To effectively understand and treat complex diseases such as cancer, mathematical and statistical modeling is essential if one wants to represent and characterize the interactions among the different regulatory components that govern the underlying decision making process. Like in any other complex decision making networks, the regulatory power is not evenly distributed among its individual members, but rather concentrated in a few high power "commanders". In biology, such commanders are usually called masters or canalizing genes. Characterizing and detecting such genes are thus highly valuable for the treatment of cancer. Chapter II is devoted to this task, where we present a Bayesian framework to model pathway interactions and then study the behavior of master genes and canalizing genes. We also propose a hypothesis testing procedure to detect a "cut" in pathways, which is useful for discerning drugs' therapeutic effect. In Chapter III, we shift our focus to the understanding of the mechanisms of action (MOA) of cancer drugs. For a new drug, the correct understanding of its MOA is a key step for its application to cancer treatments. Using the Green Fluorescent Protein technology, researchers have been able to track various reporter genes from the same cell population for an extended period of time. Such dynamic gene expression data forms the basis for drug similarity comparisons. In Chapter III, we design an algorithm that can identify mechanistic similarities in drug responses, which leads to the characterization of their respective MOAs. Finally, in the course of drug MOA study, we observe that cells in a hypothetical homogeneous population do not respond to drug treatments in a uniform and synchronous way. Instead, each cell makes a large shift in its gene expression level independently and asynchronously from the others. Hence, to systematically study such behavior, we propose a mathematical model that describes the gene expression dynamics for a population of cells after drug treatments. The application of this model to dose response data proviodes us new insights of the dosing effects. Furthermore, the model is capable of generating useful hypotheses for future experimental design.

The sex determining region Y-box 2 (sox2) gene is one of the most important transcription factors during development, particularly the development of the central nervous system (CNS). It is expressed in embryonic stem cells and later in neural stem cells, where it modulates their maintenance and differentiation. In humans, heterozygous mutations are associated with eye malformations, including anophthalmia and severe microphthalmia. Also, a subset of patients has extra-ocular phenotypes, such as hearing loss, seizures and pituitary hypoplasia. Although the roles of sox2 in embryonic stem cells and eye development are well studied, the function of sox2 in brain development and disease is still elusive. The aim of this project was to characterize a novel role for sox2 in the development of zebrafish epithalamus, which was identified from an in silico screen previously performed in our laboratory. The zebrafish epithalamus, located in the dorsal diencephalon, consists of three main structures: the pineal gland, the parapineal organ and the habenular nuclei. The pineal gland, also known as epiphysis, is a photoreceptive (in zebrafish) and neuroendocrine organ that detects light and rhythmically produces melatonin in order to regulate the circadian rhythms. The parapineal organ is located to the left side of the pineal gland and is important for the elaboration of the asymmetries observed between the left and right habenular nuclei. Finally, the bilateral habenulae are part of the dorsal diencephalic conduction system that links the forebrain with the mid- and hindbrain. The left and right habenulae show both molecular and neuroanatomical asymmetries, including differences in neuropil organization, in levels of gene expression and in the morphology and connectivity of their neurons’ projections. The relatively simple architecture of the pineal gland and the asymmetric character of the habenulae provide a useful tool for studying cell-fate determination, cell migration and establishment of brain asymmetries. In this study, we used zebrafish as a model to dissect the novel functions of sox2 in the development of the epithalamus. We showed that sox2 works synergistically with Notch pathway to negatively regulate neurogenesis within the pineal gland. The pineal gland consists of only two cell types: the photoreceptors and the projection neurons. Previous studies showed that the Notch and BMP pathways are important for the proper specification of these cells. Here, we show that sox2 normally inhibits the photoreceptor cell fate, whereas it has no effect on the number of projection neurons. Therefore, sox2 complements Notch and BMP pathways in cell-fate determination within the pineal gland. In addition, downregulation of sox2 results in abnormal parapineal organ development and disruption of the asymmetric architecture of the habenulae. A subset of sox2 morphant embryos develops right-sided parapineal organs, which is consistent with abnormal bilateral expression of the Nodal gene, pitx2 (paired-like homeodomain transcription factor 2). Also, timelapse experiments showed that migration of the parapineal cells is defective, resulting in scattered cells. The aberrant parapineal development leads to disorganization of the habenular nuclei, as shown by the abnormal neuropil arrangement and the expression of the asymmetric marker kctd12.1 (potassium channel tetramerisation domain containing 12.1).

Three 2-oxoglutarate-dependent dioxygenases required for flavonoid biosynthesis were characterized in Arabidopsis thaliana. Genes encoding flavanone 3-hydroxylase (F3H), flavonol synthase (FLS), and leucoanthocyanidin dioxygenase (LDOX) were cloned and sequenced. The predicted proteins encoded by each of these Arabidopsis genes shared high homology with all F3H, FLS, or LDOX sequences available in Genbank. Low-stringency DNA blot analysis indicated that F3H and LDOX are encoded by a single gene in Arabidopsis, while FLS may be encoded by two or three genes. RNA blot analysis was performed to determine the expression patterns of these three genes relative to previously-cloned genes encoding flavonoid biosynthetic enzymes. Light-induction experiments and analysis of regulatory mutants showed that the CHS, CHI, F3H, and FLS1 are coordinately regulated in Arabidopsis seedlings, encode enzymes acting near the beginning of the pathway, and are therefore referred to as "early" genes. The same experiments showed that DFR and LDOX are regulated distinctly from "early" genes, share similar expression patterns in response to light, and are not expressed in the ttg mutant. DFR and LDOX are therefore referred to as "late" genes due to the timing of expression in response to light and the fact that they encode enzymes acting late in flavonoid biosynthesis. To determine whether any of the previously-identified transparent testa mutants were defective in F3H, FLS, or LDOX, the chromosomal locations of these genes in the Arabidopsis genome were determined. The positions of these genes suggested that no previously-identified tt mutant was defective in the cloned FLS or LDOX structural genes, while tt6 was potentially the F3H locus. The coding region of F3H was amplified by PCR from tt6 genomic DNA and sequenced, and several point mutations were found in the coding region of this allele, three of which are predicted to result in amino acid substitutions. Polyclonal antibodies were also developed using four different purified, recombinant flavonoid enzymes as antigens. These antibodies were used to determine the pattern of accumulation of flavonoid enzymes in developing seedlings. Immunoblot analysis was also performed to determine whether mutations in genes encoding specific flavonoid enzymes or an enzyme in pathways that compete for or provide substrate for flavonoid biosynthesis (mutants defective in tryptophan or ferulic acid biosynthesis) affect the levels of flavonoid enzymes. These analyses showed that mutant seedlings which lacked specific flavonoid or tryptophan biosynthetic enzymes accumulated higher steady-state levels of other enzymes in the pathway. These results suggest that the accumulation of specific flavonoid intermediates or indole can lead directly or indirectly to higher levels of flavonoid enzymes. / Ph. D.

4 Abstract The Wnt pathway is one of the major signaling cascades contributing to multiple cellular processes during embryogenesis, and adult tissue homeostasis and regeneration. Moreover, aberrant activation of the Wnt signaling pathway is connected with development of neoplasia, notably colorectal cancer. The aim of the thesis was to identify new ways of the Wnt pathway regulation to understand better physiological as well as non-physiological mechanisms of Wnt signaling. The results are summarized in four publications. The first article deals with TROY, a member of tumor necrosis factor receptor family. We identified TROY as a Wnt target gene during our search for Wnt responsive genes in colorectal cancer cell lines. Additionally, we detected expression of Troy in tumors of two mouse models of intestinal cancer. In the healthy gut, Troy is produced in fast cycling intestinal stem cells where negatively regulates the Wnt pathway. The second study focuses on processing and posttranslational modification of murine Wnt1 and Wnt3a. Wnts are glycosylated and double acetylated by lipid adducts and our results revealed that O-linked acylation of serine is required for the subsequent S-palmitoylation of cysteine. Moreover, acylation of Wnts is connected with their signaling activity which is related to Wnt1 and...