Cardiovascular disease is among the leading causes of death in the US. It stems from the pathological buildup of plaque within the vasculature known as vascular calcification. Medial calcification, or arteriosclerosis is the buildup of plaque within the medial layer of the arteries resulting in artery wall stiffening and reduction of blood flow. Evidence suggests that the vascular smooth muscles cells (VSMCs) that line the medial layer of the arteries, undergo a phenotypic switch to osteoblast-like cells to deposit calcium while in this pathological state. The Wnt/BETA-catenin pathway could potentially play a role in the phenotypic modulation. Inhibition of the Wnt signaling pathway could be a promising approach to combat vascular calcification. Sclerostin (SOST) has been shown to be upregulated during arteriosclerosis in a manner that is indicative of the possible therapeutic potential of the protein. Therefore, we propose to confirm the role of Wnt signaling in vascular calcification and investigate the effects of SOST treatment on vascular calcification.

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Cellular processes critical to sustaining physiology, including growth, division and differentiation, are carefully governed by intricate control systems. Deregulations in these systems often result in complex diseases such as cancer. Hence, it is crucial to understand the interactions between molecular players of these control systems, their emergent network dynamics, and, ultimately, the overall contribution to cellular physiology. In this dissertation, we have developed a mathematical framework to understand two such cellular systems: an early checkpoint (START) in the budding yeast cell cycle (Chapter 1), and the canonical Wnt signaling pathway involved in cell proliferation and differentiation (Chapter 2). START transition is an important decision point where the cell commits to one round DNA replication followed by cell division. Several years of experimental research have gone into uncovering molecular details of this process, but a unified understanding is yet to emerge. In chapter one, we have developed a comprehensive mathematical model of START transition that incorporates several findings including information about the phosphorylation state of key START proteins and their subcellular localization. In the second chapter, we focus on modeling the canonical Wnt signaling pathway, a cellular circuit that plays a key role in cell proliferation and differentiation. The Wnt pathway is often deregulated in colon cancers. Based on some evidence of bistability in the Wnt signaling pathway, we proposed the existence of a positive feedback loop underlying the activation and inactivation of the core protein complex of the pathway. Bistability is a common feature of biological systems that toggle between ON and OFF states because it ensures robust switching back and forth between the two states. To study and explain the behavior of this dynamical system, we developed a mathematical model. Based on experimentally determined interactions, our simple model recapitulates the observed phenomena of bimodality (bistability) and hysteresis under the effects of the physiological signal (Wnt), a Wnt-mimic (LiCl), and a stabilizer of one of the key members of core complex (IWR-1). Overall, we believe that cell biologists and molecular geneticists can benefit from our work by using our model to make novel quantitative predictions for experimental verification. / Ph. D.

No / Wnt signaling normally functions in cell determination and proliferation and is essential for embryonic development. It does this by regulating target genes through a tightly regulated but complex signaling cascade. Overexpression of these genes due to aberrant Wnt activity can lead to uncontrolled cell growth and survival, and ultimately oncogenesis. Wnt signaling is also involved in epithelial–mesenchymal transition that contributes to tumor progression and metastasis evidence that tumor growth can be suppressed irrespective of other neoplastic promoters when the Wnt pathway is blocked and this has led to interest in its use as a therapeutic target. Recent developments in our understanding of the Wnt signaling cascade have led to research into drugs that specifically target different levels in this pathway, and the identification of β-catenin as the primary cause of dysregulated Wnt signaling has led to a number of protein knockdown strategies. Moreover, increased knowledge of the 300–400 Wnt inducible genes has provided a large untapped source of new potential therapeutic targets. Existing drugs such as nonsteroidal anti-inflammatory drugs and vitamin A and D derivatives have also shown efficacy in disrupting the Wnt signaling pathway and, together with a new generation of derivatives, they may soon be in clinical trials. This chapter details the Wnt signaling pathway, its role in different cancers, and some potential therapeutic targets that may show promise as effective cancer treatments.

Die Entwicklung von Fernmetastasen ist die Haupttodesursache bei Tumorerkrankten und der entscheidende klinisch relevante Schritt während der Tumorprogression. Die Seed and Soil Theorie von Stephen Paget besagt, dass verschiedene Tumorzellen spezifische Zielorgane während der Metatasierung bevorzugen. Während das häufigste Target der kolorektalen Karzinome die Leber ist, hat der triple-negative molekulare Subtyp von Brustkrebs die Neigung, in das Gehirn zu metastasieren. Interessanterweise spielen sowohl deregulierte EGFR (Epithelial growth factor receptor) als auch WNT Signalwege in diesen beiden Entitäten eine entscheidende Rolle. Das Ziel der Arbeit ist, die Rolle der beiden Signalwege in soliden Tumorzelllinien in Bezug auf die Tumorprogression und Kolonisation zu untersuchen. Im Rahmen der molekularen Charakterisierung der Zelllinien zeigten sich die Mammakarzinomzelllinie 410.4 und die kolorektale Tumorzelllinie CMT-93 als passende Modellsysteme für unsere Fragestellung. Anschließend wurden der EGFR und der WNT Signalweg in diesen Zellen im Sinne von gain of function und loss of function moduliert und die Auswirkungen auf Aspekte der Tumorprogression analysiert. In CMT-93 Zellen wurde ein EGFR Knockdown etabliert. Während der Knockdown keinen Einfluss auf die Proliferation hat, vermindert er die Invasion der Zellen. Somit konnte dem effizienten Knockdown eine funktionelle Wirksamkeit zugeschrieben werden. Eine EGFR Überexpression konnte sowohl in 410.4 als auch in CMT-93 Zellen etabliert werden. Die Analyse der jeweiligen Signalkaskadenweiterleitung ergab zwar Änderungen und somit eine funktionelle Relevanz, dies blieb jedoch ohne Auswirkungen auf das Invasionspotential der Zelllinien. Ein Knockdown von β-Catenin konnte in 410.4 zwar etabliert werden, blieb jedoch ohne funktionelle Auswirkungen. Eine stabile Überexpression von β-Catenin war nicht erfolgreich, da dies offenbar mit der Viabilität der Zellen interferierte. Die Relevanz des β-Catenin-abhängigen WNT Signalwegs in den beiden gewählten Zelllinien konnte somit nicht abschließend geklärt werden. Des Weiteren wurde die Bedeutung des nicht-kanonischen WNT Signalwegs via ROR2 und WNT11 untersucht. Dabei ergab sich, dass die Überexpression von WNT11 und ROR2 in 410.4 Zellen deren Invasion durch einen RHOA-abhängigen Mechnismus steigert und einen Einfluss auf den PI3K Signalweg hat. Es ist anzunehmen, dass WNT11 als downstream Target über ROR2 induziert wird und über einen positiven Feedback-Loop via ROR2 eine autokrine Stimulation ausübt.

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Coordinated inductive and morphogenetic processes of gastrulation establish the zebrafish body plan. Gastrulation includes massive cell rearrangements to generate the three germ layers and shape the embryonic body. Three modes of cell migration must occur during vertebrate gastrulation and include: epiboly, internalization of the presumptive mesendoderm and convergent extension (C&E). C&E movements narrow the germ layers mediolaterally (convergence) and elongate them anteroposteriorly (extension) to define the embryonic axis. The molecular mechanisms regulating coordinated cell migrations remain poorly understand and studying these has become of great interest to researchers. Understanding cell migration during development is highly relevant to a number of human physiological processes. Abnormal cell migration during early development can lead to congenital defects, with improper cell migration during adult life potentially leading to the invasion and metastasis of cancer. By studying cell migration events, in vivo, new insights are to be found to both the function and malfunction of key embryonic and postembryonic migratory events. The non-canonical Wnt pathway has been identified as an evolutionarily conserved signaling pathway, regulating C&E cell movements during vertebrate gastrulation. With the absence of the non-canonical Wnts (ncWnts), Wnt5 and Wnt11, during zebrafish development leading to a shorter and broader body axis with defects in elongation during segmentation resulting in undulation of the notochord. While it is clear ncWnts are necessary for C&E, many of the downstream effectors regulating these cell movements have not been defined. Previous research has shown that activation of ncWnt signaling through Wnt5 or Wnt11 results in an increase in intracellular Ca2+ during zebrafish gastrulation. To determine if the Ca2+/Calmodulin-dependent protein kinase, CaMK-II, is a potential downstream target of the Ca2+ increases during ncWnt activation, CaMK-II’s role in C&E was assessed. This study identifies camk2b1 and camk2g1 as being necessary for C&E movements, and outlines the phenotype of the overall embryo as well as individual cells of camk2b1 and camk2g1 morphants. The defects of CaMK-II morphants are specifically linked to alterations in C&E cell movements, while cell fate and proliferation are unaffected. An increase in CaMK-II activation during gastrulation produces similar C&E defects, demonstrating the specificity of CaMK-II’s activation in facilitating these highly coordinated cellular movements. We show that CaMK-II is working downstream Wnt 11 and in parallel to JNK signaling during gastrulation C&E. Overall, these data identify CaMK-II as a required component of C&E movements during zebrafish development, downstream ncWnt signaling, and altering cell migration through changes in cell shape

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The plot of this PhD thesis is dedicated to investigation of the molecular pathways and events and their disruptions in the gastrointestinal tract (further abbreviated as GIT). The major role in this part plays the Wnt signaling pathway. This marvelous multipurpose machinery is responsible for epithelia renewal from stem cells (SCs) in the stomach and intestine, and for correct zonation and function of hepatic units. Of note, the Wnt pathway directs also development of embryo as well as homeostasis of many tissues apart from GIT in all metazoans, thus its flawless function is indispensable from one's origin to death. The main part of the thesis follows canonical Wnt signaling in its physiological condition and, in contrast, with pathological disturbances. This issue can be taken by variety of means as it is described in attached publications. The first publication deals with searching for new participants of Wnt signaling and their functions and describing unique markers of SCs in the intestine. Troy, the member of tumor necrosis factor receptor (TNFR) superfamily, was identified as a novel marker of intestinal SCs by probing microarray data from chromatin immunoprecipitation obtained in cultured colorectal cancer cell lines. Moreover, we found that Troy is a Wnt target gene inhibiting the...

The human proteom diversity is caused by the ability of a single gene locus to encode more protein isoforms. The TCF/LEF genes produce a broad spectrum of protein variants, which consequently leads to a great functional diversity of the TCF/LEF proteins. The TCF/LEF transcriptional factors regulate the canonical Wnt signaling target genes. In this diploma project we focused on the Caenorhabditis elegans gene pop-1, the ortholog of the TCF/LEF genes. Using the Northern blot analysis we tried to identify alternative isoforms of the pop-1 mRNA in C. elegans. Using quantitative RT-PCR we also analyzed the pop-1 mRNA levels. Key words: canonical Wnt signaling pathway, TCF/LEF transcription factors, Caenorhabditis elegans, pop-1