No description available.

Gastroesophageal reflux disease (GERD), or acid reflux, affects 6-9 million people in the United States. It is characterized by a reflux of gastric acid and bile salts from the stomach into the esophagus, causing injuries to the esophagus known as Barrett's esophagus (BE). BE is the main risk factor for the development of esophageal adenocarcinoma (EAC), a devastating cancer in the esophagus whose molecular roots remain poorly understood. In recent years, evidence points to the esophageal epithelium itself as responsible for causing and promoting inflammation upon injury by gastric reflux, namely via an increase in inflammatory cytokine secretion. This project was focused on a cytokine of interest, Activin A, which is known for its importance during embryogenesis and stem cell differentiation. It has recently been studied for its role in inflammation and tumor formation, but not in the case of esophageal diseases. Here, we demonstrate that Activin A signaling in esophageal epithelial cells is heavily upregulated shortly after exposure to bile salts and acid. We show evidence that this upregulation causes an increase in cell migration upon a reconstituted extracellular matrix. We also provide further evidence that bile and acid injury causes epithelial cells to secrete cytokines, which drive inflammation. We show that the upregulated Activin A secretion and signaling plays an important role in promoting this inflammatory state. Finally, we provide evidence that bile salts and acid exposure, as well as increased Activin A signaling, causes esophageal epithelial cells to upregulate stem cell and transdifferentiation markers, supporting the latest theories on the origin of Barrett' esophagus stem cells as well as paligenosis.

No description available.

Currently, the only cure for liver failure is orthotopic liver transplantation. However, there are insufficient donor organs available to treat every patient on the transplant list and many die before they are able to receive a liver transplant. The bioartificial liver (BAL) device is a potential extracorporeal treatment strategy utilising hepatocytes or hepatocyte-like cells (HLCs) within a bioreactor to recapitulate normal liver function and therefore ‘bridge’ a patient with liver failure until they receive a transplant. The work in this thesis utilised tissue engineering methods to develop novel approaches to BAL device design through development and characterisation of a polymer membrane scaffold (“PX”) for hollow fibre bioreactor (HFB) culture and a HLC source generated from the transdifferentiation of pancreatic AR42J-B13 (B13) cells. A flat sheet membrane model was used for the development of asymmetrical, hydrophobic polystyrene (PS) phase inversion membranes. Oxygen plasma significantly increased PS membrane surface wettability through addition of oxygen functional groups to create an environment conducive for cell culture. The treated membrane was henceforth referred to as “PX”. The culture medium HepatoZYME+ was investigated for its ability to induce transdifferentiation of B13 cells to HLCs and maintain the hepatic phenotype. Overall, HepatoZYME+-cultured cells experienced viability loss. A diluted version, “50:50”, showed induction of the hepatic markers carbamoylphosphate synthetase-1 (CPS-1) and HNF4α, as well as a change towards a HLC morphology. When using 50:50 as a maintenance medium, transdifferentiated HLCs retained loss of pancreatic amylase and also induction of hepatic markers, with comparable serum albumin secretion to the established Dex + OSM treatment. However, culture viability in 50:50 was still compromised. Therefore, HepatoZYME+ based media were deemed unsuitable for induction and maintenance compared to Dex-based protocols. PX flat sheet membranes were able to support culture of B13 cells and also the human osteosarcoma cell line, MG63, demonstrating improved cell attachment over non-surface treated PS membranes. PX membranes supported transdifferentiation of B13 cells to HLCs, presenting with loss of pancreatic amylase, induction of the hepatic markers transferrin, GS and CPS-1 and serum albumin secretion. Furthermore, PX showed no change in mass or loss of culture surface area over 15 days in culture conditions. Together, the novel membrane material and the media formulation and feeding regime developed have strong potential to be translated to a HFB setting and guide future BAL device design.

Small molecules are important not only for treating human diseases but also for studying disease-related biological processes. This dissertation focuses on the effects of small molecules on pancreatic ductal adenocarcinoma cells. Here, I describe the discovery of two small-molecule tool compounds and their applications for interrogating the biological processes related to two distinct diseases in the human pancreas. First, BRD4770 was identified as a histone methyltransferase inhibitor through a target-based biochemical approach, and was used as a probe to study the function of methyltransferases in cancer cells. Second, BRD7552 was discovered as an inducer of Pdx1 using a cell-based phenotypic screening approach, and was used to induce the expression of Pdx1, a master regulatory transcription factor required for \(\beta\)-cell transdifferentiation. This compound is particularly interesting for the study of type-1 diabetes (T1D). The histone methyltransferase G9a catalyzes methylation of lysine 9 on histone H3, a modification linked to aberrant silencing of tumor-suppressor genes. The second chapter describes the collaborative effort leading to the identification of BRD4770 as a probe to study the function of G9a in human pancreatic cancer cells. BRD4770 induces cellular senescence and inhibits both anchorage-dependent and -independent proliferation in PANC-1 cell line, presumably mediated through ATM-pathway activation. Chapter three describes the study of a natural product gossypol, which significantly enhances the BRD4770 cytotoxicity in p53-mutant cells through autophagic cell death. The up-regulation of BNIP3 might be responsible for the synergistic cell death, suggesting that G9a inhibition may help overcome drug resistance in certain cancer cells. Ectopic overexpression of Pdx1, Ngn3, and MafA can reprogram pancreatic exocrine cells to insulin-producing cells in mice, which sheds light on a new avenue for treating T1D. The fourth chapter focuses on a gene expression-based assay using quantitative real-time PCR technique to screen >60,000 compounds for induction of one or more of these three transcription factors. A novel compound BRD7552 which up-regulated Pdx1 mRNA and protein levels in PANC-1 cells was identified. BRD7552 induces changes of the epigenetic markers within the Pdx1 promoter region consistent with transcriptional activation. Furthermore, BRD7552 partially complements Pdx1 in cell culture, enhancing the expression of insulin induced by the introduction of the three genes in PANC-1 cells. In summary, the central theme of my dissertation is to identify novel bioactive small molecules using different screening approaches, as well as to explore their effects in pancreatic ductal cells. / Chemistry and Chemical Biology

Les cellules différenciées peuvent être reprogrammées et adopter un destin cellulaire très différent. Connaître les acteurs et mécanismes qui contrôlent les processus de reprogrammation est un objectif scientifique fascinant qui éclairera notre compréhension du contrôle et du maintien de l'identité cellulaire. Notre laboratoire étudie le changement d'identité (ou transdifférenciation, TD) naturel d’une cellule épithéliale rectale (nommée Y) en motoneurone (nommé PDA) chez Caenorhabditis elegans. Dans les vers mutants pour le gène lin-15A (gène isolé dans un crible génétique du laboratoire), la cellule Y n'initie pas sa reprogrammation : Y demeure rectale. Cette protéine apparaît dans le noyau de Y juste avant le début de la TD de Y et joue un rôle clé dans l’initiation de ce processus. LIN-15A lie l’ADN et son domaine conservé en doigt de zinc (de type THAP-like) est essentiel pour initier la reprogrammation de Y. Nous nous sommes attachés à mieux comprendre le rôle de LIN-15A dans ce processus. L’inactivation de certains gènes (impliqués dans le maintien de l’identité cellulaire) permet de supprimer partiellement ou très fortement le défaut de reprogrammation de Y causé par la mutation lin-15A. Ces gènes appartiennent au groupe appelé synMuv B et ceux induisant la plus forte suppression du phénotype de lin-15A sont tous liés à la voie du rétinoblastome (RB). Dans la littérature, tous les mutants suppresseurs de défaut de PDA existant dans le mutant lin-15A présentaient une dérive de l’identité des cellules intestinales. Certains mutants de voies de réponse au jeûne chez le ver présentent également une perte du maintien de l’identité des cellules intestinales très similaire à celle induite par l’inactivation de certains gènes synMuv B. De façon très intéressante, nous avons pu observer que les vers mutants lin-15A présentent une pénétrance du défaut de PDA bien plus faible une fois privés de nourriture (au 1er stade larvaire ou au stade dauer). Certaines études laissent supposer que ces diapauses suite au jeûne entrainent une perte du maintien de l’identité cellulaire de cellules somatiques (et possiblement dans l’intestin), ce qui pourrait permettre à Y d’enclencher sa reprogrammation malgré l’absence de lin-15A, facteur clé à la levée du verrou pour initier la TD. En résumé, mes résultats ont montré que la transdifférenciation d'une cellule dépendait d'une clé moléculaire, LIN-15A, nécessaire pour lever un verrou de maintien de l'identité cellulaire dans la cellule qui va changer d'identité, et ce précisément juste avant la conversion cellulaire de Y en PDA. De façon plus générale, mes travaux ouvrent la possibilité que l'état physiologique et métabolique du ver influe sur le maintien de l'identité cellulaire. Sur le long terme, il conviendra alors de déterminer par quel biais cet état est perçu, dans quelles cellules, et comment cette information est relayée ou captée par la cellule Y, pour finalement influencer sa plasticité. / Differentiated cells can be reprogrammed to adopt a different cell identity. The discovery of factors and mechanisms controlling cellular reprogramming is a fascinating scientific goal that will shed light on the mechanisms controlling cell identity maintenance. Our laboratory studies the natural identity switch (or transdifferentiation, TD) of an epithelial rectal cell (called Y) into a motor neuron (called PDA) in Caenorhabditis elegans. In worm mutants for the gene lin-15A (isolated in a genetic screen performed in the laboratory), the Y cell does not initiate the Y reprogramming : Y stays rectal. This protein appears in the Y nucleus just before the beginning of the Y TD and plays a key role in the process initiation. LIN-15A binds DNA and its zinc finger domain (THAP-like) is essential for the initiation of Y reprogramming. Inactivation of some genes that are involved in cell identity maintenance allow a partial or strong suppression of the Y reprogramming defect due to lin-15A mutation. These genes belong to a group called synMuv B and those inducing the strongest lin- 15A phenotype suppression are all linked to the retinoblastoma (RB) pathway. In the literature, all the mutants that are PDA defect suppressors in lin-15A mutant show a switch of intestinal cell identity. Some mutants in starvation–response genes in worms show a loss of intestinal cell identity maintenance very similar to the one observed in synMuv B mutants. Interestingly, we observed that starvation (in 1st larval stage or dauer stage) induces a strong drop of the PDA defect in lin-15A mutant. Different studies suggest diapause induced by starvation trigger a loss of somatic cell identity maintenance (and possibly in intestinal cells), that could allow Y to start the reprogramming despite the lack of lin-15A, even it is a key factor to release a break and initiate the TD. In summary, my results show the cell transdifferentiation depends on a molecular key, LIN-15A, that is needed, just priorto TD initiation, to release a cell maintenance lock to allow a cell to undergo cell identity switch. My work opens the possibility that the worm physiologic and metabolic state influences cell identity maintenance. In the future, how this state is perceived has to be determined, in which cells and how this information is transmitted to Y to finally influence its plasticity.

Zelluläre Seneszenz wird als terminaler Zellzyklusarrest definiert, der mit dem Altern und funktionellen Verlust von Geweben verknüpft ist. Eine Seneszenzreaktion wird ebenso durch Onkogene und zytotoxischen Stress verursacht. Die Ausführung des Seneszenzprogramms wird durch eine zeitlich hochdynamische Aktivität von Transkriptionsfaktoren (TF) bedingt. Interessanterweise kann die Zelllinienzugehörigkeit einer Zelle durch die Expression von linien-aberranten TF überschrieben werden. Die vorliegende Arbeit untersucht Chemotherapie-induzierte Seneszenz (TIS) in Bcl-2 überexprimierenden, deshalb vor Apoptose geschützten, murinen Eµ-Myc B-Zell Lymphomen in An- oder Abwesenheit der Seneszenz-essentiellen Histonmethyltransferase Suv39h1. Analysen auf Transkriptom- und auf Proteinebene ergeben dabei, dass in einer Seneszenz-spezifischen Weise die TF AP-1, PU.1 und C/EBPβ induziert werden, welche normalerweise für die Funktion und Entwicklung myeloischer Zelllinien bedeutend sind. Dementsprechend korreliert der Seneszenzzustand mit Transkripten, Oberflächenmarkern und einer enzymatischen Funktion der myeloischen Linie. Indem die identifizierten TFs heruntergeschaltet oder überexprimiert werden, wird ihre direkte Beteiligung an der Linienuntreue der TIS Lymphome demonstriert. TIS-Kapazität wird als für den Erfolg von Krebstherapie günstige Eigenschaft betrachtet, da sie zu einem Wachstumsblock führt. Nichtsdestotrotz können sich verweilende TIS Zellen krebsbiologisch auch nachteilig auswirken. Anhand von murinen und humanen, klinisch annotieren Transkriptomdatensätzen kann hier in beiden Spezies ein myeloisch verschobenes, Linienuntreue anzeigendes Genexpressionsprofil mit einer besseren Überlebensprognose korreliert werden. Die vorliegenden Befunde legen nahe, dass die Modulation von TF Aktivitäten in Seneszenz einen potentiellen therapeutischen Angriffspunkt darstellt, um den für den Therapieerfolg nützlichen Zweig des TIS Phänotyps zu befördern. / Cellular senescence is regarded as an irreversible cell cycle arrest associated with tissue aging and its functional decline. A senescence response is also evoked by oncogenic and cytotoxic stress. The execution of the senescence program relies on a highly dynamic sequence of transcription factor (TF) activities. Interestingly, cell lineage commitment can be overridden by the expression of lineage-aberrant TFs. This thesis examines chemotherapy-induced senescence (TIS) in Bcl-2 overexpressing, thus apoptosis-protected, murine Eμ-Myc B-cell lymphomas with or without the senescence-essential histone methyltransferase Suv39h1. Transcriptome as well as protein level analyses reveal senescence-specific induction of the TFs AP-1, PU.1 and C/EBPβ which are typically crucial for myeloid lineage commitment and function. Correspondingly, the senescent state associates with myeloid lineage transcripts, surface markers and enzymatic function, reminiscent of, but not equal to a transdifferentiation phenotype. By knocking down and overexpressing the identified TFs, we demonstrate their direct involvement in the lineage infidelity of TIS lymphomas. TIS-capacity is viewed as beneficial to cancer therapy outcome due to its block on proliferation. However, lingering TIS cells can also be detrimental due to the acquisition of latent stemness properties or tumor-protective remodeling of their microenvironment. By interrogating murine and human, clinical course-annotated transcriptome data sets, an association between a myeloid-skewed, lineage infidelity indicating gene expression profile and better tumor prognosis is established in both species. The presented findings suggest that modulation of the senescent TF activities could be therapeutically exploited to foster the cancer patient-beneficial branch of the TIS phenotype.

Atherosclerotic disease is characterized by the formation of lipid-ladden plaques in artery walls. During later stages of disease, these plaques become calcified by mechanisms involving the trans-differentiation of vascular smooth muscle cells (VSMC) to osteoblast-like cells. Although vascular calcification was thought to be a passive mechanism, evidence shows that this process is heavily modulated by various cell signaling mechanisms, including CB2 endocannabinoid receptors. Previous studies have shown that known CB2 antagonists accelerate VSMCs trans-differentiation to an osteoblast-like phenotype, indicating that this receptor serves an anti-calcification signal. The goal of this investigation is to determine if a series of 11-[2-arylmethylene)hydrazono]-PBD derivatives with established CB2 binding affinity function as CB2 antagonists or agonists in a cell culture model of VSMC osteoblastic trans-differentiation. MOVAS cells were grown in standard media or osteogenic media (to induce trans-differentiation) supplemented with and without the various PBD derivatives. Following the treatment period, the extent of osteoblast-like activity was evaluated by alizarin red staining for calcium deposition. To quantify the staining present, the dye was extracted using cetylpyridinium chloride hydrate solution and then analyzed via UV-Vis spectroscopy at 570 nm. The ability of the derivatives to modulation of osteoblastic transdifferentiation of MOVAS cells was further evaluated by performing Western blot analysis for expression of Runx2, an essential transactivator of osteoblast differentiation. Results of this work determined that some of the PBD derivatives increased the calcification compared to the control, indicating that they likely act as CB2 receptor antagonists, while others decreased calcification compared to the control, indicating that they likely act as CB2 receptor agonists. Not only do these results characterize the interactions of these compounds with CB2 receptors, they demonstrate that these PBD derivatives have biological activity. These results also further implicate CB2 receptors as a regulator of VSMC cell calcification, which could lead to novel drug therapies for the treatment of atherosclerotic plaques.

<p>Oncogenic mutations in KRAS are present in approximately 95% of patients diagnosed with pancreatic ductal adenocarcinoma (<b>PDAC</b>) and are considered the initiating event during the development of pancreatic intraepithelial neoplasia (<b>PanIN</b>) precursor lesions. While it is well established that KRAS mutations can drive the initiation of pancreatic oncogenesis, the effects of oncogenic KRAS signaling on regulation of phosphatases during this process is not fully appreciated. Protein Phosphatase 2A (<b>PP2A</b>) has been implicated in suppressing KRAS-driven cellular transformation. However, low PP2A activity is observed in PDAC cells compared to non-transformed cells, suggesting that suppression of PP2A activity is an important step in the overall development of PDAC. In the current study, we demonstrate that KRASG12D induces the expression of both Cancerous Inhibitor of PP2A (<b>CIP2A</b>), an endogenous inhibitor of PP2A activity, and the PP2A target, c-MYC. Consistent with these findings, KRASG12D sequestered the specific PP2A subunit responsible for c-MYC degradation, B56a, away from the active PP2A holoenzyme in a CIP2A-dependent manner. During PDAC initiation <i>in vivo</i>, knockout of B56a promoted KRASG12D tumorigenesis by accelerating acinar-to-ductal metaplasia (<b>ADM</b>) and the formation of PanIN lesions. The process of ADM was attenuated <i>ex vivo</i> in response to pharmacological re-activation of PP2A utilizing direct small molecule activators of PP2A (<b>SMAP</b>s). Together, the results of this study suggest that suppression of PP2A-B56a through KRAS signaling can promote Myc-driven initiation of pancreatic tumorigenesis.</p>

No description available.