<p> Coordinated movement of cardiac valves controls unidirectional flow of the blood with every heart beat. Cardiac valves are composed of thin, pliable leaflets that withstand compressive tension, fluid shear stress, and bending stress as blood flows through them. The structure and the mechanical properties of the valves render them durable during the lifetime of human beings. However, changes in hemodynamic environment, inflammatory responses, and congenital valvular defects can all cause valves to undergo irreversible structural changes, one of which is calcific aortic stenosis (CAS). CAS affects 2-3% of the population over 65 years old in the western world, and the only effective treatment is valve replacement surgery. CAS is characterized by tissue stiffening and the formation of calcified nodules, the development of which is associated with abnormal differentiation of resident fibroblasts known as valvular interstitial cells (VICs). Upon tissue injury, VICs are activated to myofibroblasts which deposit excessive collagen and stiffen the matrix. Understanding how the pathogenic phenotype of VICs is regulated by cues from the matrix may lead to new therapeutic treatments for CAS. In this thesis, I examined how matrix elasticity and TGF-&beta;1 regulate VIC phenotypes. First, I characterized the VIC population from porcine aortic valves and showed that this population is relatively homogeneous. When I cultured these primary cells on different substrates, I found that poly(ethylene glycol) hydrogels mimicked the native valve matrix better than tissue culture polystyrene plates with respect to preserving the quiescent fibroblast phenotype. At the level of signaling, I demonstrated that this is mediated through an elasticity-regulated PI3K/AKT pathway. Additionally, I showed that reduced matrix rigidity redirected activated valvular myofibroblasts into dormant fibroblasts without inducing significant apoptosis. Finally, I examined the effect of TGF-&beta;1 on VIC gene expression over time with microarray-based gene expression profiling and found that TGF-&beta;1 up-regulated cell-cell contact proteins (e.g., OB-cadherin, N-cadherin) in order to regulate valvular myofibroblast activation. Collectively, my thesis work revealed novel mechanosensing mechanisms employed by VICs to respond to matrix elasticity and explored the complex interactions among multiple extracellular cues, including matrix elasticity, TGF-&beta;1 and cell-cell adhesion, to direct the cellular fate of VICs.</p>

<p> One of the greatest problems in prostate cancer management today is accurate identification of patients who require treatment for aggressive disease versus those with indolent disease who are suitable for observational strategies. Histological appearance of the tumor, called Gleason score in the prostate cancer field, is the most predictive measure currently used. However, recent studies in multiple tumor types have shown that histological appearance does not always reflect the underlying molecular phenotype of the lesion. Therefore, in prostate cancer specifically, assessment of a molecular marker of androgen receptor driven epithelial differentiation may have clinical predicative capabilities. SAM pointed domain-containing Ets transcription factor (SPDEF) is a potential AR target gene that has shown to be necessary and sufficient for epithelial cell differentiation in many tissues. Although generally associated with good prognosis, SPDEF's role in cancer in unclear. This study demonstrates, through retrospective immunohistochemical analysis, the utility of SPDEF as a predictive biomarker for patients that have an extended benefit from androgen deprivation therapy (ADT). Furthermore, dual roles of SPDEF to inhibit the initiation and supporting the progression of castrate recurrent disease through novel androgen receptor expression regulation in castrate conditions are shown. In ADT na&iuml;ve patients, SPDEF did not associate with metastatic disease or an induction of epithelial to mesenchymal transition. However, aggressive tumors tended to be larger, have greater SPDEF variability, and lack vimentin expression; a phenotype that could be explained by a partial EMT. In conclusion, SPDEF may be clinically useful to assess the epithelial phenotype of tumors, and could have utility identifying patients that will respond well to androgen deprivation therapy.</p>

<p> Recombinant adeno-associated virus (rAAV) is a widely used gene therapy vector. Although a wide range of rAAV serotypes can effectively enter most cell types, their transduction efficiencies (i.e., transgene expression) can vary widely depending on the target cell type. Integrins play important roles as co-receptors for rAAV infection, however, it remains unclear how integrin-dependent and -independent mechanisms of rAAV endocytosis influence the efficiency of intracellular virus processing and ultimately transgene expression. In this thesis, I examined the contribution of integrin-mediated endocytosis to transduction of fibroblasts by rAAV2. I found that promoting AAV2/integrin binding with Mn⁺⁺ greatly enhanced (~17-fold) rAAV2 transduction independently of cell binding and endocytosis. Subcellular localization studies of rAAV2 demonstrated that integrin activation by Mn⁺⁺ promoted AAV2 aggregation on &alpha;5 and &beta;1 integrins and recruitment of the cytosolic integrin effector protein vinculin. Focal adhesion kinase (FAK), a down stream effector of integrin signals, was essential for AAV/integrin complex endocytosis and transduction, but not AAV2 recruitment to integrins. Recruitment of FAK to AAV2/integrin complexes was increased by transiently trapping the endocytic event at the plasma membrane by pharmacologic inhibition of dynasore. This also increased the size of AAV2 clusters found beneath the cell at FAK/integrin complexes resembling immature filopodia and caused a large, FAK-dependent (75-fold) increase in AAV2 transduction. These findings support a model whereby integrin activation at the cell surface can redirect rAAV2 toward a FAK-dependent entry pathway that is more productive for cellular transduction. This pathway appears to be conserved for other rAAV serotypes that contain a capsid integrin-binding domain (AAV1 and 6).</p>

<p> Herpes Simplex Virus Type 1 is a member of the <i>alphaherpesvirinae </i> subfamily within the family <i>Herpesviridae</i>. This virus has both a lytic and latent cycle. Primary infection occurs when the virus enters epithelial cells around the mucosal lining of the nose and mouth. Within the epithelial cells, the virus undergoes an active lytic infection, causing an ulcerated blister, more famously known as a 'cold sore' or 'fever blister'. Once HSV enters the nearby sensory neurons the genome is transported to the neuronal cell body where its latency associated transcripts are activated and the virus remains in a dormant latent cycle until reactivation, when the virus is transported back down the axon to the epithelial cells at or near the site of initial infection. The Virion Host Shutoff protein is a tegument protein from HSV1 and acts as a ribonuclease, degrading both cellular and viral mRNAs, making the course of viral infection more efficient. A study by Saffran, Read and Smiley uncovered an unexpected new function of Vhs: stimulation of translation from some IRESs. An IRES is a section of mRNA with a high level of secondary structure, capable of inducing cap-independent translation. In similar experiments utilizing a bicistronic reporter transcript, I sought to discover whether or not these two functions of the Vhs protein could be de-coupled. Experiments involved dually transfecting HeLa cells with different Vhs mutants across a range of Vhs plasmid concentrations and the bicistronic reporter construct. Levels of reporter activity were measured from cell lysates 36 hours after transfections and provided a measurement of the control at the level of translation. As the cellular Bip IRES element was present between the cistrons, the 3' cistron provided a measure of IRES stimulation. The Results revealed examples of Vhs mutants in which the two activities had been separated. It is unknown what role IRES stimulation could play during Herpesvirus infection, although it is interesting to note that some HSV1 genes have IRES like elements within the 5' UTR. Future experiments can be done to investigate whether or not Vhs is actively recruiting transcription initiation factors to these IRES elements.</p>

<p> Feline oral squamous cell carcinoma (SCC) is a devastating disease that responds poorly to traditional treatment modalities. The tumor location directly impacts the patient's ability to eat and drink, and immediate intervention to alleviate clinical signs is important. To design better treatment strategies it is paramount to understand the underlying biological behavior of this poorly defined tumor. This research takes a comprehensive approach in attempt to understand this disease. A number of assays have been developed and applied to elucidate underlying biology. New imaging modalities have been used to better stage the disease and define tumor location. Finally, patients were treated with a new radiation therapy modality, stereotactic radiation therapy (SRT), and outcome was correlated with the biological assays for potential predictive value. </p><p> The goal of the prospective study described in Chapter 2 was to compare gross tumor volume measurements using ¹⁸F-FDG PET vs. those using computed tomography (CT) for SRT planning in cats with oral SCC. Twelve cats with confirmed oral SCC underwent pretreatment ¹⁸F-FDG PET/CT. Gross tumor volumes based on contrast-enhanced CT and ¹⁸F-FDG PET were measured and compared between cats. Mean PET gross tumor volume was significantly smaller than mean CT gross tumor volume in the mandibular/maxillary SCC group (n=8, <i>P</i>=0.002) and for all cats (n=12, <i> P</i>=0.006), but not for cats with lingual/laryngeal SCC (n=4, <i> P</i>=0.57). Mismatch fraction analysis revealed that most of the lingual/laryngeal patients had a large region of high-¹⁸F-FDG activity outside of the CT gross tumor volume. This mismatch fraction was significantly greater in the lingual/laryngeal group than the mandibular/maxillary group (<i> P</i>=0.028). The effect of poor spatial resolution of PET imaging was greater when the absolute tumor volume was small. Findings from this study indicated that ¹⁸F-FDG PET warrants further investigation as a supplemental imaging modality in cats with oral SCC because it detected presumed regions of primary tumor that were not detected on CT images.</p><p> For canine and feline patients with tumors in the head region, simultaneous irradiation of the primary tumor and mandibular and retropharyngeal lymph nodes (LNs) is often indicated. The purpose of this study described in Chapter 3 was to assess the reliability of a planning target volume (PTV) expansion protocol for secondary targets (LNs). </p><p> Information about the molecular biology of feline oral SCC is still limited. In Chapter 4, 22 archived tumor samples of feline oral SCC were evaluated to develop immunohistochemical assays and to determine if there was correlation to clinical parameters. Immunohistochemistry for Ki67, MVD, and EGFR was performed and scored. Patient survival information was obtained from the medical records. These molecular markers as well as MI were correlated with tumor locations and patient survival time. The 22 tumors showed wide variation in Ki67, MI, MVD, and EGFR. Tongue SCC expressed higher MVD than mandibular/maxillary SCC (<i>P</i>=0.088). </p><p> Cancer stem cell or tumor initiating cell (TIC) theory and telomere biology are actively studied fields in human head and neck (H&amp;N) cancer. In feline oral SCC, which has been advocated as a feline model for human H&amp;N cancer, our knowledge about the TIC and telomere/telomerase biology is limited. Protein expression levels of putative TIC markers of human H&amp;N cancer, CD44 and Bmi-1, were immunohistochemically evaluated for their possible role as prognostic markers in 20 patients with feline oral SCC who underwent SRT. This patient population was part of a clinical trial and information relevant to PFI and ST was available. A combined technique of fluorescent in-situ hybridization and immunofluorescent staining was used to determine telomere length ratio (fractions of very short telomere/average length telomere in the putative cancer stem cells) in the putative TICs that were positive for CD44 and Bmi-1. This was also correlated with treatment outcome. (Abstract shortened by UMI.) </p>

<p> Adult multipotent stromal cells (MSCs) may repair tissue through the action of secreted factors on endogenous stem/progenitor cells. We determined the effects of MSC-secreted factors on adult cardiac progenitor cells (CPCs). Serum-free conditioned medium (CdM) was collected from MSCs isolated by plastic adherence (MSCs) and by magnetic sorting against the p75 nerve-growth factor receptor (p75MSCs). Compared to serum-free medium (&alpha;-MEM), CdM significantly increased adult rat CPC proliferation in a concentration-dependent manner, led to phosphorylation (Tyr⁷⁰⁵) and nuclear localization of signal transducer and activator of transcription 3 (STAT3) and was blocked by both AG490, a Janus kinase 2 (Jak2)/STAT3 inhibitor, and Stattic, a specific STAT3 (Tyr⁷⁰⁵) inhibitor. Also signaling through Jak2/STAT3, MSC CdM cytoprotective factors significantly increased survival of hypoxic CPCs compared to &alpha;-MEM. Intra-arterial infusion of p75MSC CdM 24 hours after myocardial infarction (MI) in mice significantly reduced myocardial necrosis at 48 hours after MI compared to &alpha;-MEM (vehicle). Echocardiography at 1 week after MI demonstrated significantly better cardiac function in p75MSC CdM-treated mice compared to controls. Thus <i>in vivo</i> benefits of MSCs may be derived in part by the action of their secreted factors on CPCs. </p><p> Epicardial-derived cells are required for cardiac development, support myogenesis through secreted factors and participate in repair and remodeling after injury. We tested whether factors secreted by epicardial-derived precursor cells (EPDCs) would protect jeopardized ischemic myocardium after myocardial infarction and reperfusion (MI-I4R). Human epicardial progenitor cells, isolated from right atrial appendages removed during cardiac bypass surgery, were keratin-positive, epithelial in morphology and expressed TFs associated with pro-epicardium, epicardium and cardiac development. Upon progenitor cell epithelial-mesenchymal transition (EMT) into EPDCs, concentrated conditioned medium (EPI CdM) was generated. When compared to &aacute;-MEM (vehicle), intra-arterial infusion of human EPI CdM led to a reduction in infarct size of 50% in both immunodeficient and immunocompetent MI-I4R mice and improved cardiac function. These <i> in vivo</i> results were evident as early as 24 hours after MI, sustained for at least 1 month, and may derive in part through paracrine protection of jeopardized coronary microvasculature. Our results indicate that EPI CdM or a combination of its ligands may provide an effective treatment for MI.</p>

<p> The human skeleton is a dynamic organ that serves multiple functions to maintain normal physiology and health. It protects vital organs, provides support for movement, houses marrow and maintains calcium homeostasis. The skeleton is maintained by the work of two cells with opposing functions: osteoblasts, cells that synthesize organic bone matrix and osteoclasts that degrade and resorb it. These cells interact with one another in a tightly regulated process known as the bone remodeling cycle. This cycle maintains the health of bone by removing and replacing weak or damaged bone and responding to stress loads by remodeling portions of the skeleton that require reinforcement. Osteoblasts differentiate from mesenchymal stem cells and respond to hormonal stimuli by synthesizing and secreting cytokines necessary for osteoclast differentiation. Osteoblasts may become embedded within mineralized matrix, becoming osteocytes, cells that can sense changes in mechanical loading and facilitate localization of the remodeling cycle. Osteoclasts differentiate from hematopoietic stem cells (HSC) when the cell surface receptors, c-FMS and RANK, are activated by ligands produced by osteoblasts, M-CSF and RANKL respectively. In addition to c-FMS and RANK stimulation, another calcium-mediated, co-stimulatory pathway must be activated to ensure proper osteoclast differentiation. This pathway is activated by two immunoreceptors, OSCAR and TREM-2 that interact with adaptor proteins termed FcR&gamma; and DAP12 respectively. These adaptor proteins harbor immunoreceptor tyrosine-based activation motifs (ITAM), which exist on their cytoplasmic tail. Once the immunoreceptors are triggered, specific tyrosines within the ITAM motifs become phosphorylated and act as docking points for the tyrosine kinase, Syk. Once bound, Syk autophosphorylates and acts on its downstream targets. Syk dephosphorylation is, therefore, necessary to attenuate this signal to prevent over activation of osteoclasts. Recently, a novel tyrosine phosphatase, T-cell Ubiquitin ligand -2 (TULA-2) has been shown to dephosphorylate specific phosphotyrosine residues on Syk in various systems and has shown an increased specificity to dephosphorylate tyrosine 352. The goal of this project is to determine how TULA-2 mediated dephosphorylation of Syk regulates osteoclast differentiation and function. TULA-2 is a member of the TULA family of proteins, TULA and TULA-2. In spite of a significant homology and similar domain organization between TULA and TULA-2, only TULA-2 has significant phosphatase activity. Furthermore, whereas TULA is expressed only in lymphocytes, TULA-2 is expressed in most tissues albeit a higher level of expression is seen in cells of hematopoietic origin. <i>In vivo</i> analysis including Micro-computed tomography (Micro CT) and histomorphometry indicated that mice that lack both TULA and TULA-2 (DKO) have decreased bone mass compared to wild-type (WT) counterparts. An <i> in vitro</i> cell differentiation assay revealed that a larger population of osteoclast-like cells (OCL) could be cultivated from bone marrow isolated from DKO mice compared to OCL derived from WT bone marrow. An <i>in vitro</i> resorption pit assay revealed that DKO osteoclasts could resorb bone at a faster rate than WT counterparts. Additionally, over-expression of phosphatase-dead TULA-2 in WT osteoclasts increased the ability of the cells to resorb bone. At the molecular level, activation of the co-stimulatory pathway revealed increased tyrosine phosphorylation of Syk 352 in DKO pre-osteoclasts when compared to phosphorylation of Syk isolated from WT pre-osteoclasts. Cumulatively, the above data indicates that the absence of TULA-2 results in an increased signaling response leading to a larger population of hyperactive osteoclasts, which contributes to decreased bone mass in mice. These data suggest that the phosphatase activity of TULA-2 is required for negative regulation of bone resorption.</p>

<p> Ischemic heart disease impacts millions worldwide and can progress to heart failure. Percutaneous coronary intervention (PCI) is first-line therapy for patients presenting with an acute ischemic event or acute coronary syndrome (ACS). However, PCI can also worsen cardiomyocyte death, cardiac dysfunction and adverse remodeling via reperfusion injury, largely an oxidative stress-mediated insult. Novel alternative therapies for ACS have proven elusive, with no new classes of agents in years. We investigated cardiac troponin I-interacting kinase (TNNI3K), a cardiomyocyte-specific kinase, as a potential modulator of ischemia/reperfusion (I/R) injury and chronic left ventricular (LV remodeling). We found TNNI3K enhances production of mitochondrial reactive oxygen species (mROS) and induces mitochondrial dysfunction, thus increasing cardiomyocyte death and I/R injury. Moreover, TNNI3K-mediated injury is largely dependent on p38 MAPK activation. We developed a series of small-molecule TNNI3K inhibitors that reduce mitochondrial-derived superoxide generation, p38 activation, and infarct size when delivered at reperfusion to mimic ACS intervention. Moreover, although TNNI3K inhibition does not modulate the adverse remodeling that occurs after a non-reperfused myocardial infarction (MI), TNNI3K inhibition preserves cardiac function and limits chronic adverse remodeling in a model of MI with reperfusion. Taken together, TNNI3K plays an adverse role in the cardiomyocyte response to I/R, in part by driving mROS production and augmenting p38-mediated cell death specifically via reperfusion injury. Our findings reveal a previously unexplored role for TNNI3K in regulating the oxidative stress response in the heart, and support the potential for TNNI3K as a novel therapeutic target for ACS.</p>

<p> This dissertation examines the role of retinoic acid receptor activation in the post-transcriptional regulation of a fibroblast growth factor binding protein. Previous work showed that all-trans retinoic acid (ATRA) reduces mRNA expression of the angiogenic switch molecule, Fibroblast Growth Factor Binding Protein 1 (FGFBP1 or FGF-BP), independent of an effect on transcription of the FGFBP1 mRNA. I hypothesized that a retinoid-induced microRNA was involved in FGFBP1 mRNA loss. MicroRNAs (miRs) are 19-22 nucleotide (nt) single stranded non-coding RNAs that post-transcriptionally repress mature mRNA function, thereby reducing expression of their target proteins. The current dogma suggests that miRs canonically bind to the 3' untranslated region (UTR) of mRNA through a 7-nt seed-matched site. However, recent data indicate that miRs may also bind the open reading frame (ORF) of mRNAs. In this dissertation, I show that miR-27b-3p and miR-125a-5p are induced by ATRA and target FGFBP1. Overexpression of miR-27b-3p and miR-125a-5p rapidly reduced FGFBP1 mRNA levels through a target site in the open reading frame of the FGFBP1 mRNA. Both microRNAs showed specificity for regions within the ORF of FGFBP1, suggesting that these microRNAs may also be involved in inhibiting translation of the FGFBP1 protein. Next generation sequencing data from The Cancer Genome Atlas shows that loss of these microRNAs is characteristic of several epithelial cancers, including head and neck, lung, and cervical squamous cell carcinomas, suggesting a tumor suppressor role for miRs 27a-3p and 125a-5p. In total, these data suggest an important regulatory role for miRs 27b-3p and 125a-5p in the oncogenesis of squamous cell carcinomas, through modulation of FGFBP1 expression.</p>

<p> While Wnt and Hgf signaling pathways are known to regulate epithelial cell responses during kidney injury and repair, whether they exhibit functional cross-talk is not well defined. Canonical Wnt signaling is initiated by the phosphorylation of the Lrp5/6 co-receptors and culminates in stabilization of (&beta;-catenin, its translocation to the nucleus and activation of gene transcription. In the current study we demonstrate that Hgf stimulates the Met-dependent and Wnt-independent phosphorylation of Lrp5/6 at 3 separate activation motifs in murine renal epithelial cells. This Hgf-induced phosphorylation of Lrp5/6 was restricted to subconfluent, de-differentiated mouse proximal tubular (MPT) cells and down-regulated as the cells become more confluent. We then pursued the mechanism of Hgf-induced Lrp5/6 phosphorylation. This phosphorylation was found to be Gsk3-dependent and Hgf treatment stimulated the selective association of `active' (tyrosine phosphorylated) Gsk3 with Lrp5/6. In contrast, Akt-phosphorylated `inactive' Gsk3 is excluded from this association. Subsequently, we conducted studies to determine the significance of our findings. After knocking down Lrp5 and 6 in MPT cells, we found that Hgf mediated Lrp5/6 phosphorylation led to (&beta;-catenin stabilization, its nuclear accumulation and increased expression of the Wnt target gene c-myc. We also find that Hgf protects against epithelial cell apoptosis in an Lrp5/6 dependent fashion and increased Survivin gene expression. <i>In vivo </i>, the increase in Lrp5/6 phosphorylation and (&beta;-catenin stabilization on the first day after renal ischemic injury was significantly reduced in mice lacking Met receptor in the renal proximal tubule. Our results thus identify Hgf as an important transactivator of canonical Wnt signaling that is mediated by Met-stimulated, Gsk3-dependent Lrp5/6 phosphorylation.</p>