9). Global levels of SUMOylated proteins increased in EBV-positive HK1 cells when compared with their EBV-negative counterparts. However, RNA and protein levels of the SUMO machinery varied greatly, which led us to ask if the confluence of the epithelial cells affected EBV-mediated changes in cells. Results demonstrated that RNA levels of the SUMO machinery significantly increased in sub-confluent EBV-positive HK1 cells, but these changes were not as apparent at the protein level. EBV-medicated changes in the SUMO machinery were more apparent at the protein level in confluent cells. To mimic a more physiological environment, EBV-negative and EBV-positive HK1 cells were also grown using a modified air-liquid interface method to model the human airway. Results showed that the presence of EBV corresponded with increased levels of the SUMO-activating enzyme and the SUMO-conjugating enzyme. Furthermore, the pattern of SUMOylated proteins changed in EBV-positive cells when compared with their EBV-negative counterparts. Taken together, our findings demonstrate that EBV does manipulate the SUMO machinery in epithelial cells, but not to the same extent as it does in lymphocytes. Therefore, additional studies are needed to better understand the effect of EBV on global levels of SUMOylated proteins in epithelial cells, which could identify if SUMOylation inhibitors have a therapeutic potential in the treatment of EBV-positive epithelial cancers.
    • Ablation of Dorsomedial Striatum Patch Compartment Results in Modification to Reward-Driven Behaviors in Rats

      Ahn, Jamin Paul; School of Medicine
      The striatum is a neural structure that plays a critical role in cognitive functions, behavioral decision-making, and reward generation. The striatum exhibits a heterogeneous composition, containing neurons belonging to the patch compartment—which is thought to be involved in habitual reward-related behaviors—surrounded by neurons belonging to the matrix compartment—which is thought to be involved in adaptive motor control. Additionally, the striatum is further subdivided into the dorsolateral striatum (DLS) and the dorsomedial striatum (DMS), each with their own patch and matrix compartments. The DMS has been associated with goal-oriented behavior seen during the initial stages of addiction. Conversely, the DLS has been associated with habitual behaviors seen during late-stage addictive behaviors that are inflexible. It is thought that drug addiction is initially mediated by the DMS before DLS activity becomes predominant. Previously, it has been shown that the patch compartment of the DLS is necessary for development of habitual behavior, but the role of the patch compartment of the DMS is less clear. Our study intends to demonstrate that selective ablation of DMS patch compartment neurons results in a significant impact on the initial development of reward-driven behaviors during the early stages of drug seeking behavior. Since patch compartment neurons express a high level of mu-opioid receptors compared to the surrounding matrix, we used dermorphin-saporin to target patch compartment neurons in the DMS and DLS for ablation. Following infusion in the DMS or DLS with dermorphin-saporin or vehicle-only infusions, rats were trained to self-administer cocaine on a progressive ratio schedule of reinforcement, starting with fixed ratio of 1 and ending with a fixed ratio of 5. Ablation of the patch compartment of the DMS resulted in an increase in early-stage lever pressing, suggesting that the DMS patch compartment contributes to reward-driven behaviors in a way that offers support for the parallel model of DLS/DMS activity. Future studies may investigate the relationship between DMS and DLS patch/matrix compartments through measuring or inducing activity in the DMS patch while the animal undergoes behavioral training. Methods for such a study may include optogenetics, Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), or microelectrode arrays.
    • Accumulation and Toxicity of Mercury-Cyanide Complexes

      Ford IV, Earl Gilmore; School of Medicine
      Mercury is a heavy metal toxicant found in numerous occupational and environmental settings. A major source of mercury pollution in environmental settings comes from the occupational use of mercury in artisanal and small-scale gold mining (ASGM). ASGM utilizes elemental mercury (Hg0) for extraction of gold from ore, which leads to Hg0-contaminated tailings. These tailings are often reprocessed with cyanide (‾CN) to extract residual gold. Hg0 reacts with ‾CN to form mercury-cyanide (Hg(CN)) complexes, which are released into the environment with discarded tailings. These complexes create numerous environmental and health problems. To assess the disposition of Hg(CN)2 complexes in mammalian systems, wistar rats were injected with 0.5 mol/kg mercury chloride (HgCl2) or Hg(CN)2. Each injection solution contained radioactive mercury ([203Hg2+]) and/or radioactive cyanide ([14C]-NaCN). After 24 h, animals were euthanized and organ samples were collected for determination of Hg(CN)2 content and for histological analyses. To assess the disposition of Hg(CN)2 complexes in an aquatic organism, zebrafish (Danio rerio) were exposed to various concentrations of Hg(CN)2 and accumulation in both whole fish and specific organs was measured. The current data show that the disposition of Hg(CN)2 in zebrafish is similar to that when rats were exposed to HgCl2. Interestingly, when rats were exposed to NaCN alone, the uptake was significantly less than that of rats exposed to Hg(CN)2. Experiments using adult zebrafish showed that Hg(CN)2 accumulates readily in fish. Zebrafish embryos exposed to Hg(CN)2 experienced alterations in developmental processes. The current data provide important information about the handling of Hg(CN)2 complexes in mammalian systems and aquatic organisms.
    • Activity And Mechanism Of Action Of Way-150138 Against Herpes Simplex Virus Type 2

      LINDSEY, PHILLIP TY
      Resistance to currently approved drugs and a medical need to treat beta and gamma herpesviruses merit the identification and development of alternative drug therapies for herpesviral infections. Previous research has suggested that a novel class of small molecule thiourea analogs target the UL6 portal protein of HSV-1 and HSV-2, disrupting the replication cycle during the viral DNA encapsidation process. Specifically, WAY-150138 compound was shown to have notable inhibitory effect against HSV-1. The compound’s reported lack of efficacy against HSV-2 remained puzzling considering the high amount of genetic conservation shared in the portal protein between HSV-1 and HSV-2. The studies presented in this thesis sought to resolve this issue. Viral yield assays were conducted for multiple strains of HSV-1 and HSV-2 in the presence of compound, and viral titers were calculated via direct plaque assay on Vero cell monolayers. Contrary to previous studies, WAY-150138 exhibited a similar inhibitory effect against both HSV-1 and HSV-2. To further investigate the molecular mechanism of thiourea compounds that target encapsidation, the HSV-2 portal homolog, encoded by the UL6 gene, was used to generate an rb-BAC-pUL6 protein. After full-length expression of the portal protein in a recombinant baculovirus system, sucrose gradient fractionation provided samples containing enriched amounts of pUL6 for further biochemical analysis.
    • An Analysis of the Toxic Effects of Mercury Cyanide Complexes on Zebrafish

      Pittman, Elizabeth; School of Medicine
      Mercury (Hg) is a unique heavy metal toxicant that is found in numerous environmental and occupational settings. A major source of environmental Hg is from artisanal and small-scale gold mining (ASGM), whereby metallic mercury (Hg0) is used to amalgamate gold from mined ore. Hg extraction of gold is inefficient and thus, a significant amount of gold remains in the ore. The leftover Hg-contaminated tailings are often subjected to cyanidation to extract the remaining gold. During this process, mercury and cyanide form mercuric cyanide complexes that are held together with a strong, covalent Hg-carbon bond. These toxic complexes are proposed to be prevalent in terrestrial and aquatic environments around mining sites. The purpose of the current study was to determine how mercuric cyanide complexes, particularly Hg(CN)2, affect the health of aquatic organisms in contaminated environments. We used adult and larval zebrafish (Danio rerio) as model organisms for this study. Zebrafish, in various life stages, were exposed to several different concentrations of Hg(CN)2 to determine the physical and behavioral effects of this exposure. Embryos were exposed to varying concentrations of Hg(CN)2 at one-hour post fertilization (4th cell cycle) until 72 hours post fertilization when hatching begins. Concentrations ranged from 0 to 0.1 mg/L Hg(CN)2. Embryos exposed to concentrations of Hg(CN)2 above 0.08 mg/L had a lower hatching rate and survival rate. Exposure of adult fish to various concentrations of Hg(CN)2 led to significant alterations in behavior and mercury content of muscle. The current study is the first to report the way in which Hg(CN)2 affects aquatic organisms in various stages of life.
    • Bioengineering Artificial Chromosomes For The Production Of Human Mirna

      Altherr, Delon
      Glioblastoma is a devastating disease with dismal patient outcomes. With current treatment options such as chemotherapy, radiation, and resection providing little hope to patients diagnosed with glioblastoma, research has turned to mesenchymal stem cells (MSCs) as a potential cellular delivery vehicle. The ability of MSCs to cross the blood brain barrier has increased the range of prospective treatment options available to patients with diseases like glioblastoma. Gene silencing through miRNA targeted therapy is one of the treatment options available through the utilization of MSCs. MSCs naturally package and secrete exosomes with miRNAs which have been shown to inhibit glioblastoma growth through gene silencing. Up until this point, miRNA targeted therapy has been limited to current gene therapy vectors and miRNA mimics. This project proposes an alternative to these approaches through the utilization of artificial chromosomes. The central hypothesis of this work is that artificial chromosomes can be bioengineered to produce multiple miRNAs for potential therapeutic applications. In order to test this hypothesis, a murine-derived artificial chromosome (platform ACE) was bioengineered to upregulate the expression of human miRNAs let-7b and miR124-1. The overall design of the project involved two major components. First was to identify a stable expression system for production of miRNA from the platform ACE. The second component consisted of subsequent analysis of miRNA production from the platform ACE. Findings from this project suggest that inducible vectors are more stable in the production of miRNA from artificial chromosomes compared to constituently activated vectors. Additionally, this project reports the successful transcription of the engineered miRNA constructs from the platform ACE in the Chinese Hamster Ovary (CHO) engineering cell line. Although this study was successful in producing primary transcripts from the platform ACE, this project further reports the disruption of miRNA processing beyond the initial transcript to a mature miRNA product. This study is in support of previous reports that CHO cells are ineffective in the production of mature miRNA from non-endogenous sources. Furthermore, suggesting that production of mature miRNA products must occur in species specific cell lines or with species specific flanking segments. Although more research is needed in the eventual utilization of artificial chromosomes in the production of miRNA, this project proposes an alternative to the use of current gene therapy vectors and miRNA mimics. In contrast to current approaches in miRNA targeted therapy, artificial chromosomes are stably maintained and not limited in their carrying capacity. Thereby opening the door for the potential delivery of multiple biological anti-cancer therapeutics in one cell mediated therapy vehicle. The use of artificial chromosomes has a long history for the delivery of large genetic payloads as well as multiple anticancer therapeutics. Thus, the potential of this project will allow for autonomous cell mediated therapy and targeting of multiple aberrant cell processes common to the etiology of glioblastoma.
    • Characterization of Attenuated HSV-2 Mutants as Potential Vaccine Candidates Against Genital Herpes

      Garza, Bret Kevin; School of Medicine
      A prophylactic and therapeutic vaccine against herpes simplex virus type 2 (HSV-2) infection is necessary to reduce the global disease burden of HSV-2 diseases. Subunit, single-cycle, and DNA vaccines have been studied in pre-clinical and clinical trials but have not been approved mostly due to lack of sufficient efficacy. Using targeted mutagenesis, a live, attenuated HSV-2 could be a likely candidate for a protective vaccine. In this study, we constructed and characterized two novel HSV-2 mutant strains, TKBAC /∆UL24 and TKBAC /∆UL39, that have loss-of-function in genes that are associated with viral pathogenesis. Bacterial artificial chromosome (BAC) technology and recombineering were used for construction of these strains and their revertants, TKBAC /UL24R and TKBAC /UL39R. ∆UL24 and ∆UL39 have internal deletions and TKBAC (the parent virus) has an insertion within the viral thymidine kinase gene consisting of the 7.5 kb BAC sequence. Sequencing of ∆UL24 and ∆UL39 confirmed the expected deletions with the HSV-2 UL24 and UL39 genes while the flanking regions remained intact. BAC DNAs were digested with select restriction endonucleases and fractionated by agarose gel electrophoresis. All constructs showed a similar digestion pattern compared to the parent strain, TKBAC, suggesting the mutant genomes were stable. Mutagenized BACS were used to create infectious virus stocks after transfection into Vero cells. We demonstrated that TKBAC /∆UL24 and TKBAC /∆UL39 were replication-competent in Vero cells. An HSV-2 UL24 mutant was shown to be a potential attenuated vaccine candidate in previous studies. The double mutant containing disruption of the TK gene would provide for an even safer attenuated vaccine candidate that would be less like to reactivate from latency and cause disease. Replication characteristics including a reduction in plaque size for TKBAC /∆UL39 suggested that this virus was crippled in vitro, while TKBAC /∆UL24 demonstrated similar replication characteristics to the parent strain. In vitro plaque reduction assays and viral yield assays against acyclovir suggest that TKBAC /∆UL39 is more sensitive to acyclovir compared to the parent strain, based on a lower IC50. Based on previous studies with individual TK or UL39 mutants, the double mutant should be even more deficient in the establishment of and reactivation from latency. Based on these results, TKBAC /∆UL24 and TKBAC /∆UL39 should be considered for further preclinical evaluation (in animal models) as viable candidates for a protective, safe prophylactic and therapeutic vaccine.
    • Compensatory Hypertrophy Enhances Renal Uptake Of Mercury

      Matta, Kayla Elizabeth
      Approximately 15 % of the adult population in the United States has been diagnosed with some degree of chronic kidney disease (CKD). CKD is characterized by a progressive and permanent loss of functioning nephrons. Following this loss, the remaining functional nephrons undergo compensatory changes including increased renal blood flow, increased single nephron glomerular filtration rate (SNGFR), and cellular hypertrophy. We hypothesize that compensatory hypertrophy of proximal tubules leads to an increase in the uptake and accumulation of xenobiotics and toxicants, such as mercury (Hg). Mercury is a ubiquitous environmental toxicant to which humans are exposed through various routes. Patients with CKD may be more susceptible to Hg and thus, it is important to understand how Hg is handled in the kidneys of these patients. We hypothesize that hypertrophied proximal tubular cells take up more Hg and are also are more sensitive to the toxic effects of Hg. To test this hypothesis, we used New Zealand White rabbits and Wistar rats. Cellular uptake of Hg, as a conjugate of glutathione (GSH; GSH-Hg-GSH), was measured at the basolateral membrane of isolated non-perfused proximal tubules from control and nephrectomized rabbits. Differences in mercury uptake at the cellular level were measured in control and hypertrophied S2 segments of proximal tubules by measuring several biochemical parameters of GSH-Hg-GSH uptake. Expression of selected enzymes was assessed in unexposed control and hypertrophied tubules using quantitative Polymerase Chain Reaction (qPCR). Effects of Hg on the entire kidney were measured in kidneys from rats injected intravenously with HgCl2 (0.5 µmol/kg/2 ml). Specific laboratory techniques utilized were the Glutathione Colorimetric Detection Assay, TBARS (Thiobarbituric acid reactive substances) Assay, qPCR, and Western Blot. Collectively, our findings show that hypertrophied tubules take up and accumulate more Hg than normal tubules. Also, hypertrophied cells appear to be more sensitive to the toxic effects of Hg than normal cells. These data provide important information regarding the altered handling of mercuric ions in patients with renal insufficiency due to chronic kidney disease.
    • 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.
    • 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.
    • DKK1's Potential Role As A Biomarker In Pancreatic Adenocarcinoma

      Igbinigie, Eseosaserea Grace
      Dickkopf-1 (Dkk1)’s dysregulation has been implicated in the pathogenesis of a variety of cancers. It is part of the Dkk family of proteins that includes Dkk2, Dkk3 and Dkk4. This family of secreted proteins shares similar conserved cysteine domains and inhibits the Wnt/b-catenin pathway by causing the degradation of �?�-catenin, thereby stopping cell proliferation. Dkk1 has also been previously shown to affect the CKAP/Akt pathway to increase Akt phosphorylation and promote cell proliferation. To determine the location and pathway that Dkk1 may regulate in pancreatic cancer cells, we performed immunofluorescence assays on Suit-2 cells. The results showed that Dkk1 is mainly located in the nucleus with a small percentage of the proteins in the cytoplasm. For Dkk1’s potential receptors, CKAP4 was found to have a similar staining to Dkk1 while Lrp6 was found to be evenly spread through the nucleus and cytoplasm. Further staining with the Wnt/�?�-catenin downstream protein, �?�-catenin, showed that it was colocalized with Dkk1 in the nucleus indicating that Dkk1’s presence did not inhibit its ability to translocate into the nucleus. Further studies into the cause of Dkk1’s inability to degrade and stop �?�-catenin’s translocation that causes increased cell proliferation is needed.
    • Effects Of T2r Activation On Different Lung Sensory Signaling

      Emamifar, Nick
      Pharmaceuticals are commonly bitter tasting and therefore may activate bitter taste receptors (T2Rs), which are canonically expressed in the taste buds of the tongue. An accumulation of recent evidence has suggested T2Rs carrying out a diverse set of non-tasting functions that are critical to the maintenance of homeostasis in extraoral locations throughout the body which give rise to the possibility of new medications to target these receptors. Using whole-cell patch-clamp recordings, the present study aims to illustrate how the bitter tasting pharmaceuticals, through the activation of T2Rs, modulate the neuroplasticity and therefore the function of lung sensory neurons. Three different airway irritants were used to activate pulmonary sensory receptors: zinc, an agonist for TRPA1; low pH or acid, an activator for both acid-sensing ion channels (ASICs) and TRPV1; and ATP, an agonist for P2X purinoceptors. Our results show that pretreatment with T2Rs activator chloroquine (0.01, 0.1 and 1 mM, 90 s) concentration-dependently potentiates zinc (30 µM, 3−16 s)-evoked TRPA1 currents and markedly inhibits ATP (0.3 or 1 µM, 4−6 s)-evoked P2X currents, whereas affects acid (pH 5.5 and pH 6.5, 4−16 s)-evoked inward currents differently: an inhibition for the slow inactivation ASIC-like current and a potentiation for the TRPV1-like current as well as the fast-inactivation ASIC current. Our results demonstrate that T2R activation in lung afferents has distinct modulatory effects on various ion channels that are sensitive to different airway irritants.
    • Epstein-barr Virus Viral Protein Lmp1 Increases Rap1 Sumoylation, Enhancing Telomere Maintenance During Tumorigenesis

      Cramblet, Wyatt Tyler
      Cancer remains the second leading cause of death in the USA, and there are multiple avenues of study to decipher how to combat the disease [1]. One approach in cancer treatments is to attack the tumor’s ability to replicate indefinitely. Normally healthy cells can only replicate a finite number of times before reaching their senescence limit due to loss of telomere DNA. During tumorigenesis, cells must gain the ability of maintaining chromosomal telomeres through continuous cycles of replication. Two mechanisms by which telomeres can be maintained are through activation of the telomerase complex and alternate lengthening of telomeres (ALT), a telomerase-independent mechanism that uses homologous recombination to maintain telomere length. Epstein-Barr Virus (EBV), a ubiquitous ϒ-herpesvirus, establishes a life-long latent infection within the host, and latent EBV infections are linked to the development of several epithelial and lymphoid malignancies. The principal viral oncoprotein is latent membrane protein-1 (LMP1). Early during the establishment of latency, EBV relies on ALT for maintaining telomeres [2]. However, telomerase-dependent telomere maintenance is observed in EBV-associated lymphomas. Understanding how and when the switch from ALT/telomerase-independent to telomerase-dependent telomere maintenance occurs could open new paths to the prevention or treatment of EBV-associated lymphomas. We focused on the Human Repressor Activator Protein 1 (RAP1) because it is involved in both telomerase-independent and telomerase-dependent telomere maintenance. We hypothesize that LMP1 CTAR3 induces the SUMOylation of RAP1, which contributes to LMP1-mediated oncogenesis through the maintenance of telomeres initially by ALT processes before switching to telomerase-dependent telomere elongation. Here we show conditions for telomerase-independent telomere maintenance improve when LMP1 induces the SUMOylation of PML and increases formation of PML nuclear bodies. We also show LMP1 blocks the interaction of PML with RAP1 while also increasing RAP1 association with SUMO proteins and telomerase activity. RAP1 point mutants that prevent the increased association of RAP1 with SUMO proteins also demonstrated a decrease in telomerase activity in conjuncture with increased association with PML. This data confirms that LMP1 contributes to maintaining telomeres, which not only aids EBV latency but also tumorigenesis. By better understanding how LMP1 affects telomere maintenance via its interaction with RAP1 we may discover novel therapies to prevent LMP1-mediated tumorigenesis.
    • Ethanol Induced Dysregulation Of The Psycho-neuro-immune Network

      Abney, Sarah Elizabeth
      Alcohol is one of the most harmful substance to the user and others that can be readily abused compared to other drugs (heroin, methamphetamine, etc.). In this study, we evaluated the roles of specific cytokines in the pathology of acute alcohol consumption in animal models. Two groups of NIH Swiss mice were treated IP with 1.8 g/kg (group A) and 3.0 g/kg of ethanol (group B) for 3 days. Controls (group C) were treated with saline. Tissue specific gene expression of key signal transducers, inflammatory cytokines, and their receptors (R) were evaluated with qPCR. Systemic cytokine levels were evaluated with flow-cytometry. Significantly higher gene expression of STAT-4, -5A -6 was observed in brain tissues of Group A animals compared to Group B. Compared to control animals, Group A animals exhibited increased gene expression of the T-bet and CXCL2(MIP2) in brain tissue. Splenic tissue of ethanol treated animals showed significant decreased gene expression of STATs, but increased gene expression of inflammatory cytokines TNFα and CXCL2. Plasma level of IL1 and CCL2(MCP1) were significantly higher in the Groups A and B compare to control animals. Subsequent study animals were treated similarly with ethanol agonist, Beta caryophyllene oxide (BCPO; Group O), to counteract alcohol-induced substance seeking behavior. BCPO treatment led to significant increase of plasma levels of TNFα, IFNγ, CCL2, IL23, and IL10 compared to Group A; and TNFα, IFNγ, IL10 and IL23 compared to Group C. This data suggests that alcohol consumption even with minimal doses leads to systemic inflammation and unequally affects systemic and tissue-specific cytokine equilibrium.
    • Glp-1r Agonists Improve Cerebrovascular Integrity And Vascular Cognitive Impairment And Dementia (vcid) Beyond Glycemic Control Via Recovery Of Brain Pericyte Function In Diabetic Mice

      Bailey, Joseph Martin
      We have previously shown that diabetes causes pericyte-dysfunction that leads to loss of vascular integrity and vascular-induced cognitive impairment and dementia (VCID). Glucagon-like peptide-1(GLP-1), used in the management of type-2 diabetes mellitus, improve cognitive of diabetic patients beyond glycemic control, yet the mechanism is unknown. In the present study, we hypothesis that GLP-1 agonist improves VICD through prevention of diabetes-induced pericytes dysfunction in a non-glucose dependent way. Methods: Control and diabetic mice were randomly assigned for saline or Exendin-4 (GLP-1 agonist 30 ng/kg/day), delivered through osmotic pump over 28 days. Vascular integrity was assessed by measuring cerebrovascular neovascularization indices (Vascular density, tortuosity, and branching density). Cognitive functions were evaluated with Barnes maze and Morrison Water maze. Human brain microvascular pericytes, HBMPCs, were grown in high glucose 25 mM/ sodium palmitate 200 uM (HG/Pal) to mimic diabetic conditions. HBMPCs were treated with/out Exendin-4 and assessed for oxidative stress and angiogenic properties. Results: Diabetic mice treated with GLP-1 agonist showed a significant reduction in all cerebral pathological neovascularization indices (P<0.05). Exendin-4 vascular protective effects was accompanied by significant improvement of the learning and memory functions of diabetic mice (P<0.05). Our results showed that HBMPCs expressed the GLP-1 receptor. Stimulation of HBMPC with GLP-1 against under diabetic conditions restored pericyte functions, decreased diabetes-induced inflammation, oxidative stress, and migration. (P<0.05). Conclusion: Our results provide novel evidence that GLP-1 agonist produces neurovascular protective effects in part through targeting pericytes. Restoration of pericyte functions in diabetes represent a novel therapeutic target for diabetes-induced vascular remodeling and VCID.
    • Manipulation of the SUMO Activating Enzyme (SAE) by the Viral Oncoprotein, LMP1

      McKinnon, Ashton Taylor; School of Medicine
      As a ubiquitous virus, Epstein-Barr virus (EBV) infects 95% of the global population. Although typically asymptomatic, its latency has been shown to cause a variety of different types of cancers, such as Burkitt’s lymphoma and Hodgkin’s lymphoma. Latent Membrane Protein-1 (LMP1) has been shown to be the principle oncoprotein of EBV by facilitating dysregulation of many pathways through both direct interactions and downstream modulation. This project focuses on the direct interactions of LMP1 with the SUMOylation process. SUMOylation is characterized by the addition of a Small Ubiquitin-like Modifier (SUMO) to a target protein. This helps to facilitate cellular growth and proliferation, protein stabilization and turnover, amongst other cellular processes. We hypothesize that LMP1 directly manipulates the SUMO Activating Enzyme/SUMO E1 (SAE) through a variety of mechanisms. This project investigates the interactions of SAE and LMP1 utilizing immunoprecipitations. Our results show that SAE modulation occurs through interactions in a C-Terminal Activating Region (CTAR) -2 dependent manner. Another aspect is understanding the effect LMP1 has on SAE stabilization. This is done by treating LMP1-expressing cells and non-expressing cells with cycloheximide over a set time course. Western blot data shows a steady decrease in the amount of SAE present in non-LMP1-expressing cells, while SAE remains relatively stable in LMP1-expressing cells. Lastly, the lab is interested in how LMP1 affects protein trafficking. This utilizes immunofluorescence to tag proteins of interest in whole cells and visualize them using confocal microscopy. In addition, we use cellular compartmentalization to detect cytosolic and nuclear proteins in comparison to whole cell lysates. Immunofluorescence results show more SAE present in the cytosol of LMP1-expressing cells, while in negative counterparts, we see SAE primarily in the nucleus. This is confirmed in western blot data from compartmentalization experiments. Our findings suggest that LMP1 is capable of interacting with a variety of different proteins, including direct interactions with enzymes involved in the SUMOylation process, specifically SAE. Future work aims to elucidate how LMP1 can affect other enzymes in the SUMOylation process.
    • Methamphetamine Induced Immune Dysregulation

      Andrzejak, Sydney
      Methamphetamine (METH) use can induce and contribute to the development of neurodegenerative disorders. In this study, we evaluated the roles of specific cytokines in the pathology of acute and chronic methamphetamine usage in vitro and in vivo. An immortalized rat astrocyte CTX-TNA2 cell line was used for a model of immune cells in central nervous system. Cells were treated with methamphetamine hydrochloride, dopamine hydrochloride as a reference point, and lipopolysaccharides (LPS) as a model of immunogenic stimulant. Our in vivo experimental design utilized female NIH-swiss mice that underwent a seven-day treatment period. The control mice were administrated 0.9% saline, and our treated mice were administrated 12 mg/kg of (+)- methamphetamine through intraperitoneal injections. Cells and tissue specific gene expression of key signal transducers, cytokines, and their receptors were evaluated with qPCR. Systemic cytokine levels were evaluated with flow-cytometry. The results suggest that METH acts locally to the brain tissue to cause a shift toward inflammation by increasing cytokine, receptor and signal transducer of interleukin-6 (IL-6) and interleukin-17 (IL-17) pathways. While suppressing systemic cytokine production leading to an imbalance in Th-X paradigm.
    • Optimization Of Protein Purification Paths For Clover-ruby2 Fluorescent Proteins For In Vivo Kinetic Model Development

      Fussell, Robert Charles
      Chromophores are photoactive molecules that can be excited by light and return to ground state by fluorescence. When two different fluorescent proteins are tethered together, an excited donor can transfer energy to the acceptor via a process called Förster Resonance Energy Transfer, FRET, if certain conditions are met. FRET results in the acceptor becoming excited as well, leading to emission at a specific wavelength. The phenomenon is useful for several different experiments such as protease assays, in which cleavage of the protein linking the donor and acceptor abolishes FRET as the pair separates. This mechanism would prove useful when comparing affinity and turnover rates of proteases on the fluorescent protein labeled surface loops of G protein coupled receptors, GPCR, in future investigations. However, the accuracy of the data can be affected by protein interactions that can inflate or deflate detectable FRET. There has been little research in the different methods of purification and their effects on the purity and functionality of the fluorescent proteins. To determine the effects by different chromatography columns, different purification protocols were devised. In addition, several experiments on the fluorescent proteins alone were done to determine the factors such as light, temperature, salinity, and concentration that contributed towards degradation and aggregation. Results identified the most successful protocol. Application of a G-25 desalting column followed by an ion exchange and gel filtration columns produced an almost pure substrate and the most functional mRuby-2. Additionally, maturation for 6 hours in low salt conditions allowed for optimized mRuby-2. Maintaining concentrations of the FRET pair at 1.5μM to 2.5μM provided concise quantity control data. The protocol can be utilized for in vitro vs in vivo model, to accurately determine the effectiveness of enzymes on GPCR surface loop.
    • Potential Mechanisms Of Inorganic Mercury Intoxication In Rat Kidney Cells

      Orr, Sarah Elizabeth
      Mercury is a ubiquitous toxic metal that is found in the environment. Different forms of mercury can be ingested, inhaled, and/or absorbed dermally. Exposure to mercury occurs typically through the ingestion of organic mercuric compounds in contaminated seafood and/or the inhalation of mercury vapor from mercury-containing dental amalgams, broken mercury-containing thermometers, or industrial sources such as coal-fired power plants. Mercury intoxication occurs in target cells of organs, such as kidneys, following significant exposure to one of the different chemical forms of mercury. Due to the adverse health effects of mercuric ions in humans, a thorough analysis of the mechanisms by which mercury initiates cellular intoxication in target cells is necessary for the development of effective therapeutic strategies. The purpose of this study was to identify the specific intracellular mechanisms that lead to the toxic effects of inorganic mercury (Hg2+) in target cells. Exposure to mercury can lead to significant alterations in cytoskeletal structure, calcium availability, membrane permeability, oxidative stress, and autophagy. These alterations were studied and assessed using a variety of laboratory techniques including cell culture, laser-scanning confocal microscopy, and quantitative PCR. Additionally, the protective properties of alpha lipoic acid were analyzed using similar methods. By experimentally examining each of these aspects, we have established a more complete understanding of intoxication and cellular injury induced by inorganic mercury in proximal tubular cells.
    • Predictive Analysis of the Immunosuppressive Functionality of Human Bone Marrow Derived Mesenchymal Stem Cells as Cellular Therapeutics

      lipat, ariel joy mann; School of Medicine
      Human Mesenchymal Stem/Stromal Cells (MSCs) of bone marrow carry immunomodulatory and regenerative properties and are being tested as a cellular therapy for inflammatory and degenerative disorders. They are involved with the paracrine secretion of anti-inflammatory cytokines and chemokines and the promotion of anti-inflammation in tissue microenvironments by dampening inflammatory T-cells. However, the mechanism of action of MSCs on T-cells has yet to be understood. Here we aim to identify the pattern of chemokine secretion in human bone marrow MSCs and their regulation and functions on T-cell responses and immune suppression. MSCs were derived from healthy human bone marrow aspirates. MSC secretome was collected systematically under defined cell densities and subjected to multiplex secretome analysis with or without exogenous stimulation to identify inherently secreted MSC chemokines. MSC derived chemokines’ immunosuppressive role on T-cells was further determined with a PBMC and MSC coculture and siRNA chemokine transfection strategies. MSC secretome was further tested on human peripheral blood mononuclear cells (PBMCs) derived from blood and early phosphorylation of signaling molecules in T-cells were specifically analyzed utilizing PhosflowTM technology in flow cytometry. Of thirty tested chemokines nine (CXCL16, CCL2, CXCL6, CCL7, CXCL1, CCL13, CCL5 CXCL2 and CCL1) are secreted inherently by MSCs suggesting that MSC potency and immunosuppressive potential can be determined by the presence of these chemokines. In addition, MSC mediated blocking of T cell proliferation predominantly inversely correlates with chemokines. Knockdown of chemokines have demonstrated that MSC sourced inherent chemokines do not actively play a role in T cell suppression and thus are the bystander predictors of T cell suppression. The present analysis of MSC’s matrix chemokine responses can be deployed in the advanced potency determination of MSCs. As well, little difference was seen between chemokine levels from intestinal organoid secretome samples from IBD and non-IBD cultures. Seven signaling molecules [PLCγ1, PLCγ2, PKCα, JNK, P38 MAPK, Erk ½, pAkt (pS473)] were analyzed for phosphorylation events in T-cells when stimulated with MSC secretome. Our results provided evidence that MSC derived chemokines and secretome predicts T-cell suppression. These mechanistic understandings will help us to improve MSC based cellular therapy.