Gopher tortoise (Gopherus polyphemus) populations have experienced precipitous declines from habitat loss, and human and disease related mortality. The goal of this study was to characterize the health status of free-ranging Louisiana gopher tortoises. Gopher tortoises were collected during two distinct trapping seasons: fall (August-October 2002) and spring (April-June 2003). Captured tortoises were given a physical exam and the carapace and plastron length and width, weight, and body temperature were recorded. Blood was collected from the subcarapacial vein and submitted for the following testing procedures: complete blood count, plasma chemistry, infectious disease serology (Mycoplasma), and toxicologic screen (copper, mercury, zinc, and lead). Fifty-nine tortoises were captured during the study. Fifty-seven (97%) of the tortoises were adult animals and two (3%) were juveniles. Twenty (34%) of the tortoises were captured in the fall and thirty nine (66%) were captured in the spring. There were thirty male (53%) and twenty-seven female (47%) tortoises captured between the two trapping periods. The gender of the hatchling tortoises could not be determined. Complete blood counts, plasma biochemistry analyses and toxin screens were performed on fifty seven adult tortoises. There were several differences detected in the white blood cell count between trapping season. The white blood cell count and heterophil count were significantly higher in the spring compared to the fall. Significant gender differences were observed for potassium, calcium, and phosphorus. Marked seasonal variation was observed with glucose, uric acid, and GGT. Only creatinine kinase levels differed significantly between sites. There was a difference in the mercury and copper levels between gender. Only copper differed significantly between season. Overall, 26% of the tortoises were serologically suspect or positive for Mycoplasma agassizii. There was no difference in parasite shedding between gender, site of capture, or season. The parasites identified in these tortoises were consistent with findings in gopher tortoises throughout their range. In general, our findings suggest that these tortoises are in good health.

Osteoarthritis is arguably the most common ailment in both dogs and people in the developed World. Treatment for osteoarthritis is currently symptomatic. Development of therapies designed at stopping the progression of osteoarthritis, require a method of evaluating efficacy. Objective analysis by measuring joint metabolism via clinical trials of the cross-over design is currently not possible with the methods utilized. Extracellular matrix degradation is a hallmark of osteoarthritis. The matrix metalloproteinases are major degradative enzymes. There are currently no commercially available assays to measure canine matrix metalloproteinases. Human and canine matrix metalloproteinases are highly homologous. The use of antibodies and assays designed to detect human matrix metalloproteinases may also detect canine molecules. A commercially available human enzyme-linked immunosorbent assay (ELISA) was tested against canine samples without success. Casein zymography detected the presence of canine enzymes weighing the same as human matrix metalloproteinase 3 (MMP-3) and matrix metalloproteinase 13 (MMP-13). Inhibition of the casein degradation was achieved by the addition of ethylenediamine tetra-acetic acid (EDTA). The presence of a protein of the correct molecular weight with the ability to digest casein that is inhibited by EDTA is only circumstantial and does not definitively identify the enzymes. Enzyme-assisted immunoelectroblotting (Western blotting) utilizing human polyclonal antibodies was unsuccessful at positively identifying the canine molecule.

Endothelin-1 (ET-1) has been implicated in allergic type of respiratory inflammatory diseases in various species of animals including horses. This peptide elicits its actions by acting through endothelin-A (ET-A) and endothelin-B (ET-B) receptor sub-types. In this project, we have hypothesized that endothelin receptors (both ET-A and ET-B) are altered in terms of affinity and expression, in the lungs of summer pasture-associated obstructive pulmonary disease (SPAOPD)-affected horses. Objective of this dissertation was to determine the alterations in the affinity and expression of endothelin receptors in the lungs of healthy and SPAOPD-affected horses. To pursue our hypothesis, we have employed pharmacological, immunohistochemical and molecular studies. Totally 33 horses were used in this study. All the horses were examined and grouped in to 16 healthy and 17 SPAOPD-affected, based on clinical evaluation, clinical scoring, pulmonary function testing and broncho-alveolar lavage fluid (BALF) analysis. Horses were then euthanatized, and tissue specimens were immediately collected from all lung lobes. In pharmacological studies, cumulative concentration response curves and pA2 values were determined and compared in both groups of horses. The pA2 value of ET-B receptors was significantly greater in the SPAOPD-affected horses when compared with healthy horses. In immunohistochemical studies, expression of these receptors was determined in the bronchial smooth muscles and epithelium of both groups of horses. The percentage of immunostaining was significantly greater in the smooth muscles of SPAOPD-affected horses when compared with healthy horses. In molecular studies, by employing RT-PCR, western blotting and realtime PCR, expression of these receptors has been determined and compared. The molecular expression of ET-B receptors was siginifcantly greater in the peripheral lungs of SPAOPD-affected horses when compared with healthy horses. Specific antagonists, primers and polyclonal primary antibodies of ET-A or ET-B receptors were used for these three studies. On the whole, it can be concluded that ET-B receptors show a clear tendency of up-regulation in the lungs of SPAOPD-affected horses. These findings help us in the better understanding of the pathogenesis of this progressive, career-ending disease.

In the last twenty years, interferon-α (IFN-α) has gained success as an immunotherapy for treating such cancers as hairy cell leukemia, malignant melanoma, and renal cell cancer. Our goal was to improve the effectiveness of IFN-α therapy by genetically modified the IFN-α gene to encode a tumor-targeting peptide fused to a functional IFN-α protein. To ensure the targeting peptide worked, a genetically modified reporter gene encoding a secreted alkaline phosphatase (SEAP) gene and different mini-peptides were used to determine distribution and targeting ability. The DNA fragment encoding the most effective peptide was selected to modify the IFN-α gene construct for therapeutic trials. This fusion gene encoded the peptide with the amino acid sequence of C-D-G-R-C, and demonstrated a higher localization of the genetically modified gene product in the tumor local area. Tumor volume and animal survival was measured over several weeks to compare the anti-tumor effects of the IFN-α to CDGRC-IFN-α treatments. Results indicate an increase in therapeutic efficacy due to treatment with the CDGRC-IFN-α gene over the wild-type IFN-α gene. Flow cytometry was performed and it was determined that both of the tumor targeted gene products, CDGRC-SEAP and CNGRC-SEAP share a high affinity for the receptor, Aminopeptidase N (CD13). In order to determine the mechanism responsible for the enhanced anti-tumor effect by CDGRC-IFN-α gene therapy, the T cell infiltration, subsequent CTL activity, and tumor vessel density were confirmed through immunostaining. An increase in number of CD8⁺ T cells was seen, as well as an increase in activity of cytotoxic T cells. Decreased vessel density in CDGRC-IFN-α treated animals suggest that this therapy enhanced anti-angiogenisis. A high level of non-specific activity was detected in the CTL assay, suggesting involvement of other immune cells, such as NK cells. Overall, this study describes the first example of using a genetically modified immunostimulatory gene encoding tumor-targeted IFN-α for treating tumors. This novel concept may have the potential for increasing therapeutic efficacy of several current cancer treatments.

Cancer metastasis is a frequent manifestation of malignant melanoma progression. Successful invasion into distant organs by tumor cells must include attachment to microvessel endothelial cells, and degradation of extracellular matrix. Heparan sulfate proteoglycans are ubiquitous macromolecules associated with cell surface and extracellular matrix of a wide range of cells and tissues. Heparanase is an extracellular matrix degradative enzyme which degrades the heparan sulfate chains of heparan sulfate proteoglycans. To investigate effects of changes in heparanase gene expression in metastatic melanoma cells, we constructed adenoviral vectors containing the full-length human heparanase cDNA in both sense (Ad-S/hep) and anti-sense orientations (Ad-AS/hep). We demonstrated increased heparanase expression and activity in melanoma cell lines following Ad-S/hep infection by Western blot analyses and heparanase activity assay. Conversely, heparanase content was significantly inhibited following infection with Ad-AS/hep. Alteration of heparanase protein expression by these adenoviral constructs correlated with invasive cellular properties in vitro and in vivo. Unexpectedly, overexpression of heparanase inhibited brain tumor formation in vivo possibly by extensive remodeling of the extracellular matrix which in turn modifies growth factor signaling and activity. Finally, cell-surface heparan sulfate is also known to inhibit or promote tumorigenesis depending on size and composition. We proposed that heparanase generates bioactive heparan sulfate chains from the melanoma cell-surface that modify biological activities associated with vascular endothelial growth factor, a molecule essential for brain metastasis. Heparanase-degraded melanoma cell-surface heparan sulfate stimulated migration, but not proliferation of melanoma in vitro. It also enhanced angiogenesis in vivo, independent of vascular endothelial growth factor activity, an unexpected finding. Interestingly, melanoma cell-surface heparan sulfate did not have an observed effect on endothelioma migration in vitro. We also attempted to characterize the melanoma cell-surface heparan sulfate isolated by heparanase degradation by ion-pair high pressure liquid chromatography. This method proved to be not sensitive enough to detect nanogram quantities of HSGAG present in our samples.

Environmental contamination with airborne particles has been a human health concern for many years. Epidemiologic studies in urban communities have linked ambient particle exposure to various health effects, including chronic obstructive pulmonary disease, lung cancer, and several cardiovascular disease conditions. The pathogenesis of these conditions with respect to ambient particle exposure is complex because ambient particles are complex in composition. The particles vary greatly in origin, size, surface area, and elemental composition; and a given particle type, such as those generated by petrochemical (gasoline, diesel, industrial substrate) combustion, may be coated with many other compounds, including polynuclear aromatic hydrocarbons (PAHs). Our laboratory group had previously characterized the generation of PAHs from incomplete combustion of the high volume petrochemical 1,3-butadiene (BD) and briefly described the biological effects of BDs incomplete combustion product, butadiene soot (BDS), in vitro. The studies presented here represent a continuation of these initial studies, where we first characterize BDS with respect to particle size distribution and assembly, PAH composition, and elemental content of BDS ultrafine particles. We also describe in vitro assays demonstrating that BDS ultrafine particles can transport and transfer adsorbed organic constituents directly to target respiratory cells, without uptake of the particles by the cells. Next, we demonstrate that combustion-derived PAHs adsorbed onto BDS particles are concentrated in lipid droplets of respiratory system cells and that, in vitro, these PAHs activate xenobiotic metabolism pathways. We also present an in vivo analysis of bronchoalveolar lavage fluid (BALF) with inflammatory cell infiltrates, histopathological evidence of inflammation and particle retention, and gene expression analysis revealing upregulation of several cytokines and AhR-responsive biotransformation enzymes. Finally, we present ultrastructural evidence that BDS particles can be internalized by bronchoepithelial cells in vitro and phagocytosed by alveolar macrophages in vivo. These studies were designed to characterize and promote BDS as both a model mixture and a real-life example of a petrochemical product of incomplete combustion with the potential both for environmental contamination and for contributing to health problems.

The ABC transporter P-glycoprotein (Pgp) and membrane permeability are determinant factors for absorption and disposition of xenobiotics. These studies investigated the effects of potential modulators of Pgp and/or membrane function on the disposition, bioavailability and bioaccumulation of environmentally relevant pharmaceuticals, tetracycline and ivermectin, and the carcinogen benzo[a]pyrene in catfish. The pesticide dieldrin and surfactant linear alkylbenzene sulfonate (LAS) were selected as mixture components thought to have potential interactions with these determinants of disposition. Initial in situ experiments demonstrated dieldrin and tetracycline both significantly inhibited biliary excretion of Pgp prototypic substrate Rhodamine-123 in isolated perfused livers. Further, dieldrin (20µM) reduced movement of 3H-tetracycline (8.8nM) into bile (55%) to a greater extent than Pgp prototypic competitive inhibitor verapamil. In contrast to inhibitory effects in situ, a 4-week dietary dieldrin preexposure (0.1mg/day/kg body weight) increased plasma clearance (17%) and reduced tissue concentrations of 3H-tetracycline equivalents (parent and metabolites) for a single intra-aortic administration of 3H-tetracycline (8.31µg/kg body weight) in catfish. A 23% increase in the immunoreactive Pgp level in the hepatic membranes following chronic dietary dieldrin exposure was correlated with in vivo changes in disposition. Additional in situ studies demonstrated LAS treatments (1, 5, and 20µM) reduced movement of Rhodamine-123 (1µM) into bile in isolated perfused livers (18.6, 38.1 and 66.7%, respectively). Fluorescent anisotropy measurements of the corresponding hepatic membranes showed a 29.7% decrease (increase in membrane fluidity) at the 1µM LAS concentration, with little additional change evident at higher concentrations. In sequential in vivo experiments, following six daily diet administrations of 3H-ivermectin (10 µg/day/kg body weight) or 3H-benzo[a]pyrene (40 µg/day/kg body weight) starting on day 7 during a twelve-day waterborne LAS exposure (0, 100 and 300µg/l), 3H-ivermectin and 3H-benzo[a]pyrene equivalent remaining in catfish and their blood and tissue concentrations increased in a dose-dependent fashion with increasing LAS concentrations. The first study indicates dieldrin inhibited Pgp transport at the high concentration with the inductive effect upon Pgp expression predominating at the low concentration exposures in vivo. Findings of the second study suggest that LAS at environmental concentrations altered membrane permeability and/or transporter function so to increase bioaccumulation of other xenobiotics from the diet.

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.