Large, heavily scaled tomocerid springtails (Collembola) are abundant in eastern forests, and are important components of the detrital food web. The genus Pogonognathellus predominates in the southern Appalachians While a number of well-delimited tomocerid species have been described, others have vague morphological boundaries and appear to be species complexes. Before this study began in 2005, four species were known to occur in Great Smoky Mountains National Park (GSMNP) and the surrounding Appalachians (P. bidentatus, dubius, elongatus, flavescens). Also occurring throughout the area is Tomocerus lamellifera, a widespread and distinctive species. The goals of this project were to correlate scale patterns and colors, ground color, morphology, and DNA sequences for separation of species; to describe any new species detected; and to redescribe known species. Scale patterns and molecular sequences have not been previously studied in North American Tomoceridae. We collected 432 specimens from diverse localities and maintained them in culture containers. After a specimen molted, it was photographed to capture the pristine scale pattern and color, then preserved in 100% ethanol (EtOH). The preserved specimen then was re-photographed for ground color (most scales are dislodged in preservative). Selected specimens were dissected and the various appendages (legs, mouthparts, furcula) were slide-mounted for morphological analysis. The torso was used to obtain DNA, from which the 5’-3’ exoribonuclease II gene was amplified, sequenced, and analyzed phylogenetically. Freshly collected specimens of P. flavescens from the type locality (Sweden), preserved in 100% EtOH, were provided by Arne Fjellberg and included in the analysis. Tomocerus minor and Harlomillsia oculata (Oncopoduridae) were used as outgroups for the phylogenetic analysis. A phylogenetic tree based on the molecular sequences was used to indicate relationships that then were tested with morphological characters, chaetotaxy, scale pattern, and color. Putative P. flavescens from North America were not grouped with Swedish specimens, raising the possibility that P. flavescens does not occur in North America. Pogonognathellus nigritus, previously synonymized with P. elongatus, was determined to be a valid species, and P. elongatus itself was found to be mis-described in most accounts. Two new species were identified: one from a cave in GSMNP which is closely related to a California cave species, and a second from several forest localities (i.e. GSMNP and Mount Mitchell, NC). A group of specimens ascribed to the flavescens/dubius complex also appear to represent several undescribed species, but more specimens are needed for further analysis.

Thesis (Ph. D.)--Texas A&M University, 2005. / "Major Subject: Plant Pathology" Title from author supplied metadata (automated record created on Sep. 15, 2006.) Vita. Abstract. Includes bibliographical references.

Thesis (Ph. D.)--Oklahoma State University, 2006. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--Oklahoma State University, 2006. / Vita. Includes bibliographical references.

Amyotrophic lateral sclerosis (ALS) is a relentlessly progressive and fatal motor neuron disease, characterized by loss of motor neurons in the cortex, brainstem and spinal cord. Clinical management is plagued by a lack of biomarkers and effective treatment. In spite of numerous scientific advancements, molecular mechanisms involved in its initiation and progression remain an enigma. At the mechanistic level, ALS is considered multifactorial. Extracellular signals may modulate nuclear events with a possible consequence being the reactivation of cell cycle-related genes and protein alterations in the terminally differentiated motor neurons. In the first specific aim, we hypothesized that re-entry of post-mitotic motor neurons into the cell cycle, concurrent with altered activity or distribution of transcription factors will result in apoptosis of motor neurons during ALS. To address this hypothesis, we utilized archived human autopsy material from the cortical and spinal cord regions of ALS and age-matched control cases. We conclude that surviving ALS motor neurons in these regions exhibited increased levels of G1 to S phase regulators (Cyclin D1, CDK4, hyperphosphorylated -pRb and E2F-1). It also revealed two intriguing results: (i) E2F-1, a transcription factor, was cytoplasmic and (ii) increased nuclear p53 was noted in spinal motor neurons but absent in neurons of the motor cortex. In addition there was increased protein levels of apoptotic death markers (BAX, FAS, Caspases) and DNA fragmentation. Therefore we have identified a potential role for cell cycle proteins in an apoptotic mode of motor neuron death in ALS. In the second specific aim we hypothesized that a mass spectrometry-based proteomics approach will identify diagnostic biomarkers and molecular targets for drug discoveries. We used cerebrospinal fluid (CSF) from ALS and control subjects to identify and validate a biomarker panel specific to ALS. Furthermore, utilizing peptide map fingerprinting and tandem mass-spectrometry, we have identified three of the protein peaks to be a carboxyl-terminal fragment of neurosecretory chaperone protein 7B2 (3.44kDa), Cystatin C (13.3kDa) and monomer of transthyretin (13.78kDa). Taken together, this body of work furthers the understanding of both the mechanisms leading to selective motor neuron loss in ALS and paves the way for diagnostics and therapeutics.

Brain derived neurotrophic factor (BDNF) promotes cell survival, proliferation, differentiation, and enhances neurotransmission. In several neurodegenerative diseases, including Parkinsons disease (PD), BDNF mRNA and protein are altered in regions of pathology. The cellular response to BDNF is mediated by trkB. There are two trkB receptor isoforms abundantly expressed in the brain, full-length (fl) and truncated trkB (tc). TrkB.fl is a tyrosine kinase receptor that activates intracellular signaling cascades. Although the extracellular and transmembrane domains of trkB.tc are 100% homologous to trkB.fl, its intracellular domain is unique and lacks the catalytic amino acids. TrkB.fl functions as a signaling receptor while trkB.tc binds to and internalizes BDNF. The intracellular function of trkB.tc is unclear because it lacks the cytoplasmic signaling domain. The purpose of this dissertation was to identify a novel role for the truncated trkB receptor in mature neurons of the central nervous system. Our study had two goals: 1) to investigate the hypothesis that trkB.tc facilitates the endocytic sorting of BDNF and 2) to investigate the changes in trkB.tc protein distribution in PD. Finally, we correlated the changes in trkB.tc distribution in PD with its potential role in BDNF transport. Our organelle studies revealed co-localization of trkB.tc and internalized BDNF within endosomes, showing that the two proteins were transported as a complex. This protein complex is maintained within recycling endosomes. Although we did see co-localization of trkB.tc and internalized BDNF within lysosomes, it was not as extensive as the sorting of BDNF to recycling vesicles endosomes. Immunofluorescence studies of human autopsy striatum and substantia nigra revealed that trkB.tc and trkB.fl are differentially distributed in the control and PD brains. Furthermore, changes in the distribution of both trkB isoforms are seen in PD and correspond to regions of pathology. We conclude that upregulation of striatal trkB.tc in PD is an early response to neurodegeneration and regulates the effects of BDNF. In summary, trkB.tc facilitates the intracellular sorting of internalized BDNF to recycling endosomes. The altered distribution of trkB.tc in PD suggests enhanced trkB.tc transport, and potentially BDNF transport. This may enhance the neuroprotective effects of BDNF in PD.

Idiopathic pulmonary fibrosis (IPF) is a debilitating disease that involves a severe reduction in respiratory function, essentially culminating in the loss of the ability to sufficiently breathe. Current therapies are largely ineffective, and many of the molecular details of the pathogenesis of IPF remain unknown. Thus, new therapeutic targets need to be identified. We investigated a possible role for the receptor for advanced glycation end products (RAGE) in the pathogenesis of IPF. RAGE is a multiligand member of the immunoglobulin superfamily of cell surface receptors. It is generally associated with cellular perturbation in that RAGE-ligand interactions initiate a signaling cascade that ends with the activation of the pro-inflammatory transcription factor NF-kappaB. In most adult healthy tissues RAGE is expressed at low levels, but it is highly upregulated at sites of various pathologies, where it is thought to act as a propagation factor for disease. Notably, the exception to low RAGE expression in healthy adult tissues is the lung, the organ shown to have the highest RAGE transcript levels in humans and rats. This suggests that RAGE may have a role in lung homeostasis, implying that RAGE serves a function in the lung distinct from that in other tissues. RAGE has a secreted isoform, sRAGE, that acts as a decoy receptor. In humans, sRAGE is a product of alternative splicing. In contrast, we found that sRAGE in mice is produced by proteolytic truncation of cell surface RAGE. The focus of this project was to investigate the hypotheses that RAGE and sRAGE regulation are altered during the pathogenesis of pulmonary fibrosis, and that this alteration is a key step in the pathogenesis of pulmonary fibrosis. To address these hypotheses, we utilized the bleomycin and asbestos mouse models of pulmonary fibrosis as well as human IPF tissues. We found that in both animal models and in IPF tissues, RAGE and sRAGE protein levels are significantly reduced during the pathogenesis of pulmonary fibrosis. Observations from RAGE knockout mice suggest that absence of pulmonary RAGE is itself pro-fibrotic. These studies reveal RAGE as a novel therapeutic target in the pathogenesis of idiopathic pulmonary fibrosis.

Two major issues hinder the application of microarray based gene expression profiling in clinical laboratories as a diagnostic or prognostic tool. The first issue is the sheer volume and high-dimensionality of gene expression data from microarray experiments, which require advanced algorithms to extract meaningful gene expression patterns that correlate with biological impact. The second issue is the substantial amount of variation in microarray gene expression data, which impairs the performance of analysis method and makes sharing or integrating microarray data very difficult. Variations can be introduced by all possible sources including the DNA microarray technology itself and the experimental procedures. Many of these variations have not been characterized, measured, or linked to the sources. In the first part of this dissertation, a decision tree learning method was demonstrated to perform as well as more popularly accepted classification methods in partitioning cancer samples with microarray data. More importantly, results demonstrate that variation introduced into microarray data by tissue sampling and tissue handling compromised the performance of classification methods. In the second part of this dissertation, variations introduced by the T7 based in vitro transcription labeling methods were investigated in detail. Results demonstrated that individual amplification methods significantly biased gene expression data even though the methods compared in this study were all derivatives of the T7 RNA polymerase based in vitro transcription labeling approach. Variations observed can be partially explained by the number of biotinylated nucleotides used for labeling and the incubation time of the in vitro transcription experiments. These variations can generate discordant gene expression results even using the same RNA samples and cannot be corrected by post experiment analysis including advanced normalization techniques. Studies in this dissertation stress the concept that experimental and analytical methods must work together. This dissertation also emphasizes the importance of standardizing the DNA microarray technology and experimental procedures in order to optimize gene expression analysis and create quality standards compatible with the clinical application of this technology. These findings should be taken into account especially when comparing data from different platforms, and in standardizing protocols for clinical applications in pathology.

Pulmonary fibrosis is the collective name for disorders characterized by excessive deposition of interstitial collagen in the lung. Although the molecular mechanisms underlying pulmonary fibrosis are poorly understood, current evidence implicates both oxidant/antioxidant and protease/antiprotease imbalances in disease development. Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme protective against the development of pulmonary fibrosis in experimental models. EC-SOD localization in the lung is regulated by a heparin-binding domain conferring affinity for the extracellular matrix. This domain is susceptible to proteolytic removal, allowing EC-SOD to diffuse from the matrix. While the in vivo protease of EC-SOD has not been identified, it is known that members of the matrix metalloproteinase (MMP) family of proteases are upregulated in pulmonary fibrosis and could contribute to EC-SOD diffusion from the matrix. Furthermore, latent MMPs can be activated by oxidants, indicating that loss of EC-SOD from the matrix could lead to increased MMP activity. It was hypothesized that the depletion of EC-SOD from the lung and interrelated increase in MMP activity contribute to pulmonary fibrosis development. To examine this hypothesis, a mouse model of pulmonary fibrosis initiated by asbestos fibers (asbestosis) was developed. This injury caused depletion of EC-SOD from the lung parenchyma. Simultaneously, EC-SOD accumulated in the airspaces entirely due to release from airspace inflammatory cells. Depletion from lung matrices may be important since EC-SOD knockout mice develop worse inflammation and fibrosis after asbestos exposure. The metalloproteinases, MMP-2 and MMP-9, were upregulated after asbestos exposure. MMPs appeared to be important in asbestosis development, as global pharmacologic inhibition of MMPs decreased disease severity. MMP inhibition also reduced airspace EC-SOD accumulation, indicating a role for MMPs in EC-SOD localization. EC-SOD knockout mice treated with asbestos did not have significantly different MMP-2 and -9 activity compared to wild type mice. However, in bleomycin injury, knockout mice had increased airspace MMP-9, indicating a role for EC-SOD in the regulation of this protease. In summary, our data provides strong evidence for contributory roles for both EC-SOD and MMPs in the development of pulmonary fibrosis and additionally provides novel insights into EC-SOD regulation in the lung.

Prostate cancer is the most common cancer in males, and the second leading cause of cancer deaths in American men. Most of the mortality associated with this disease is a result of widespread dissemination of tumors cells from the primary tumor mass. In order for metastasis to occur, the cancer cell must overcome multiple barrier which include development, neovascularization, intravastion, adherence or attachment, extravasation, and ectopic growth. As dissemination from the primary tumor mass is a rate-limiting step during metastasis, tumor cells undergo an epithelial to mesenchymal transition (EMT) to acquire enhanced invasiveness and increased motility. A key step within EMT is a loss of cadherin mediated cell-cell adhesion. Unfortunately, current understanding of the regulatory mechanism of this decreased cell-cell adhesion is poorly understood. Herein this work utilizes the LHRH antagonist Cetrorelix to investigate the regulation of E-cadherin expression in invasive prostate cancer cells. We provide direct evidence that E-cadherin expression can be reinstated upon abrogation of EGFR signaling via LHRH antagonist Cetrorelix or specific inhibitors of EGFR signaling thereby limiting the invasiveness of these cells. In concert, we developed a microscale liver perfusion culture system that provides a tissue-relevant environment to assess metastasis behavior of human prostate cancer cell line DU-145 in the liver capillary bed as a model system. This system offers the currently unavailable features of real time observation of in vivo microenvironment with the manipulation of in vitro cultures. Within this system we were able to observe three dimensional growth and invasion of prostate cancer cells juxtaposed to hepatic tissue, revealing an exceptionally defined cell border at the interface of prostate cancer cells and hepatic tissue. Although not completed defined within this system, we hypothesize that exists heterotypic cell-communication between prostate cancer cells and hepatocytes. The very distinct cell border observed within our liver microreactor, coupled with our previous findings of reexpression of E-cadherin expression lead us to investigate the involvement of E-cadherin in this heterotypic communication. Consequently, prostate cancer cells utilize E-cadherin at the point of initial adherence to parenchymal hepatocytes (heterotypic interaction) and throughout the development of the metastatic tumor mass (homotypic interaction). Our observed expression pattern of E-cadherin has not been reported before. These findings constitute a new paradigm in the adhesiveness or lack there of in cancer cells during tumor invasion. The differentiation or redifferentiation (EMT) of the cancer cell during the pathophysiological events of metastasis is likely a characteristic of adaptability to the microenvironment. The term epithelial mesenchymal transition (EMT) only summates the dedifferentiation of epithelial cells to escape the primary tumor, although we have provided evidence of phenotypic reversion. Therefore we provide the impetus that Epithelial Mesenchymal Transition (EMT) should be renamed Meschenymal Epithelial reverting Transition (MErT) to underscore the dynamics of the cancer cell progression.