Tumor initiation and progression are dependent on both aberrant gene expression in tumor cells and the communication between tumor cells and its micro- and systemic microenvironment. Many tumor suppressor genes and oncogenes have been characterized to suppress or promote tumor growth, but fewer genes in tumors are well-characterized as interacting with immune cells in the host to promote or inhibit tumor growth. The interleukin (IL) 27 receptor WSX1 is expressed in immune cells and induces an IL27-dependent immune response. Opposing this conventional dogma, our initial results reveal a much higher level of WSX1 expression in multiple types of epithelial tumor cells when compared to normal epithelial cells. These revelations suggest a role for WSX1 in tumor development, and thus a possible target in cancer immune-therapy. Using genetically modified tumor cells, our studies show that the expression of WSX1 in tumor cells regulates the communication between tumor and host cells resulting in two different consequences. In both the cervical cell line TC1 and the squamous carcinoma cell line AT84, overexpression of WSX1inhibited tumorigenicity both in vivo and in vitro. Sensitizing NK cell-mediated surveillance through upregulation of NKG2D ligands in tumor cells is the underlying mechanism by which WSX1 inhibits tumor growth. Further investigations into other cell lines, such as colon cancer (CT26) and Lewis Lungs Carcinoma (LLC), confirmed the role of WSX1 as a tumor suppressor in vitro. In contrast to the role that WSX1 plays in the aforementioned cells, aggressive LLC and melanoma AGS tumor cells expressing WSX1 grow faster than the control cohorts. These studies reveal that the principal mechanism by which WSX1 promotes tumor growth is the inhibition of T cell proliferation and production of the effector cytokine IFNγ both in the tumor microenvironment and distal lymphatic tissues. Our evidence reveals that this effect is initiated via direct tumor cell and immune cell contact. This important observation reveals a new pathway of tumor-host interaction, which will ultimately lead to better strategies in immune therapy to reverse tumor tolerance.

Hypertension is a major predisposing factor for the development of cardiovascular and renal diseases. The renin-angiotensin system (RAS) plays a pivotal role in the pathogenesis of cardiovascular diseases such as hypertension, myocardial infarction, heart failure, and stroke. Angiotensin II (Ang II), the effector peptide of the RAS, activates a wide spectrum of signaling responses via the Ang II type-I receptor that mediate its physiological control of blood pressure, thirst and sodium balance. For the past two decades, increasing evidence has demonstrated that the circulatory RAS and local/tissue RAS components (heart and brain) may contribute to the development of hypertensive response. Currently, hypertension is considered a low-grade inflammatory condition induced by interaction of the RAS with various pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α). Several in vitro and in vivo studies suggest the existence of a cross-talk between Ang II and TNF-α, implying an important role for TNF-α in blood pressure regulation. However, the functional importance of TNF-α in Ang II-induced response is unclear. In this dissertation, we examined the hypothesis that TNF-α is involved in the Ang II-induced hypertensive response and explored the interaction of Ang II and TNF-α in the heart and brain in the pathogenesis of hypertension. To examine this interaction, the effects of chronic administration of Ang II was evaluated in TNF-α knockout mice to dissect out the role played by TNF-α in the Ang II-induced effects. Additionally, the role of reactive oxygen species and the transcription factor nuclear factor-kappaB (NF-κB), were examined in this interaction between Ang II and TNF-α. Furthermore, to understand the role of central control of blood pressure via the hypothalamic paraventricular nucleus, an important cardiovascular regulatory center in the brain, we studied the effect of TNF-α blockade and Angiotensin Converting Enzyme 2 overexpression within the brain on blood pressure control. Overall, these studies demonstrate a functional interaction between the RAS and TNF-α in hypertension and the possible roles of oxidative stress and NF-κB in mediating the Ang II-induced hypertensive response. These findings provide an important clue in our quest for understanding the pathophysiology of hypertension and other cardiovascular diseases.

Advances in cancer therapies continue to be improved, yet cancer continues to be one of the deadliest diseases in the world. Harnessing the power of the bodys immune system to attack cancer is a promising strategy that can further improve therapies for neoplastic diseases. As part of this strategy, cytokines such as interleukin (IL) 2 and interferon á are currently accepted cancer treatments, and other cytokines such as IL12 and GM-CSF also show potential as new treatments. Clinical trials with these cytokines have shown less than acceptable therapeutic efficacy and toxicities, but tumor-targeting motifs can improve these effects. Both antibodies and peptides specific for tumor antigens have been used in recombinant protein and gene therapy systems to increase the intratumoral cytokine accumulation and decrease systemic toxicities. Still, these treatments have not been capable of overcoming the obstacles for clinical acceptance. The hypothesis tested in this dissertation is that inserting tumor-targeting peptide coding sequences into IL12 plasmid DNA will create a novel systemic gene therapy approach which will increase the antitumor efficacy and decrease toxicity for cancer treatments. To accomplish this goal, a reporter gene mediated screening strategy was developed to identify a peptide which can target multiple tumor models. While preparing this method, it was discovered that these peptides can have a strong effect on the activity of the conjugated reporter gene. Once this strategy was finalized, the peptide VNTANST was found to increase the intratumoral accumulation of the reporter gene in five tumor models including a human xenogeneic model. The VNTANST coding sequence was then inserted into an IL12 plasmid to examine the antitumor efficacy. In breast adenocarcinoma, squamous cell carcinoma, and colon carcinoma models, VNTANST-IL12 plasmid DNA treatments distal from the tumor site increased tumor inhibition and, in two models, prolonged survival. Also, these treatments reduced the development of metastatic lung tumors in a spontaneous metastatic model. As expected, these tumor-targeted IL12 treatments decreased the level of liver toxicity compared to wild-type treatments. The receptor for VNTANST was identified as vimentin, which is a potentially powerful target for human cancers.

The Transient Receptor Potential Melastatin 4 protein (TRPM4) is a member of the TRP family of ion channels that is expressed in both electrically excitable and non-excitable cells. Functional studies revealed that TRPM4 significantly impacts Ca2+ signals in both immune and pancreatic β-cells, which is important for cellular processes such as hormone secretion. However, its role in glucagon secreting α-cells has not been reported. Type 2 Diabetes Mellitus is often associated with increased glucagon levels; yet, the exact mechanism controlling its secretion is not known. In pancreatic α-cells, an increase in intracellular Ca2+ concentration causes glucagon secretion. We hypothesize that TRPM4 is important for glucagon secretion in α-cells by controlling intracellular Ca2+ signals. In this study, we investigated TRPM4 expression in the α-cell lines INR1G9 (hamster) and αTC1-6 (mouse) and characterized the channel using the patch-clamp technique. By RT-PCR we identified TRPM4-transcripts in both cell lines examined. Furthermore, patch-clamp recordings with increasing intracellular Ca2+ concentrations resulted in a dose-dependent activation of TRPM4-like currents. The greatest depolarizing currents were obtained with 3μM Ca2+ concentration. The current-voltage relationship (I/V) resembled those previously described for TRPM4. In addition, we demonstrated the voltage dependency of the channel, where negative potentials inhibited and positive potentials increased channel activity. Finally, replacement of Na+ ions in the extracellular solution with N-methyl-D-glucamine significantly reduced the inward currents and caused a hyperpolarizing shift in the I/V, which affirms that the channel is Na+ permeable. These data demonstrate that TRPM4 is present and functional in pancreatic α-cells and suggest a potential role for the channel in glucagon secretion and glucose homeostasis. The role of TRPM4 in glucagon secretion was assessed using a stable TRPM4 knockdown αTC1-6 cell line. Calcium-imaging and glucagon secretion experiments revealed a relationship between the decreased intracellular Ca2+ concentration and glucagon secretion in TRPM4 knockdown cells compared to controls. These results indicate that depolarization by TRPM4 plays an important role in glucagon secretion and perhaps glucose homeostasis. Elucidation of the glucagon secretion pathway could lead to a treatment for hyperglucagonemia associated with Type 2 Diabetes.

This dissertation includes a comprehensive current review of reversible electroporation (EP) and other related physical gene transfection techniques; an overview of results of electrochemogene therapy (ECGT) used to treat naturally occurring spontaneous neoplasms in dogs; and the results of comprehensive, pre-clinical toxicology testing of electrogene therapy (EGT) of a tumor-targeted version of interleukin-12 (IL-12) in mice. Intralesional bleomycin (BLM) and feline interleukin-12 (fIL-12) DNA injection combined with trans-lesional EP resulted in complete cure of two recurrent oral squamous cell carcinomas and an acanthomatous ameloblastoma in a series of six cases of spontaneous neoplasia in pet dogs. The three remaining dogs, which had no other treatment options, had partial responses to ECGT. One of these dogs had mandibular melanoma with pulmonary and lymph node metastases; one dog had cubital histiocytic sarcoma with spleen metastases; and one had soft palate fibrosarcoma. Treatment of all six dogs was associated with minimal side effects, was easy to perform, was associated with repair of bone lysis in cured dogs; improved the quality of life for dogs with partial responses; and extended overall survival time. For the purpose of meeting pre-clinical safety requirements for an Investigational New Drug filing, we assessed the safety of tumor-targeted interleukin-12 (ttIL-12) when administered by EGT in C3H/HeJ mice by identifying an initial safe dose for human dose escalation schemes, toxicity target organs, markers of toxicity, and toxicity reversibility. Dystrophic cardiac calcification in older, 5 ìg ttIL-12-treated mice was the only serious toxicity. Based on these results and the lack of any effect on wound healing when combined with surgery, low-intensity EGT with ttIL-12 appears to be safe and well tolerated as both a single treatment modality and when combined with surgical tumor resection.

Hypertension affects 50 million Americans and remains the second leading cause of renal failure in the United States. Current pharmacological and non-pharmacological approaches to treat hypertension have proven effective, but the complexities of the disease and its renal effects warrant the need for new treatments. The hypothesis of this dissertation was that pharmacological or non-pharmacological approaches to reducing inflammation and oxidative stress would prevent hypertension-induced renal injury in the spontaneously hypertensive rat (SHR). In the first study, we blocked the inflammatory transcription factor, nuclear factor-kappa B (NF-ĸB), with pyrrolidine dithiocarbamate in the SHR kidney. In treated SHR, blood pressure decreased, renal hemodynamics were preserved, and oxidative stress and inflammation were attenuated at both the cytosolic and mitochondrial levels; suggesting a role for NF-ĸB in potentiating hypertension-induced renal injury. In the second study, we examined the effects of aerobic exercise training on renal oxidative stress and inflammation. Exercised SHR exhibited normalized blood pressure and renal hemodynamics. These effects were attributed to lower NF-ĸB activity and decreased oxidative stress in the SHR kidney. In the third and fourth studies, we examined the effects of diet modification by use of blueberry-enriched diets, since blueberries have one of the highest antioxidant capacities of any fruit or vegetable tested to date. In the third study, we fed stroke-prone SHR high salt and a blueberry-enriched diet for 2 days, 6 weeks, or 12 weeks, and examined renal parameters. The SHR fed the blueberry diet for the 6- or 12-week periods demonstrated lower oxidative stress, lower blood pressure, and preservation of renal hemodynamics. These effects were likely due to a hormetic effect of the blueberries themselves, since rats fed blueberries for 2 days demonstrated higher oxidative stress. In the final study, we added blueberries to a stroke-permissive diet, which accelerates renal damage in SHR. Rats were fed diets for 10 weeks. Rats fed the control diet had severe hypertension, severe oxidative stress, and severe inflammation as evidenced by NF-ĸB activation, and exhibited signs of renal failure. Rats fed the blueberry supplemented diet exhibited decreases in blood pressure, oxidative stress, and inflammation, and also had preserved renal structure and function.

Research suggests that the cyclic AMP (cAMP) signaling pathway is implicated in the development of alcoholism. Previous work in our laboratory has demonstrated that alcohol enhances the activity of adenylyl cyclase (AC) in an isoform specific manner; human type 7 AC (AC7) is most enhanced by ethanol as measured by cAMP accumulation assay in whole cells. We hypothesize that alcohol enhances AC activity by directly interacting with the protein and that alcohol effects on AC can be studied using recombinant AC expressed in bacteria. Our objectives include: 1) design and optimization of the conditions for protein expression and protein purity for recombinant AC proteins; 2) identification of the importance of each cytoplasmic domain to the alcohol effect through chimeric analysis; and 3) investigation of the interaction between alcohol and AC stimulators such as MnCl2, forskolin, and Gsá through concentration-response experiments. To examine these objectives, we expressed in bacteria, and purified recombinant AC proteins carrying the C1a and/or C2 domains of AC2, AC7, and AC9. We present the optimal conditions for the expression and purification of multiple recombinant AC proteins and show that purified recombinant AC proteins retain enzymatic activity and alcohol responsiveness. Through chimeric analysis, we found that the C1a and C2 domains both contribute to the alcohol effect on AC7, however, AC7 C1a may play a stronger role. We also find that recombinant AC responds to alcohol differently under varied conditions of AC activation by MnCl2, forskolin, and Gsá. Through concentration-response experiments we found that there is some interaction between alcohol and forskolin or Gsá, but alcohol does not appear to be competing with forskolin or Gsá at an allosteric site. Overall the results suggest that alcohol interacts with AC proteins directly, independent of stimulants examined, and causes a conformational change, which results in either enhancement or inhibition depending on stimulation conditions.

Mesenchymal stem cells are multipotent cells that can differentiate into many cell types. However, the molecular mechanism controlling this process remains unclear. We utilized rat dental follicle stem cells (rDFSCs) and human adipose derived stem cells (hASCs) to study the mechanisms controlling osteogenesis and adipogenesis. Elevations in the intracellular Ca2+ concentration are a phenomenon commonly observed during stem cell differentiation but cease after the process is complete. The Transient Receptor Potential Melastatin 4 (TRPM4) is an ion channel that controls Ca2+ signals in excitable and non-excitable cells. However, there are no studies on TRPM4 in stem cells. In another study, we investigate the mechanism by which arginine vasopressin (AVP), a neuropeptide hormone secreted mostly from the posterior pituitary gland increased Ca2+ signals and inhibited adipogenesis in hASCs. The overall goal of our studies is to investigate the effect of TRPM4 and AVP on stem cell differentiation and Ca2+ signaling. First, we identified TRPM4 gene expression and its characteristics such as Ca2+-activated, voltage dependent and monovalent conducting properties in rDFSCs. Molecular suppression of TRPM4 transformed the normal agonist-induced first and secondary phases of Ca2+ signals into a gradual and sustained increase which enhanced osteogenesis but inhibited adipogenesis in rDFSCs. Next, we examined TRPM4s impact on Ca2+ signals and adipogenesis in hASCs, which is a more suitable stem cell type for adipogenic studies. Suppression of the TRPM4 diminished the histamine-induced Ca2+ signals mainly via H1 receptors. The increases in intracellular Ca2+ were due to influx via voltage-dependent Ca2+ channels of the L-type (Cav1.2) and release from the endoplasmic reticulum (ER). Lastly, we determined the role of AVP on adipogenesis in hASCs. These cells were responsive to AVP stimulation by increasing intracellular Ca2+ via V1a receptors, Gq-proteins and the PLC-IP3 pathway. Both Ca2+ release from the ER and influx from the extracellular space contribute to the Ca2+ signals. AVP supplementation to the differentiation media decreased the number of adipocytes during adipogenesis. The effect of AVP on adipocyte formation was reversed by the V1a receptor blocker V2255. In conclusion, TRPM4 and AVP control Ca2+ signals which affect stem cell differentiation.

Nucleotide excision repair (NER) is a multi-step cellular process that removes bulky and/or helix-distorting DNA lesions, such as UV induced cyclobutane pyrimidine dimers (CPDs) and bulky chemical adducts. Transcription coupled repair (TCR) is a subpathway of NER dedicated to rapid removal of lesions in the transcribed strand of actively transcribed genes. The TCR mechanism in bacteria has been relatively well elucidated. However, TCR in eukaryotic cells appears to be extremely complicated. The exact nature of the TCR signal and the mechanism of the transcription-repair coupling have been long-standing enigmas. This dissertation focused on how the TCR repressors and facilitators interplay with RNA polymerase II (RNAP II) to carry out TCR in yeast Saccharomyces cerevisiae. By site-specific incorporation of the unnatural amino acid p-benzoyl-L-phenylalanine, we mapped interactions between Spt5 and RNAP II in S. cerevisiae. Through its KOW4-5 domains, Spt5 extensively interacts with Rpb4/7. Spt5 also interacts with Rpb1 and Rpb2, two largest subunits of RNAP II, at the clamp, protrusion and wall domains. Deletion of Spt5 KOW4-5 domains decreases transcription elongation and derepresses TCR. Our findings suggest that Spt5 is a key coordinator for holding the RNAP II complex in a closed conformation that is highly competent for transcription elongation but repressive to TCR. We also demonstrated that E1103G mutation of Rpb1, the largest subunit of RNAP II, which promotes transcription bypass of UV-induced CPDs, increases survival of UV irradiated yeast cells but attenuates TCR. In contrast, G730D mutation of Rpb1, which abolishes transcription bypass of CPDs, enhances TCR. Our findings suggest that transcription bypass of lesions attenuates TCR but enhances cell tolerance to DNA lesions. Efficient stalling of RNAP II is essential for efficient TCR. Sen1 is an RNA/DNA helicase that has been shown to mediate termination of noncoding RNAs and some mRNAs. Like deletion of Rad26 or Rpb9, the Sen1 N-terminal deletion (1-975 residues) increases the UV sensitivity of the GGR-deficient cells. Moreover, the Sen1 N-terminal deletion decreases TCR in rad7Δ and rad7Δ rad26Δ cells but not that in rad7Δ rpb9Δ cells. Our findings suggest that the N-terminal domain of Sen1 contributes to Rad26-independent TCR.

Post-traumatic stress disorder (PTSD), a trauma- and stressor-related disorder, is a condition that can develop in response to life-threatening situations. According to the Diagnostic and Statistical Manual of Mental Disorders 5 (DSM-5), a diagnosis of PTSD necessitates exposure to a life-threatening event, intrusive recollections, avoidance of associated stimuli, hyperarousal, and a significant social impairment. All of these symptoms must persist for at least 30 days and not be due to illness, medication, or substance abuse. To date, no definitive diagnostic biomarkers have been identified for PTSD. Recent research, however, points toward physiological abnormalities in the brain, hypothalamic-pituitary-adrenal (HPA) axis, and immune system that may be partially responsible. Many chronic conditions such as hypertension, heart failure, and metabolic syndrome perpetuate in a state of increased inflammation and oxidative stress, exacerbating their pathophysiology. In many psychiatric conditions such as depression and anxiety disorders, neurotransmitter modulation may play a critical role in their pathogenesis. Based upon the literature and work from our laboratory, we hypothesized that similar pathophysiological mechanisms may play a role in PTSD development. We tested our theory by creating a PTSD-like syndrome in rats with the use of a predator exposure/psychosocial stress animal model. We then conducted a series of in vivo and ex vivo experiments in an attempt to discover the roles of inflammation, oxidative stress, and neurotransmitter modulation in PTSD development. First, we evaluated inflammation and oxidative stress in the brain, adrenal glands, and blood in response to the predator exposure model. We then analyzed neurotransmitter modulation in the hippocampus and prefrontal cortex. Next, we investigated the anti-inflammatory and neuromodulating effects of the histone deacetylase inhibitor (HDACi) valproic acid (VA) on inflammation/oxidative stress and neurotransmitters. Finally, we employed the selective-serotonin reuptake inhibitor (SSRI) sertraline to ascertain why SSRIs have historically been ineffective in treating PTSD. Taken together, our findings indicate inflammation, oxidative stress, and aberrant neurotransmitter profiles may play a significant role in PTSD development and progression. In addition, VA may prove to be a legitimate pharmacologic alternative in PTSD treatment, as SSRIs may increase the noradrenergic response and actually exacerbate anxiety in a clinical setting.