The versatility of Ca2+ as a messenger regulating a myriad of signalling events requires that the concentration of Ca2+ ions in the cytoplasm be highly regulated. Capacitative Ca2+ entry (CCE) or store-operated Ca2+ (SOC) entry, whereby the depletion of intracellular Ca2+ stores induces the influx of Ca2+ across the plasma membrane, plays a crucial role in Ca2+ signalling. Despite the recent advances in elucidating the entry pathway, its molecular identity, biophysical properties and store-depletion signal remains undefined. Thapsigargin (TG), a sarcoplasmic/endoplasmic reticulum Ca2+ A TPase pump (SERCA), inhibitor induces passive depletion of the internal Ca2+ stores and triggers CCE. The universality of this signal has been widely accepted and TG has proven to be a valuable tool in studying CCE. The neuronal cell line NG 115 -401 L lacks the TG activated Ca2+ influx pathway. Agonists of the ryanodine receptor (RyR); chlorom- cresol (CMC), polychlorinated biphenyl 95 (PCB), ryanodine, caffeine, and that of the inositol-1 ,4 ,5-trisphosphate receptor (IP3R), bradykinin, effectively couple to the activation of Ca2+ influx in these cells. The Ca2+ influx signal due to these agonists can be inhibited by SOC blockers such as La3+, Zn2+, Ni2+ and SF&F 96365. Thapsigargin, CMC and PCB95 share the same Ca2+ releasable pools in the 401 L cells. Our data thus suggests that the channels present in the 401 L cells are likely to be receptor-activated channels rather than the store-depletion activated channels. Cell viability studies show that thapsigargin (25 nM) can decrease viability by 75% within 24 hrs and the RyR agonist caffeine decreased viability to <60% within 24hrs. CMC, PCB95 and ryanodine also were cytotoxic at higher doses. Nuclear fragmentation patterns and activation of caspase-3 in thapsigargin and caffeine-treated cells suggest the induction of apoptosis within 12 hrs of treatment. The treated cells were shown to generate nitric oxide, a potential apoptosis inducing agent.

In the present study, the ease of rearing, short generation time and molecular research tools available for the zebrafish model (Danio rerio, Hamilton) were exploited to investigate transcriptional regulation in relation to feeding, photoperiod and experimental selection. Chapter 2 describes transcriptional regulation in fast skeletal muscle following fasting and a single satiating meal of bloodworms. Changes in transcript abundance were investigated in relation to the food content in the gut. Using qPCR, the transcription patterns of 16 genes comprising the insulin-like growth factor (IGF) system were characterized, and differential regulation between some of the paralogues was recorded. For example, feeding was associated with upregulation of igf1a and igf2b at 3 and 6h after the single-meal was offered, respectively, whereas igf1b was not detected in skeletal muscle. On the other hand, fasting triggered the upregulation of the igf1 receptors and igfbp1a/b, the only binding proteins whose transcription was responsive to a single-satiating meal. In addition to the investigation of the IGF-axis, an agnostic approach was used to discover other genes involved in transcriptional response to nutritional status, by employing a whole-genome microarray containing 44K probes. This resulted in the discovery of 147 genes in skeletal muscle that were differentially expressed between fasting and satiation. Ubiquitin-ligases involved in proteasome-mediated protein degradation, and antiproliferative and pro-apoptotic genes were among the genes upregulated during fasting, whereas satiation resulted in an upregulation of genes involved in protein synthesis and folding, and a gene highly correlated with growth in mice and fish, the enzyme ornithine decarboxylase 1. Zebrafish exhibit circadian rhythms of breeding, locomotor activity and feeding that are controlled by molecular clock mechanisms in central and peripheral organs. In chapter 3 the transcription of 17 known clock genes was investigated in skeletal muscle in relation to the photoperiod and food content in the gut. The hypothesis that myogenic regulatory factors and components of the IGF-pathway were clock-controlled was also tested. Positive (clock1 and bmal1 paralogues) and negative oscillators (cry1a and per genes) showed a strong circadian pattern in skeletal muscle in anti-phase with each other. MyoD was not clock-controlled in zebrafish in contrast to findings in mice, whereas myf6 showed a circadian pattern of expression in phase with clock and bmal. Similarly, the expression of two IGF binding proteins (igfbp3 and 5b) was circadian and in phase with the positive oscillators clock and bmal. It was also found that some paralogues responded differently to photoperiod. For example, clock1a was 3-fold more responsive than clock1b. Cry1b did not show a circadian pattern of expression. These patterns of expression provide evidence that the molecular clock mechanisms in skeletal muscle are synchronized with the molecular clock in central pacemaker organs such as eyes and the pineal gland. Using the short generation time of zebrafish the effects of selective breeding for body size at age were investigated and are described in chapter 4. Three rounds of artificial selection for small (S-lineage) and large body size (L-lineage) resulted in zebrafish populations whose average standard length were, respectively, 2% lower and 10% higher than an unselected control lineage (U-lineage). Fish from the L-lineage showed an increased egg production and bigger egg size with more yolk, possibly contributing to the larger body size observed in the early larval stage (6dpf) of fish from this lineage. Fish from S- and L-lineage exposed to fasting and refeeding showed very similar feed intake, providing evidence that experimental selection did not cause significant changes in appetite control. Investigation of the expression of the IGF-axis and nutritionally-response in skeletal muscle after fasting and refeeding revealed that the pattern of expression was not different between the selected lineages, but that a differential responsiveness was observed in a limited number of genes, providing evidence that experimental selection might have changed the way fish allocate the energy acquired through feeding. For example, a constitutive higher expression of igf1a was recorded in skeletal muscle of fish from the L-lineage whereas igfbp1a/b transcripts were higher in muscle of fish from the S-lineage. These findings demonstrate the rapid changes in growth and transcriptional response in skeletal muscle of zebrafish after only three rounds of selection. Furthermore, it provides evidences that differences in growth during embryonic and larval stages might be related to higher levels of energy deposited during oogenesis, whereas differences in adult fish were better explained by changes in energy allocation instead of energy acquisition. In chapter 5 the main findings made during this study and their impact on the literature are discussed.

The Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor and member of the basic helix-loop-helix Per/ARNT/Sim (bHLH/PAS) family of chemosensors and developmental regulators. As a member of the PAS domain family of transcription factors responsive to exogenous signals, the AhR exerts influence on many processes relating to cellular fate. The activation of AhR is widely associated with toxic endpoints related to dioxin exposure. However, the AhR also activates endogenous gene programs related to development, cellular growth, and differentiation. The AhR is able to bind a variety of ligands, leading to a wide range of biological outcomes. Recent reports have shown that the AhR can mediate tumor suppressive effects. As a ligand-activated transcription factor, the AhR has the potential to actuate a variety of transcriptional programs that are dependent on the AhR ligand. Our central hypothesis is that AhR ligands can be identified that are capable of initiating tumor suppressive functions of the AhR. We utilized complementary cell-based and in silico virtual screening approaches to identify potential AhR ligands. We developed homology models of the AhR ligand-binding domain (LBD) for virtual ligand screening (VLS) of small molecule libraries. This led to the identification of new AhR ligands 5,7- dihydroxyflavanone!and 5-hydroxy-7-methoxyflavone. Additional small molecule libraries were screened in parallel that led to identification of flutamide as a putative AhR ligand. Flutamide is clinically approved for the treatment of prostate cancer due to its ability to antagonize androgen receptor mediated transcription. We investigated the biological effects of flutamide in AhR positive cancer cells that do not express the androgen receptor and found that flutamide inhibited the growth of HepG2 cells. Suppression of AhR expression reversed the anti-proliferative effects of flutamide. We tested 15 structural analogs of flutamide, including the flutamide metabolite 2-hydroxyflutamide for activation of AhR transcriptional activity. Flutamide is unique in its ability to activate the AhR, and suppresses hepatoma cell growth. These data suggests that flutamide-induced AhR transcriptional activity is required to initiate the tumor suppressive effects. We examined changes in cell cycle checkpoint proteins after flutamide treatment and discovered increased expression of cell cycle inhibitory proteins p27[superscript Kip1] and p15[superscript INK]. We also found that transforming Growth Factor β1 (TGFβ1), which regulates both p27[superscript Kip1] and p15[superscript INK], is upregulated by flutamide. We demonstrate that TGFβ1 is upregulated by flutamide in an AhR-dependent manner and is required for suppression of proliferation by flutamide. We identify specific and unique transcriptional signatures of the AhR upon activation by flutamide, that are distinct from the potent AhR agonist 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD). In summary, we characterize flutamide as an AhR ligand and demonstrate its AhR-dependent tumor suppressive effects in hepatoma cells. We provide the first direct evidence that AhR regulates TGFβ signaling in a ligand dependent manner. We demonstrate that the AhR-induced downstream transcriptional signature and subsequent biological effects are specific to the AhR ligand. Our studies have broad impact for characterizing the AhR as a new therapeutic target in hepatocellular carcinoma. / Graduation date: 2013 / Access restricted to the OSU Community at author's request from March 28, 2013 - March 28, 2015

Vegetative propagation allows the amplification of selected genotypes for research, breeding, and commercial planting. However, efficient in vitro regeneration and genetic transformation remains a major obstacle to research and commercial application in many plant species. Our aims are to improve knowledge of gene regulatory circuits important to meristem organization, and to identify genes that might be useful for improving the efficiency of in vitro regeneration. In this thesis, we have approached these goals in two ways. First, we analyzed gene expression during poplar (Populus) regeneration using an AffymetrixGeneChip® array representing over 56,000 poplar transcripts. We have produced a catalog of regulated genes that can be used to inform studies of gene function and biotechnology. Second, we developed a GUS reporter system for monitoring meristem initiation using promoters of poplar homologs to the meristem-active regulatory genes WUSCHEL (WUS) and SHOOTMERISTEMLESS (STM). This provides plant materials whose developmental state can be assayed with improved speed and sensitivity. For the microarray study, we hybridized cDNAs derived from tissues of a female hybrid poplar clone (INRA 717-1 B4, Populus tremula x P. alba) at five sequential time points during organogenesis. Samples were taken from stems prior to callus induction, at 3 days and 5 days after callus induction, and at 3 and 8 days after the start of shoot induction. Approximately 15% of the monitored genes were significantly up-or down-regulated based on both Extraction and Analysis of Differentially Expressed Gene Expression (EDGE) and Linear Models for Microarray Data (LIMMA, FDR<0.01). Of these, over 3,000 genes had a 5-fold or greater change in expression. We found a very strong and rapid change in gene expression at the first time point after callus induction, prior to detectable morphological changes. Subsequent changes in gene expression at later regeneration stages were more than an order of magnitude smaller. A total of 588 transcription factors that were distributed in 45 gene families were differentially regulated. Genes that showed strong differential expression encoded proteins active in auxin and cytokinin signaling, cell division, and plastid development. When compared with data on in vitro callogenesis from root explants in Arabidopsis, 25% (1,260) of up-regulated and 22% (748) of down- regulated genes were in common with the genes that we found regulated in poplar during callus induction. When ~3kb of the 5' flanking regions of close homologs were used to drive expression of the GUSPlus gene, 50 to 60% of the transgenic events showed expression in apical and axillary meristems. However, expression was also common in other organs, including in leaf veins (40% and 46% of WUS and STM transgenic events, respectively) and hydathodes (56% of WUS transgenic events). Histochemical GUS staining of explants during callogenesis and shoot regeneration using in vitro stems as explants showed that expression was detectable prior to visible shoot development, starting 3 to 15 days after explants were placed onto callus inducing medium. Based on microarray gene expression data, a paralog of poplar WUS was detectably up-regulated during shoot initiation, but the other paralog was not. Surprisingly, both paralogs of poplar STM were down-regulated 3- to 6-fold during early callus initiation, a possible consequence of its stronger expression in the secondary meristem (cambium) than in shoot tissues. We identified 15 to 35 copies of cytokinin response regulator binding motifs (ARR1AT) and one copy of the auxin response element (AuxRE) in both promoters. Several of the WUS and STM transgenic events produced should be useful for monitoring the timing and location of meristem development during natural and in vitro shoot regeneration. / Graduation date: 2008

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Recent work has shown that specific ALDH isoenzymes can contribute to the underlying pathology of different diseases. Many ALDH isozymes are important in oxidizing reactive aldehydes resulting from lipid peroxidation, and, thus, help maintain cellular homeostasis. Increased expression and activity of ALDH isozymes are found in many human cancers and are often associated with poor prognosis. Therefore, the development of inhibitors of the different ALDH enzymes is of interest as means to treat some of these disease states. Here I describe the results of assays designed to characterize the site of interaction and the mode of inhibition for the unique compounds that function as inhibitors of aldehyde dehydrogenase 2 and determine their respective IC50 values with intent to develop structure-activity relationships for future development.

Indiana University-Purdue University Indianapolis (IUPUI). / The abysmal five year pancreatic cancer survival rate of less than 6% highlights the need for new treatments for this deadly malignancy. Cytotoxic drugs normally target rapidly dividing cancer cells but unfortunately often target stem cells resulting in toxicity. This warrants the development of compounds that selectively target tumor cells. An inhibitor of the mitotic kinase, MPS1, which has been shown to be more selective towards cancer cells than non-tumorigenic cells, shows promise but its effects on stem cells has not been investigated. MPS1 is an essential component of the Spindle Assembly Checkpoint and is proposed to be up-regulated in cancer cells to maintain chromosomal segregation errors within survivable limits. Inhibition of MPS1 kinase causes cancer cell death accompanied by massive aneuploidy. Our studies demonstrate that human adipose stem cells (ASCs) and can tolerate higher levels of a small molecule MPS1 inhibitor than pancreatic cancer cells. In contrast to PANC-1 cancer cells, ASCs and telomerase-immortalized pancreatic ductal epithelial cells did not exhibit elevated chromosome mis-segregation after treatment with the MPS1 inhibitor for 72hrs. In contrast, PANC-1 pancreatic cancer cells exhibited a large increase in chromosomal mis-segregation under similar conditions. Furthermore, growth of ASCs was minimally affected post treatment whereas PANC-1 cells were severely growth impaired suggesting a favorable therapeutic index. Our studies, demonstrate that MPS1 inhibition is selective towards pancreatic cancer cells and that stem cells are less affected in vitro. These data suggest MPS1 inhibition should be further investigated as a new treatment approach in pancreatic cancer.

Indiana University-Purdue University Indianapolis (IUPUI) / Photoreceptors are a class of sensory neuronal cells that are deleteriously affected in many disorders and injuries of the visual system. Significant injury or loss of these cells often results in a partial or complete loss of vision. While previous studies have determined many necessary components of the gene regulatory network governing the establishment, development, and maintenance of these cells, the necessary and sufficient profile and timecourse of gene expression and/or silencing has yet to be elucidated. Arduous protocols do exist to derive photoreceptors in vitro utilizing pluripotent stem cells, but only recently have been able to yield cells that are disease- and/or patient-specific. The discovery that mammalian somatic cells can be directly reprogrammed to another terminally-differentiated cell phenotype has inspired an explosion of research demonstrating the successful genetic reprogramming of one cell type to another, a process which is typically both more timely and efficient than those used to derive the same cells from pluripotent stem cell sources. Therefore, the emphasis of this study was to establish a novel system to be used to determine a minimal transcriptional network capable of directly reprogramming mouse embryonic fibroblasts (MEFs) to rod photoreceptors. The tools, assays, and experimental design chosen and established herein were designed and characterized to facilitate this determination, and preliminary data demonstrated the utility of this approach for accomplishing this aim.

Indiana University-Purdue University Indianapolis (IUPUI) / Cervical cancer is caused by the persistent infection of human papilloma virus (HPV) in the cervix epithelium. Although effective preventative care is available, the widespread nature of infection and the variety of HPV strains unprotected by HPV vaccines necessitate a better understanding of the disease for development of new therapies. A major tumor suppressing mechanism is the inhibition of cell division by tissue structure; however, the underlining molecular circuitry for this regulation remains unclear. Recently, the Yap transcriptional co-activator has emerged as a key growth promoter that mediates contact growth arrest and limits organ size. Thus, we aimed to uncover upstream signals that connect tissue organization to Yap regulation in the inhibition of cervical cancer. Two events that disrupt tissue structure were examined including the loss of the tumor suppressor LKB1 and the expression of the viral oncogene HPV16-E6. We identified that Yap mediates cell growth regulation downstream of both LKB1 and E6. Restoration of LKB1 expression in HeLa cervical cancer cells, which lack this tumor suppressor, or shRNA knockdown of LKB1 in NTERT immortalized normal human dermal keratinocytes, demonstrated that LKB1 promotes Yap phosphorylation, nuclear exclusion, and proteasomal degradation. The ability of phosphorylation-defective Yap mutants to rescue LKB1 phenotypes, such as reduced cell proliferation and cell size, suggest that Yap inhibition contributes to LKB1 tumor suppressor function(s). Interestingly, LKB1’s suppression of Yap activity required neither the canonical Yap kinases, Lats1/2, nor metabolic downstream targets of LKB1, AMPK and mTORC1. Instead, the scaffolding protein NF2 was required for LKB1 to induce a specific actin cytoskeleton structure that associates with Yap suppression. Meanwhile, HPV16-E6 promoted Yap activation in all stages of keratinocyte differentiation. E6 activated the Rap1 small GTPase, which in turn promoted Yap activity. Since Rap1 does not mediate differentiation inhibition caused by E6, E6 may play a role in promoting cell growth through Rap1-Yap activation rather than preventing growth arrest through the disruption of differentiation. Altogether, the LKB1-NF2-Yap and E6-Rap1-Yap pathways represent two examples of a novel phenomenon, whereby the structure of a cell directly influences its gene expression and proliferation.

Indiana University-Purdue University Indianapolis (IUPUI) / While breast cancer is the second most commonly diagnosed cancer worldwide, its causes and natural history are not well defined. The female mammary organ is unique in that it does not reach full maturity until the lactation cycle following pregnancy. This cycle entails extensive growth and reorganization of the primitive epithelial ductal network. Following lactation, these same epithelial cells undergo an equally extensive program of apoptosis and involution. The mammary gland's sensitivity to pro-growth and pro-apoptotic signals may partly explain its proclivity to develop cancers. For epithelial cells to become transformed they must lose intracellular organization known as polarity as differentiated epithelial tissues are refractory to aberrant growth. One essential component of epithelial to mesenchymal transition is the intrinsic capacity of cells to repurpose polarity constituents to promote growth. Recently, a novel mechanism of organ size control has been shown to repurpose the apical junctional associated protein Yap into the nucleus where it functions as a transcriptional coactivator promoting growth and dedifferentiation. The focus of my work has been on a family of adaptor proteins termed Amots that have been shown to scaffold Yap and inhibit growth signaling. Specifically, I have shown that the 80KDa form of Amot, termed Amot80, acts as a dominant negative to the other Amot proteins to promote cell growth while reducing cell differentiation. Amot80 was found to promote the prolonged activation of MAPK signaling. Further, Amot80 expression was also found to enhance the transcriptional activity of Yap. This effect likely underlies the ability of Amot80 to drive disorganized overgrowth of MCF10A cells grown in Matrigel̈™. Overall, these data suggest a mechanism whereby the balance of Amot proteins controls the equilibrium between growth and differentiation within mammary epithelial tissues.