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.

The study of dermal wound healing has long been used to elucidate the cellular and molecular processes guiding the connective tissue response to injury. Of particular interest are the mechanisms by which soluble mediators, including inflammatory signals, guide fibroblast activity within the wound bed. This thesis addresses the role of prostaglandin E2 (PGE2) in the regulation of fibroblast activities relevant to restoration of tissue structure and function. Although PGE2 has been previously shown to play an important role in various wound healing steps, its precise contribution to the overall outcome of dermal repair is unclear. Using three well defined human dermal fibroblast phenotypes this study demonstrates that while PGE2 signaling during dermal repair triggers pro-inflammatory cascades, its effects on fibroblast activities are putatively anti-fibrotic. Specifically, exogenous PGE2 decreases the migratory and contractile potential of dermal fibroblasts through destabilization of the actin cytoskeleton and inhibits endogenous collagen synthesis. While PGE2 effects on fibroblast activity are largely conserved across phenotypes, fetal fibroblasts maintain a quantitatively diminished response to PGE2-induced alterations of cytoskeletal dynamics. Upon further analysis, this effect was shown to be representative of a larger intrinsic fibroblast phenotype. Fetal dermal fibroblasts were shown to maintain elevated rates of migration and contraction, as part of a generalized hyperactive dynamic state. Surprisingly, this phenotype was found to be sufficiently robust so as to persist despite changes in substrate and environmental constraints. In light of this finding, one additional approach was used to ascertain the robustness of the fetal fibroblast. Transplantation of fetal dermal fibroblasts into an adult wound environment was used to assess whether the intrinsic fetal fibroblast phenotype can survive the multitude of events comprising adult wound healing. While results are preliminary, this approach does present a useful tool for future studies aimed at elucidating the precise fetal fibroblast phenotype and its contribution to overall wound healing response.

Cell migration is a complex biophysical event that is dysregulated in a variety of human diseases including cancer. The ability of tumor cells to migrate enables cancer dissemination causing significant mortality thus making it an important therapeutic target. Motility is exhibited epigenetically by activation of numerous signaling pathways that transmit extracellular cues to the final effectors of cell movement. Such signaling switches are a part of larger and highly complex signaling (proteomic) networks that are under the control of numerous activators or inhibitors. Although majority of the proteins that are 'required' during cell motility have been identified, it is yet unclear wherein they fit within the signaling network to govern motility. Thus, a 'systems biology' approach is needed to understand the complex interplay of signaling cascades in mediating cell motility so that better therapeutic targets can be defined. We utilized a mathematical modeling approach, called decision tree analysis to map the interplay between five key signaling proteins known to regulate vital biophysical processes of fibroblast motility downstream of EGF receptor activation. Interestingly, our model identified myosin light chain (MLC) mediated cell contractility as a crucial node for maximal motility. Even more non-intuitively the decision tree model predicted that subtotal inhibition of MLC can actually increase motility. Confirmatory experiments with fibroblasts and cancer cells have shown that to be the case. Since the model proposed that total abrogation of contractility can limit cell migration, we asked if such an intervention can limit tumor invasion. Since PKCδ is implicated in EGF receptor mediated transcellular contractility, we abrogated PKCδ using pharmacological (Rottlerin) and molecular (RNAi) interventions. Such depletion of PKCδ reduced migration as well as invasiveness of prostate carcinoma cells predominantly by decreasing their contractility through myosin light chain (MLC). Additionally, activation of PKCδ correlated with human prostate cancer progression as assessed by immunohistochemistry of prostate tissue sections. In summation our studies illustrate the importance of quantitative (total versus subtotal) disruption of key signaling nodes in mediating a desired cell response. Novel computational modeling approaches are needed to identify newer molecular switches from existing proteomic networks that can be explored, using classical experimental methods, as therapeutic targets.

Legionella pneumophila (Lp.), the causative agent of Legionnaires’ disease, has been well known for infecting and replicating inside airway macrophages. Previous results from our laboratory indicated that Lp. attaches to non-polarized lung epithelial cells by binding and activating E-cadherin and β1-integrin. However, in the polarized alveolar epithelia these receptors are segregated from the apical cell surface by tight junctions (TJs). Our results showed that Lp. behaves as an opportunistic pathogen that efficiently targets disrupted, but not integrated epithelial cell monolayer. In such conditions, Lp. can reach its host-cell receptors and consequently invade and replicate intracellularly, producing large amount of bacterial progeny. These results and clinical evidence suggest the contribution of damaged airway epithelia to Legionellosis by providing a replication reservoir for the bacteria.

Legionella pneumophila (Lp.), the causative agent of Legionnaires’ disease, has been well known for infecting and replicating inside airway macrophages. Previous results from our laboratory indicated that Lp. attaches to non-polarized lung epithelial cells by binding and activating E-cadherin and β1-integrin. However, in the polarized alveolar epithelia these receptors are segregated from the apical cell surface by tight junctions (TJs). Our results showed that Lp. behaves as an opportunistic pathogen that efficiently targets disrupted, but not integrated epithelial cell monolayer. In such conditions, Lp. can reach its host-cell receptors and consequently invade and replicate intracellularly, producing large amount of bacterial progeny. These results and clinical evidence suggest the contribution of damaged airway epithelia to Legionellosis by providing a replication reservoir for the bacteria.

There is hardly a cellular process that is not regulated in some way by phosphoinositides, which makes biochemical and physiological studies of these lipids extremely important. PI 3-kinases are key regulators of phosphoinositide metabolism and have been shown to affect a large variety of cellular responses. The key products of PI 3-kinases that have functional activity in higher eukaryotic cells are PI(3,4,5)P₃ and PI(3,4)P₂. PI(3,4,5)P₃ is universally accepted as one of the most important second messengers in signal transduction. However, our knowledge of the functions of PI(3,4)P₂ as a lipid second messenger is much less precise. In this dissertation, work was undertaken to elucidate the regulation of PI(3,4,5)P₃ and PI(3,4)P₂ production and downstream signaling in B cells. Cells with membrane targeted exogenous SHIP were utilized to manipulate phosphoinositide levels. The relationship of PI(3,4,5)P₃ and PI(3,4)P₂ levels to downstream PKB phosphorylation and activation was studied. PI(3,4,5)P₃ and PI(3,4)P₂ levels were found to closely correlate with PKB phosphorylation levels at Thr308 and Ser473, respectively. In addition, PI(3,4)P₂ levels determine the PKB activity in the cytosol; while PI(3,4,5)P₃ levels determine PKB activity at the plasma membrane. Different doses and different forms of B cell receptor (BCR) agonists were used for stimulation. PI 3-kinase activation was studied carefully following stimulation with low doses of anti-BCR antibody and F(ab')₂ fragments. Low concentrations of F(ab')₂ fragments produced higher levels of PI(3,4,5)P₃ than did a high concentration of F(ab')₂ fragments. Downstream PKB signaling was studied in these models. Similar conclusions were drawn from both SHIP over-expressing BJAB cells and dose-dependent BCR stimulations. We speculated that phosphoinositides’ regulation of the kinetics of PKB phosphorylation at Ser473 and Thr308 might be mediated by additional proteins. Investigation of plasma membrane-associated PKB showed that it formed a protein complex of around 400KD, which we attempted to characterize further with respect to PKB phosphorylation and association with lipids. In conclusion, phosphoinositide production is intricately regulated in vivo to control downstream signaling. The levels of PI(3,4)P₂ and PI(3,4,5)P₃ have precise and profound effects on PKB and other molecules such as TAPP and Bam32. This study has contributed new insight into the PI 3-kinase signaling pathway from the aspect of phosphoinositide lipid function.