Indiana University-Purdue University Indianapolis (IUPUI) / Pancreatic ductal adenocarcinoma (PDAC), which comprises 85% of pancreatic cancers, is the 4th leading cause of cancer death in the United States with a 5-year survival rate of 8%. While human PDACs (hPDACs) are hypovascular, they also overexpress a number of angiogenic growth factors and receptors. Additionally, the use of anti-angiogenic agents in murine models of PDAC leads to reduced tumor volume, tumor spread, and microvessel density (MVD), and improved survival. Nonetheless, clinical trials using anti-angiogenic therapy have been overwhelmingly unsuccessful in hPDAC. On the other hand, pancreatic neuroendocrine tumors (PNETs) account for only 2% of pancreatic tumors, yet they are very vascular and classically angiogenic, respond to anti-angiogenic therapy, and confer a better prognosis than PDAC even in the metastatic setting. In an effort to compare and contrast the angiogenic transcriptomes of these two tumor types, we analyzed RNA-Sequencing (RNA-Seq) data from The Cancer Genome Atlas (TCGA) and found that a pro-angiogenic gene signature is present in 35% of PDACs and that it is mostly distinct from the angiogenic signature present in PNETs. The pro-angiogenic PDAC subgroup also exhibits a transcriptome that reflects active TGF-β signaling, less frequent SMAD4 inactivation than PDACs without the signature, and up-regulation of several pro-inflammatory genes, including members of JAK signaling pathways. Consequently, targeting the TGF-β receptor type-1 kinase with SB505124 and JAK1/2 with ruxolitinib blocks proliferative crosstalk between human pancreatic cancer cells (PCCs) and human endothelial cells (ECs). Additionally, treatment of the KRC (oncogenic Kras, homozygous deletion of Rb1) and KPC (oncogenic Kras, mutated Trp53) genetically engineered PDAC mouse models with ruxolitinib suppresses murine PDAC (mPDAC) progression only in the KRC model, which shows superior enrichment and differential expression of the human pro-angiogenic gene signature as compared to KPC tumors. These findings suggest that targeting both TGF-β and JAK signaling in the 35% of PDAC patients whose cancers exhibit an pro-angiogenic gene signature should be explored in a clinical trial.

It is now clear that human and animal exposure to estrogenic compound occurs through several sources. This include: i) naturally occurring endogenous estrogens, ii) exogenous or intentional estrogens for prophylactic (e.g. oral contraceptive) and therapeutic (e.g. as replacement therapy for ovulation in nulliparous women and in menopausal women, and in some men suffering from prostate cancer) purposes, iii) accidental via estrogenic chemical exposure (e.g. pesticides, industrial byproducts) and phytoestrogens (e.g. soybeans). It has long been recognized that estrogen, a female sex hormone, functions not only on the reproductive system, but also on various other systems including the immune system. Estrogens are thought to be of both physiologic and pathologic importance. Female in general, have better immune capabilities than males, a phenomenon attributed to the action of sex hormones on the immune system. There is also a female-gender bias in susceptibility to autoimmune diseases. Estrogens have been linked either directly or indirectly to the etiology and pathogenesis of various female-predominant autoimmune diseases. Estrogens have also been linked to the onset of cancer, and conditions where the immune system often malfunctions. Estrogen affects the functions of both B and T cells, possibly by regulating such factors as lymphokine gene expression and/or cellular death by apoptosis. However, the functioning of both B and T cells under the influence of long-term exposure to estrogen has not been fully understood. The primary aim of this thesis was to investigate the effect of long-term exposure to 17β-estradiol on lymphokine and bcl-2/fas (proto-oncogenes) mRNA expression. We evaluated the effects of estrogen on the expression of genes for lymphokines, which are essential for the immune response. It is hypothesized that estrogen may regulate the immune system by modifying the expression of lymphokine genes and/or genes that regulate apoptosis. The results demonstrated that long-term 17β-estradiol exposure reduced the viability of lymphocytes when compared to lymphocytes from placebo-treated mice. IL-2 and IFN-g mRNA was consistently higher in ConA-stimulated lymphocytes from estrogen-treated mice (P < 0.05). The mRNA for TGF-β₁ lymphokine was also increased but was not consistent at all time points of incubation. The expression of IL-4 mRNA was not noticeably affected by estrogen treatment of mice. Long-term exposure to 17β-estradiol appear to have some influence on the mRNA expression of proto-oncogenes fas and bcl-2 in splenic and thymic T lymphocytes. There was a trend of increased bcl-2 mRNA expression in estrogen-treated mice compared to placebo-treated mice, whereas the mRNA expression of fas gene appeared to be lower compared to controls. Overall, these findings suggest that 17β-estradiol may selectively influence lymphokine and proto-oncogene mRNA expression. These results suggest that the one mode of modulation of the immune response by 17β-estradiol may be through alterations in the lymphokine and proto-oncogene expression. Since estrogen-treatment markedly induces atrophy of the thymus and diminishes the cellularity of the lymphoid organs (e.g. Spleen), it became necessary to perform multiple assays on the same cells, particularly lymphokine and apoptosis gene expression. A secondary objective of this thesis was to investigate whether lymphocytes, which have undergone proliferation in Lympho-Pro™ assay (Alamar Blue assay), could be utilized for further analysis. In this regard, we found that a non-radioactive assay that utilizes Alamar Blue had significant advantages over the conventional ³H-thymidine incorporation assay. By using cells from estrogen and placebo-treated mice in the Alamar Blue assay, we found that this assay not only allowed determination of lymphocyte proliferation, but also the assessment of mRNA expression, cytogenetics, apoptosis and immunophenotyping of the same lymphocytes. / Master of Science

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Indiana University-Purdue University Indianapolis (IUPUI) / Ovarian cancer (OC) is a lethal disease due to metastasis and chemoresistance. Our laboratory previously reported that tissue transglutaminase (TG2) is overexpressed in OC and enhances OC peritoneal metastasis. TG2 is a multifunctional protein which catalyzes Ca2+-dependent cross-linking of proteins. The purpose of this study was to explore the mechanism by which TG2 is upregulated in OC and its role in OC progression. We demonstrated that transforming growth factor (TGF)-β1 is secreted in the OC milieu and regulates the expression and function of TG2 primarily through the canonical Smad signaling pathway. Increased TG2 expression level correlates with a mesenchymal phenotype of OC cells, suggesting that TGF-β1 induced TG2 promotes epithelial-to-mesenchymal transition (EMT). TG2 induces EMT by negatively regulating E-cadherin expression. TG2 modulates E-cadherin transcriptional suppressor Zeb1 expression by activating NF-κB complex, which leads to increased cell invasiveness in vitro and tumor metastasis in vivo. The N-terminal fibronectin (FN) binding domain of TG2 (tTG 1-140), lacking both enzymatic and GTPase function, induced EMT in OC cells, suggesting the interaction with FN involved in EMT induction. A TGF-β receptor kinase inhibitor, SD-208, blocked TGF-β1 induced TG2 upregulation and EMT in vitro and tumor dissemination in vivo, which confirms the link between TGF-β1 and TG2 in EMT and tumor metastasis. TG2 expression was correlated with the number and size of self-renewing spheroids, the percentage of CD44+CD117+ ovarian cancer stem cells (CSCs) and with the expression level of stem cell specific transcriptional factors Nanog, Oct3/4, and Sox2. These data suggest that TG2 is an important player in the homeostasis of ovarian CSCs, which are critical for OC peritoneal metastasis and chemoresistance. TG2 expression was also increased in CSCs isolated from human ovarian tumors, confirming the implication of TG2 in CSCs homeostasis. Further, we demonstrated that TG2 protects OC cells from cisplatin-induced apoptosis by regulating NF-κB activity. We proposed a model whereby TGF-β-inducible TG2 modulates EMT, metastasis, CSC homeostasis and chemoresistance in OC. These findings contribute to a better understanding of the mechanisms of OC metastasis modulated by TG2.

Background During development, endothelial cells acquire mesenchymal-like properties to migrate and facilitate normal vascular formation. This process of transformation is known as endothelial to mesenchymal transition (EndMT) and has also been implicated in diseases like vascular pathologies contributing to endothelial inflammation, atherosclerosis and tumour angiogenesis. The Angiomotin family of scaffold proteins play a role in transducing mechanical force at cell junctions. Of this family, Angiomotin-Like 2 (AmotL2) localises to endothelial cell junctions and was recently found to play a role in regulating endothelial cell mechanosensing and inflammation. Methods/Materials Primary human endothelial cell lines (HUVEC) were cultured and manipulated in vitro to investigate the role of AmotL2 in EndMT. Lentiviral short hairpin RNA interference was employed in AmotL2-loss-of-function studies, (produced using HEK - Human Embryonic Kidney - cells) to generate knockdown(kd) cells. Western blotting (WB) was used to assess AmotL2 depletion and changes in protein expression of key EndMT markers. qPCR was performed to look at the same at a transcriptional level. Immunofluorescent staining and confocal imaging were performed to validate WB and qPCR results as well as to study protein localisation. Results AmotL2 was found to regulate Snail1 and N-cadherin at both protein and mRNA levels. Morphological findings displayed the AmotL2kd cells to be elongated, deviating from the regular cobblestone morphology observed in control cells. An increase in scaffold protein levels was observed in the AmotL2 kd samples. Similar results were seen in qPCR data where increased mRNA expression was observed in the AmotL2 kd samples for the same targets. On analysis of IF image data, more nuclear staining was observed in the kd samples. qPCR analysis done on samples treated with TGF-β, exhibited an increase in mRNA expression of targets involved in the EndMT pathway in the treatment samples against the controls. Conclusion The results suggest that AmotL2 plays a role in EndMT by affecting the transcription factors and proteins involved in the pathway, which leads to changing morphology and behaviour of the cells. Looking into more targets involved in EndMT may give us a better understanding of how this process leads to diseases like atherosclerosis and tumour angiogenesis.

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Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Die Narbenbildung des Hautorgans stellt für die gegenwärtige Medizin weiterhin eine schwierige Aufgabe dar. Die frühzeitige Beeinflussung des Wundheilungspro- zesses hin zu einer verminderten oder narbenlosen Heilung scheint von entschei- dender Bedeutung. Ein vielversprechender Ansatz ist die präoperative Laserthe- rapie und dadurch erzeugte Hitzeschockantwort. Auf molekulare Ebene kommt es u.a. zur Expression von Hitzeschockproteine. Die vorliegende in-vitro Studie beschäftigte sich mit der laserinduzierten Hochregulation des Hitzeschockproteins 70 in den epidermalen Schichten. Hierfür wurden drei nicht ablative Lasersysteme mit insgesamt 12 verschiedenen Parametereinstellungen verwendet (1.540-nm Er:Glass- , 755-nm Alexandrit-, 1.064-nm Nd:YAG-Laser). Mithilfe eines humanen Hautexplantatmodells sollte unter gleichbleibenden Bedingungen Zeitpunkt und Konzentration der maximal induzierten Hsp70-Expression sowie epidermale Schä- digungen dargestellt werden. In der verfügbaren Literatur waren hierzu nur begrenzt Daten vorhanden. Alle drei Lasersysteme zeigten signifikante Hsp70-Expressionen. Der Zeitpunkt der maximalen Hsp70-Expression konnte zwischen Tag 1 und 3 festgehalten werden. Dabei zeigten die Lasersysteme unterschiedliche Hsp70- Maxima und unterschiedliche Epidermisschädigungen. Die Ergebnisse ließen schlussfolgern, dass eine potenzielle präoperative Narbenprävention tendeziell ein Tag vor dem chirurgischen Eingriff und mit den stärkeren Parametereinstellungen des 1.064-nm Nd:YAG Lasers durchgeführt werden sollte.