Fetal stress has been linked to adult atherosclerosis, obesity, and diabetes. Epidemiology studies have associated fetal exposure to maternal smoking and post-natal exposure to environmental tobacco smoke (ETS) with increased asthma risk. We tested the hypothesis, in a mouse model of asthma, that ETS exposure in utero alters airway function and respiratory immune responses in adult offspring. Pregnant BALB/c mice were exposed daily to ETS or filtered air (AIR). Neonatal gene expression was assessed. Offspring inhaled aerosolized ovalbumin (OVA) or saline in weeks 7-8. Regardless of whether they inhaled OVA or saline, mice were sensitized by OVA injections in weeks 11 and 13 followed by OVA aerosol challenge in weeks 14-15. At weeks 6, 10, and 15, we assessed OVA-specific serum immunoglobins, bronchoalveolar lavage cells and cytokines, lung and nasal histopathology, lung gene expression, and airway hyperresponsiveness (AHR). Neonatal mice demonstrated slight but potentially critical differences in gene expression related to their exposure to ETS in utero. At 6 weeks, there were no significant differences between mice exposed to ETS in utero and those exposed to AIR in utero. At 10 weeks, following OVA aerosol, mice exposed to ETS in utero displayed greater AHR than mice exposed to AIR in utero (Ñ = 0.05), unaccompanied by changes in histopathology, cytokine profile, or antibody levels. However, there were significant differences in gene expression between these 10 week groups. At 15 weeks, mice that had inhaled saline in weeks 7-8 developed airway inflammation: eosinophilia (Ñ = 0.05), IL-5 (Ñ = 0.05) and AHR (Ñ = 0.05) were greater in mice exposed to ETS in utero vs. mice exposed to AIR in utero. Mice that had inhaled OVA in weeks 7-8 demonstrated no airway inflammation after sensitization and challenge and those exposed to ETS in utero had suppressed immune and inflammatory responses. Consistent with other findings at 15 weeks, there were significant differences in gene expression between mice receiving ETS exposure in utero and those receiving AIR exposure in utero. ETS exposure in utero exacerbates subsequent adult responses to initial allergen exposure and causes altered gene expression, especially with additional lung perturbation.

Despite advanced therapeutic strategies for post-myocardial infarction (MI) patients, many ultimately develop congestive heart failure (CHF), rendering the disease a major cause of death in the United States. MI is associated with an acute increase in sympathetic nervous system activity, becoming persistent in CHF patients. Increased pro-inflammatory cytokines (PICs) following MI are implicated in the pathogenesis of CHF. The increase in tumor necrosis factor (TNF), a primary PIC, correlates closely with heart disease severity. Moreover, central PIC production increases post-MI, and can affect the brains cardiovascular regulatory regions that control sympathoexcitation. Therefore, understanding how PICs modulate sympathoexcitation is important for development of new therapeutics. Recent studies underscore the importance of central NADPH oxidases in the pathogenesis of hypertension. However, the role of central NADPH oxidase-induced reactive oxygen species (ROS) production in the development of CHF remains limited. In this dissertation, the hypothesis that central PICs induce ROS production and modulate sympathoexcitatory neurons of the paraventricular nucleus (PVN) is explored through an array of selective animal models combined with novel technologies for sympathoexcitation and cardiovascular function assessment. The effect of the TNF blocker, pentoxyfylline, was investigated on the expression of the catalytic subunits of NADPH oxidase (Noxs) in the PVN neurons and on the sympathetic activity in CHF rats. Additionally, effects of TNF inhibition on central nitric oxide were explored, as this ROS restrains sympathoexcitation. More specifically, central TNF was inhibited to understand the interaction between superoxide and nitric oxide in the PVN neurons during CHF. TNF knock-out mice were also used to study the effect of TNF on volume overload associated with CHF. Finally, to understand the role of peripheral TNF on the PVNs sympathoexcitatory neurons, and to exclude the effects of neurohormones in CHF, human recombinant TNF was injected 5-days systemically to achieve the levels observed following MI in conjunction with ROS and angiotensin II type-1 receptor blockers. These studies provide new evidence that TNF induces oxidative stress in the PVN through an AT1R mediated mechanism in CHF, and offers new insight into the sympathoexcitatory mechanisms in the brain possibly involved in the pathogenesis of CHF.

Nucleotide excision repair (NER) is a conserved DNA repair mechanism capable of removing a variety of helix-distorting lesions, such as UV-induced cyclobutane pyrimidine dimers (CPDs). NER can be grouped into two pathways: global genomic NER (GGR), which refers to repair throughout the genome, and transcription coupled NER (TCR), which refers to a repair mechanism that is dedicated to the transcribed strand (TS) of actively transcribed genes. In yeast S. cerevisiae, Rad7, Rad16, and Elc1 are specifically required for GGR. TCR is believed to be initiated by RNA polymerase II (Pol II) stalled at a lesion in the TS of a gene. Rad26, the yeast homolog of the human CSB protein, and RPB9, a nonessential subunit of Pol II, play important roles in TCR. However, the exact mechanisms of NER in eukaryotic cells are still elusive. By using yeast S. cerevisiae as a model organism, this dissertation focused on the functional mechanisms of transcription factor Tfb5, transcription elongation factors Spt4 and Spt5, and the putative yeast transcription repair coupling factor (TRCF) Rad26 in NER, especially in TCR pathway. Tfb5, the tenth subunit of the transcription/repair factor TFIIH, is implicated in one group of the human syndrome trichothiodystrophy (TTD). We found that Tfb5 plays different roles in different NER pathways in yeast. Tfb5 is essential for GGR and Rpb9 mediated TCR. However, Tfb5 is partially dispensable for Rad26 mediated TCR, especially in GGR deficient cells. Spt4 and its interacting partner Spt5 cooperatively suppress TCR only in the absence of Rad26, regardless of the presence of Rpb9. The phosphorylation of C-terminal repeat (CTR) domain of Spt5 by the Bur kinase plays an important role in the suppression. Immunoprecipitation results indicate that Rad26 dynamically associates with Pol II and restrains the binding of Spt4/Spt5 to Pol II. ATPase activity of Rad26 is required for facilitating TCR and for restraining the binding of Spt4/Spt5 to Pol II. Finally, we proposed that Rad26 enhances TCR by restraining the binding of suppressors Spt4/Spt5 to Pol II. These findings provide new insights into the functional mechanisms of Tfb5, Spt4/Spt5 and Rad26 in NER, especially in TCR.

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.

Posterior pedicle screw and rod fixation has become standard in the treatment of oncological resections requiring stabilization, deformity correction and unstable thoracolumbar fractures. Given the high mechanical stress at the points of highest instability, some clinicians have utilized dual rods on each side to augment the construct. The added advantage of this type of construct has not been previously evaluated in-vitro. The goal of this study is to evaluate the biomechanical advantage of a dual rod construct in the thoracolumbar spine, using a burst fracture cadaveric model. Methods: Seven fresh human cadaveric (T9-L3) spines were tested in normal conditions, after an iatrogenic T12 burst fracture, and successively after laminectomy and standard two rod pedicle screw stabilization (two level above and two below) and two different dual rod overlapping constructs. Pure moment torque was applied quasistatically, while 3D motion was measured optoelectronically. Thoracolumbar range of motion was measured during flexion, extension, left / right lateral bending, and left / right axial rotation. Results: All constructs significantly stabilized the simulated burst fracture in all modes of testing. There was no statistically significant difference, however, in the ability to restrict motion between the 3 different constructs, either from T10-L2, or across the fracture segment of T11-L1. Conclusions: There does not appear to be a biomechanical advantage to using dual rods over standard single rods for immediate fixation in an unstable segment. Whether dual rods protect the construct against long-term failure is not yet known.