Terminal uridylation of RNA 3ꞌ ends has recently been recognised as an important biological regulatory mechanism. The human terminal uridyl transferase ZCCHC11 was shown previously to uridylate and thereby regulate replication-dependent histone mRNAs and several miRNA precursors and mature miRNAs. Studies in cancer cell lines, mouse models and patient samples suggest that ZCCHC11 is a potential oncogene. However, very little is known about regulation of its expression and subcellular localisation. In this study, the regulation of the expression of ZCCHC11 was investigated, especially in relation to the roles played by the untranslated regions of its mRNA. The second aim of this study was to investigate the subcellular localisation of ZCCHC11, both under physiological conditions and under cellular stress. The results presented show that ZCCHC11 expression is regulated negatively through its 5ꞌ and 3ꞌUTRs. Upstream open reading frames in the 5ꞌUTR decrease its translation, while regulation through the 3ꞌUTR involves the combined activity of positive and negative factors. In this study it was shown that HuR positively regulates ZCCHC11 expression, whereas TTP contributes to its negative regulation. An homologous enzyme, ZCCHC6, negatively regulates ZCCHC11 expression. Regulation through the 3ꞌUTR of ZCCHC11 is particularly marked following S-phase arrest. ZCCHC11 expression increases under cellular stress and both UTRs take part in this regulation. Regulation under cellular stress also involves an alteration in sub-cellular localisation. ZCCHC11 is predominantly cytoplasmic in unstressed cells, but re-localises to p-bodies and stress granules under cellular stress. Mutating one of three zinc knuckle motifs within ZCCHC11 significantly decreased differential co-localisation with stress granule marker TIA-1 under oxidative stress. Taken together, the data presented provide insights into the post-transcriptional regulation of this post-transcriptional regulatory protein and its subcellular localisation, especially under cellular stress.

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.

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.

Many signals involved in pathophysiology are controlled by hypoxia-inducible factors (HIFs), transcription factors that induce expression of hypoxia-responsive genes. HIFs are highly conserved master regulators of oxygen homeostasis. These factors are post-translationally regulated by a family of oxygen-dependent HIF hydroxylases, whose members include four prolyl 4-hydroxylases (PHD1-4) and an asparaginyl hydroxylase (FIH-1). All HIF hydroxylases require molecular oxygen, Fe(II), ascorbate, and 2-oxoglutarate as cofactors. We hypothesized that alterations in subcellular localization may provide an additional point of regulation for the HIF pathway in response to hypoxia. Most of these enzymes are abundant in resting liver, an organ which is itself unique due to its physiologic oxygen gradient, and they can exist in both nuclear and cytoplasmic pools. In this study, we analyzed the localization of endogenous HIFs and their regulatory hydroxylases in primary rat hepatocytes cultured under hypoxia-reoxygenation conditions. We observed an absence of nuclear HIF-1á activation in hypoxic hepatocytes, even though several known HIF target genes were upregulated, suggesting that HIF-2á and HIF-3á are the predominant isoforms in liver. We show that in hepatocytes, hypoxia-reoxygenation targets HIF-1á to the peroxisome rather than the nucleus, where it co-localizes with the von Hippel Lindau protein (VHL) and the HIF hydroxylases. Confocal immunofluorescence microscopy demonstrated that the HIF hydroxylases can translocate from the nucleus to the cytoplasm in response to hypoxia, with increased accumulation in peroxisomes upon reoxygenation. These results were confirmed via immuno-transmission electron microscopy and Western blotting. Surprisingly, in resting liver tissue, peri-venous localization of the HIF hydroxylases was detected, consistent with areas of low oxygenation. This was in contrast to nuclear HIF-1á, which was undetectable in a number of liver injury models. In conclusion, these studies establish the peroxisome as a highly relevant site of subcellular localization and function for the endogenous HIF pathway in hepatocytes.