Thesis (Ph.D.)--Indiana University, Dept. of Biology, 2005. / Source: Dissertation Abstracts International, Volume: 66-08, Section: B, page: 4033. Adviser: Lynda F. Delph. Title from dissertation home page (viewed Oct. 10, 2006).

Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2006. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3571. Adviser: A. Lane Rayburn. Includes bibliographical references. Available on microfilm from Pro Quest Information and Learning.

<p> Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the third leading cause of cancer death in the United States. Twin cohort studies indicate that inherited susceptibility accounts for approximately 35% of all CRC cases, but only 5-6% of CRC cases can be attributed to known functional mutations. We were the first to identify a germline mutation in Transforming Growth Factor Beta Receptor 1 (<i>TGFBR1</i>) that is also somatically acquired in tumors, a 9 bp in frame deletion within exon 1 (rs11466445), which results in a receptor with decreased TGF-&beta; signaling properties. The observed association between this hypomorphic variant and cancer risk led us to hypothesize that constitutively decreased TGF-&beta; signaling may contribute to the development of CRC. </p><p> In this dissertation, we developed a novel mouse model of <i>Tgfbr1 </i> haploinsufficiency (<i>Tgfbr1</i>^+/&minus;) and found that <i>Tgfbr1</i>^+/&minus; mice were twice as likely as <i>Tgfbr1</i>^+/+ mice to develop CRC. We subsequently identified two human haplotypes associated with constitutively decreased <i>TGFBR1</i> expression and CRC risk and found that decreased <i> TGFBR1</i> expression is strongly associated with three SNPs: rs7034462, rs11466445 and rs11568785. Further examination of <i>TGFBR1</i> haplotype tagging SNPs suggests that the <i>TGFBR1</i> rs7034462-TT is a novel moderate penetrance risk genotype, which has high penetrance among African Americans, the ethnic group with the highest risk for CRC. Our results provide strong support for the novel notion that rs7034462-TT is a potentially clinically relevant CRC susceptibility genotype that may identify individuals at high risk of dying from CRC.</p>

Gnathostome novelties include jaws, paired appendages, and true teeth. Dlx genes encode transcription factors indispensable for embryonic development of these novelties. Gnathostomes possess at least 6 Dlx genes organized in 3 bi-gene clusters, a physical arrangement which is proposed to affect their expression though shared enhancer elements. I studied the Dlx genes of Cyclostomes, the evolutionary sister group of Gnathostomes. I identified 4 novel members of the Dlx gene family in hagfish (Eptatretus stoutii), and confirmed the presence of 6 Dlx genes in lamprey ( Petromyzon marinus). I found that Cyclostomes have only 1 gene cluster and several orphan genes. This lack of conserved arrangement is coincident with an absence of conserved Dlx enhancers. Irregardless, some regulatory conservation is still apparent as lamprey non-coding DNA is able to drive dlx specific expression patterns of reporter genes in zebrafish. Therefore Cyclostome and Gnathostome Dlx are both organized and regulated differently. The causes and consequences of these changes in Chordate evolution are discussed.

<p> Leaf senescence is the final stage of leaf development where older leaves undergo an active degenerative process. This highly coordinated event is characterized by a cascade of differential gene expression resulting in senescence upregulated and senescence downregulated genes. Cytosine methylation, a mechanism of epigenetic control, has been shown to play a role in regulating gene expression. Gene body cytosine methylation is correlated with transcriptional activation while promoter cytosine methylation is correlated with transcriptional repression. Evidence from previous work suggests CG methylation (^mCG) in promoter regions plays a role in repressing gene expression and that a correlation between demethylation and mRNA induction is most likely within 500 bp up- and downstream of TSSs. The purpose of this study is to investigate the correlation between promoter cytosine methylation and transcriptional repression by identifying potential cytosine methylation-regulated senescence upregulated genes (CMR-SURGs). Four candidate CMR-SURGs were identified from previously generated RNA-seq data and an online cytosine methylome. We hypothesized that the four CMR-SURGs would display a correlation between mRNA induction and loss of promoter ^mCG. mRNA expression was measured by real-time qPCR, and cytosine methylation was quantified by bisulfite treatment of genomic DNA followed by PCR, cloning, and sequencing of PCR products. These data however, showed that only <i>UGT78D1</i> displayed a negative correlation between promoter cytosine methylation and age-related mRNA induction.</p><p>

<p> The integrity of the mechanisms that control gene transcription during development and in post-natal life is essential to maintain tissue homeostasis and impede the development of genetic diseases such as cancer. Inheritance of a defective hyperpatahyroidism 2 (<i>HRPT2</i>) allele, an essential regulator of gene transcription, predisposes individuals to a constellation of symptoms ranging from endocrine abnormalities to parathyroid adenomas and jaw bone tumors (HPT-JT). In order to elucidate the function of the <i> HRPT2</i> gene and the pathogenesis that results upon spontaneous inactivation in familial cases of parathyroid cancer and HPT-JT, mouse models were generated that allow for deletion of <i>Hrpt2</i> within different stages and tissues during development. We have used the <i>Hrpt2</i> flox mouse model to delete <i>Hrpt2</i> in mesenchymal progenitor cells as well as committed, terminally differentiated osteoblasts and osteocytes. Whereas loss of <i>Hrpt2</i> in mesenchymal progenitors was embryonic lethal, genetic deletion of <i>Hrpt2</i> in mature bone forming cells led to increased bone mass and bone strength. However, <i>Hrpt2 </i> conditional knockout bones had increased cortical porosity and osteocyte apoptosis associated with increased osteoblast specific gene expression. This work suggests that <i>Hrpt2</i> is required for cell proliferation and differentiation and acts as a transcriptional repressor in terminally differentiated cell types.</p><p> Control of gene transcription defines cell identity and fate. Ascorbic acid (AA, also known as vitamin C) is an essential vitamin for humans and is well known for its role in collagen synthesis. AA acts as a cofactor for TET enzymes, which hydroxylate methylated cytosines. Here, we characterize how 7 days of AA treatment causes changes in gene transcription, 5-hydroxymethylcytosine deposition, and the active chromatin marks H3K4me3 and H3k27ac in MC3T3-E1 murine pre-osteoblasts cells, initiating cell differentiation and expression of the osteoblast phenotype. Though 5hmC deposition was not specific for only highly expressed genes, it was highly enriched at transcriptional start sites and CpG islands. While H3K4me3 was mostly unchanged, H3K27ac was predictive of driving gene expression. This work suggests that AA causes dramatic changes to the epigenome through epigenetic modifiers to impact cell differentiation. </p><p>

This dissertation develops and applies several statistical and computational methods to the analysis of Next Generation Sequencing (NGS) data in order to gain a better understanding of our biology. In the rest of the chapter we introduce key concepts in molecular biology, and recent technological developments that help us better understand this complex science, which, in turn, provide the foundation and motivation for the subsequent chapters. In the second chapter we present the problem of estimating gene/isoform expression at the allelic level, and different models to solve this problem. First, we describe the observed data and the computational workflow to process the data. Next, we propose frequentist and bayesian models motivated by the central dogma of molecular biology and the data generating process (DGP) for RNA-Seq. We develop EM and Gibbs sampling approaches to estimate gene and transcript-specic expression from our proposed models. Finally, we present the performance of our models in simulations and we end with the analysis of experimental RNA-Seq data at the allelic level. In the third chapter we present our paired factorial experimental design to study parentally biased gene/isoform expression in the mouse cerebellum, and dynamic changes of this pattern between young and adult stages of cerebellar development. We present a bayesian variable selection model to estimate the difference in expression between the paternal and maternal genes, while incorporating relevant factors and its interactions into the model. Next, we apply our model to our experimental data, and further on we validate our predictions using pyrosequencing follow-up experiments. We subsequently applied our model to the pyrosequencing data across multiple brain regions. Our method, combined with the validation experiments, allowed us to find novel imprinted genes, and investigate, for the first time, imprinting dynamics across brain regions and across development. In the fourth chapter we move from the controlled-experiments in mouse isogenic lines to the highly variant world of human genetics in observational studies. In this chapter we introduce a Bayesian Regression Allelic Imbalance Model, BRAIM, that estimates the imbalance coming from two major sources: cis-regulation and imprinting. We model the cis-effect as an additive effect for the heterozygous group and we model the parent-of-origin detect with a latent variable that indicates to which parent a given allele belongs. Next, we show the performance of the model under simulation scenarios, and finally we apply the model to several experiments across multiple tissues and multiple individuals. In the fifth chapter we characterize the transcriptional regulation and gene expression of in-vitro Embryonic Stem Cells (ESCs), and two-related in-vivo cells; the Inner Cell Mass (ICM) tissue, and the embryonic tissue at day 6.5. Our objective is two fold. First we would like to understand the differences in gene expression between the ESCs and their in-vivo counterpart from where these cells were derived (ICM). Second, we want to characterize the active transcriptional regulatory regions using several histone modifications and to connect such regulatory activity with gene expression. In this chapter we used several statistical and computational methods to analyze and visualize the data, and it provides a good showcase of how combining several methods of analysis we can delve into interesting developmental biology.

Nearly fifty percent of North American myocarditis cases are associated to coxsackievirus group B, type 3 (CVB3) infection. CVB3 infection of mice provides a useful model to study pathogenic mechanism of myocarditis. The objective of this study is to test the hypothesis that susceptibility, during the acute CVB3 infection, is under polygenic control including H2 as well as the non H-2 genes. To identify differential parameters of the disease, and if they are influenced by the H-2 haplotype, several phenotypic traits were characterized. Three inbred strains of mice and two congenic strains were infected with CVB3. Differences in survival, body weight loss, quantification of myocarditis and quantification of sarcolemmal disruption were found by comparing three sets of mice sharing the same H-2 haplotype but not the same background. It was determined that host susceptibility to CVB3-induced myocarditis is mainly controlled by "background" genes. Moreover, because there is a naturally occurring variability among inbred mice, ten inbred strains of mice were used for the genetic analysis of four CVB3-induced phenotypes: survival, body weight loss, heart viral load and quantification of sarcolemmal disruption. It was concluded that the strains could be divided into three groups: the highly resistant, the resistant to intermediate strains and the highly susceptible strains. This phenotypic data on commonly used and genetically diverse inbred mouse strains sets up the platform for a detailed analysis of the genetic basis of susceptibility to CVB3.

In this thesis, I use 10 populations of Drosophila melanogaster, each fixed for a different visible recessive mutation to investigate two outstanding issues in sexual selection research. First, to quantifty indirect benefits of female mate choice and explore the nature of genotype x environment interactions for different fitness components, I estimated the effects of these mutations on male sexual fitness and productivity in the population's ancestral laboratory environment, as well as a novel food environment. Indirect benefits in the ancestral environment were lacking, suggesting that a good genes process is not acting. Cross-environment correlations were not conducive to indirect benefits following colonization of a novel environment. Second, to investigate the contribution of variable search effort to variance among males in mating success, I conducted mate choice trials to compare the relative mating success of mutant males in small vs. large arenas. Sexual selection against mutant males was stronger when search effort was included than when it was excluded, indicating that varying ability to find mates may increase the strength of selection against deleterious alleles.

Iron is one of the most important micronutrients used by living organisms. Iron is frequently a limiting nutrient for plant growth, and plants are a major source of iron for human nutrition. The most prominent symptom of iron deficiency in plants is interveinal chlorosis, or yellowing between the veins, which appears first in the youngest leaves. Iron deficiency anemia (IDA) is the number one human nutritional deficiency worldwide. In order to solve the problem of iron deficiency, it is desirable to breed plants that have increased iron in those parts that are consumed by humans. To do this, we must first understand the molecular basis of Fe uptake, transport, and storage in plants. In soil, iron is quickly oxidized to Fe(III), and Fe(III) is relatively insoluble, thus difficult for plants to obtain. Our lab has been working on metal ion homeostasis mechanisms in plants and the ultimate goal of our research is to understand the mechanisms by which plants maintain the correct levels of iron, zinc and copper in each cell and tissue. The Yellow Stripe-like (YSL) family of proteins has been identified based on sequence similarity to maize Yellow stripe 1 (YS1). YS1 transports Fe(III) that is complexed by phytosiderophores (PS), strong Fe(III) chelators of the mugineic acid family of compounds. Non-grass species of plants neither make nor use PS, yet YSL family members are found in non-grass species including Arabidopsis thaliana. YSLs in non-grasses have been hypothesized to transport metals that are complexed by nicotianamine (NA), an iron chelator that is structurally similar to PS and which is found in all higher plants. In this dissertation, Arabidopsis YSL1 and YSL3 are demonstrated to be important in iron transport and also responsible for loading Fe, Cu, and Zn from leaves into seeds. Arabidopsis YSL4 and YSL6 are demonstrated to be involved in iron transport and metal mobilization into seeds. The transport function of Arabidopsis YSL1 and YSL2 are shown be partially overlapping to the function of Arabidopsis YSL3 in vegetative structures, but distinct in reproductive organs. Arabidopsis YSL3 and YSL6 are shown to have distinct functions in planta.