Receptor-like kinases (RLKs) make up one of the largest gene families in Arabidopsis thaliana. These genes are required for various biological processes, including response to biotic stress, cell elongation, cell proliferation, and cell fate patterning. An emerging theme in Arabidopsis and other plants is that networks of RLKs are required to regulate a specific process throughout development involving spatial and temporal regulation of transcription factors. However, there are still many RLKs (>50%) with no known function.Several RLKs regulate epidermal development by contributing to early embryonic epidermal maintenance or to epidermal differentiation. In my first analysis, I characterize the role of two related RLKs GASSHO1 (GSO1) and GSO2 in epidermal differentiation. gso1 gso2 double mutants initially form an epidermis during embryogenesis, but analysis of post-embryonic root development indicates the mis-expression of epidermal-specific genes. Three previously characterized RLKs that are involved in epidermal development are also involved in meristem maintenance. In order to decipher the RLK gene networks controlling epidermal development and meristem maintenance, it is necessary to identify additional RLKs involved in both of these processes. I further identified roles for GSO1 and GSO2 in maintaining root growth and root apical meristem (RAM) activity. A future goal will be to elucidate the networks of RLKs, including GSO1 and GSO2 in regulating epidermal and RAM development.The development of the vasculature in plants is controlled by a vascular meristem, the procambium. Oriented cell divisions from the procambium produce phloem, to the periphery, and xylem, to the center of the plant. In a reverse genetic screen to determine to roles of the remaining RLKs with unknown function, we identified the RLK XYLEM INTERMIXED WITH PHLOEM1 (XIP1) that is required for vascular development. We show XIP1 is required for regulating the differentiation of the phloem and for the organization of xylem vessel elements. Our analysis indicates that XIP1 is part of a vascular meristem network, further emphasizing the importance of social networks of RLKs regulating a specific process in development.

The phylo-geography and population genetics of three Crotalus species in Southern Arizona (C. atrox, C. cerastes, and C. scutulatus) were examined using mitochondrial DNA genes and nuclear microsatellite DNA markers. My focus was twofold: (1) the phylo-geography and population structure in Southern Arizona and (2) possible genetic signatures of population fragmentation by linear barriers on rattlesnakes populations at Picacho Peak. My results show genetic signatures of geneflow restrictions in one species (C. atrox) which coincide with Interstate 10. I did not observe similar genetic effects in C. cerastes or scutulatus, possibly caused by smaller sample sizes and marker numbers. I found limited phylo-geographic and population genetic structure for all three species in Southern Arizona indicating large interconnected populations. This study provides wildlife management with a powerful genetic toolset and provides important baseline data for future assessment and monitoring efforts of important predators and their populations in the Sonoran desert habitat.

An early event in animal development is the formation of the three primary germ layers that define the body plan. During gastrulation, cells migrate through the primitive streak of the embryo and undergo changes in morphology and gene expression, thus creating the mesodermal and endodermal cell layers. Gastrulation requires expression of Fibroblast Growth Factor (FGF), Wnt, and Platelet-Derived Growth Factor (PDGF). Embryos treated with FGF inhibitors fail to gastrulate, as cell migration is completely halted. During gastrulation, 44 microRNAs are expressed in the primitive streak of G. gallus embryos, and six (microRNAs -let7b, -9, -19b, -107, -130b, and -218) are strongly upregulated when FGF signaling is blocked. The abundance of these six FGF-regulated microRNAs is controlled at various stages of processing: most are regulated transcriptionally, and three of them (let7b, 9, and 130b) are blocked by the presence of Lin28B, an RNA-binding protein upregulated by FGF signaling. These microRNAs target various serine/threonine and tyrosine kinase receptors. We propose a novel pathway by which FGF signaling downregulates several key microRNAs (partially through Lin28B), upregulating gene targets such as PDGFRA, which permits and directs cell migration during gastrulation. These findings add new layers of complexity to the role that FGF signaling plays during embryogenesis. FGF signaling is also required for the formation of the heartfields, and has an overlapping pattern of expression with BMP (Bone Morphogenetic Protein). A microarray experiment using inhibitors of FGF and BMP found that thousands of genes in pre-cardiac mesoderm are affected by FGF signaling, BMP signaling, or a cooperative effect of the two. The promoter regions of similarly regulated genes were queried for over-represented transcription factor binding sites or novel DNA motifs. Cluster analysis of over-represented sites determined candidate transcriptional modules that were tested in primary cardiac myocyte and fibroblast cultures. About 75% of predicted modules in FGF-upregulated genes proved to be functional enhancers or repressors. Functional enhancers among FGF-upregulated genes contained clusters of CdxA and NFY sites, and increased transcription in the presence of a constitutively active FGF receptor.

Cell polarity is an important regulator of cellular processes and is vital in helping to prevent metaplasia and tumorigenesis. There are three many polarity complexes that regulate and maintain epithelial cellular polarity. The Par and Crumbs complexes locate to the apical membrane of the cell, while the Scribble complex is located basolaterally. Of the Scribble complex components, the polarity protein Hugl1, also known as Mgl1 in mice, is especially important in helping to maintain apical basolateral and planar polarity, and is lost in multiple types of cancer. When Hugl1 expression is lost in epithelial cells, it results in a mesenchymal phenotype. We now show that the loss of Hugl1 fundamentally shifts the cellular phenotype and specifically alters EGFR trafficking and signaling. Loss of Hugl1 results in the nuclear translocation of Taz and Slug, increased migration, and the mislocalization of EGFR (Epidermal Growth Factor Receptor), driving cellular growth. Hugl1 regulates the expression of multiple cell identity markers and its loss results in stem cell characteristics, including the increased expression of CD44, and a decrease of CD49f and CD24 expression. The loss of Hugl1 also results in increased growth in soft-agar and prolonged survival when transplanted into NOD-SCID mice; its loss also results in EGF-dependent migration which aids in increasing mammosphere survival. Furthermore, isolated EGFR mislocalization via a point mutation (P667A) also drives these same phenotypes, including activation of Akt and Taz nuclear translocation, indicating the importance of Hugl1 in the regulation of EGFR localization and its signaling. In mice, the loss of total Mgl1 is lethal within days of birth due to hydrocephaly and results in the formation of rosette like structures in the brain that are reminiscent of neuroectodermal tumors. We designed a targeted Mgl1 knockout in the mammary epithelial cells using the Cre/Lox system to evaluate the effects of Mgl1 loss in murine mammary gland development and tumorigenesis. The loss of Mgl1 expression in mice inhibits ductal outgrowth, increases side branching and epithelial layers, and results in the mislocalization of EGFR. While overt mammary tumors did not develop, some individuals did develop hyperplastic nodules that could progress into cancer. The knockdown of Hugl1 in vitro and Mgl1 in vivo reveal how the loss of polarity and presence of Hugl1 results in cancer stem cell characteristics, increased migration, and abnormal signaling due to the mislocalization of EGFR. While these changes result in metaplasia and a potential pre-cancerous state, the loss of Hugl1 alone is not enough to drive the cancer progression, indicating that other mutations or factors are necessary for the development of breast cancer. Because of the key role polarity plays in the prevention of breast cancer development we investigated if the addition of Hugl1 back into breast cancer cells could revert the cancerous cells to a normal epithelial phenotype. Most of the breast cancer cells transfected with Hugl1 expression did not survive, indicating that the re-expression of polarity regulators forces cancer cells to die. The small percentage of cells that did survive re-expression of Hugl1 had retarded growth in soft agar and a decrease in EGFR expression. Together, these data indicate that Hugl1 expression and EGFR activity are closely related and that Hugl1 is required for the proper localization and signaling of EGFR. When Hugl1 is lost, EGFR is mislocalized and fails to be degraded properly, promoting pre-neoplastic changes.

Recent research effort (measured in number of publications) on genes is biased towards genes that have been studied heavily in the past. Some factors for why this occurs is that many of these historically studied genes are important for survival or there are more tools available that make genetic studies of them much more accessible. Studies of research effort on \textit{Saccharomyces cerevisiae} genes characterized with genetic or protein interactions found that there is an aversion to studying lesser-known genes in networks. As well, in a study of three human protein families, many of the genes that have recently been discovered to have association with complex disease, through methods such as genome wide association studies (GWAS), are understudied in the present compared to the small number of historically heavily studied genes. In this study we explore possible causes of and diversion from this preferential bias with gene conservation and human genes being disease-associated. We find there is some evidence of conservation driving biases in research effort for essential genes in \textit{Saccharomyces cerevisiae}, but inconclusive evidence in other organisms. We look for effects of disease association through Mendelian and complex diseases in a historical, pre-GWAS, and contemporary, post-GWAS, context. Within both contexts we find that Mendelian disease genes may drive preferential study bias. For contemporary research effort we utilize a model of publication rates and find that there are individual GWAS genes that tend to be investigated more than predicted compared to non-GWAS genes. It appears that the proportion of GWAS genes that had highly unexpected increases in publication rate compared to model predictions rose fairly quickly but has been declining. Our analysis suggests that GWAS has had a small impact on what genes some scientists study despite preferential study biases. However GWAS gene-disease association's impact on research effort appears to be declining, possibly due to scientists not being as interested in GWAS results as time goes on.

A recurring theme in biology is the aggregation of mRNA-protein complexes (mRNPs) into higher order assemblies. Often these complexes play important roles in the regulation of gene expression, but the function of the conserved cytoplasmic mRNP assemblies - P bodies and stress granules, is not known. It is believed that the misregulation of granule assembly is related to disorders like Amyotrophic Lateral Sclerosis and Frontotemporal Lobe Degeneration. Determining the complete composition of these granules may hold the key to understanding the function and mechanism of assembly of these granules. This work describes multiple approaches taken to identify new protein and mRNA components of P bodies and stress granules. New members of the P body and stress granule proteome reveal a role for these granules in diverse cellular processes including signal transduction, transcription and metabolism. Additionally, a new stress granule resident complex - the CCT complex, was also identified as a novel regulator of granule disassembly. This work also describes the first purification scheme for stress granules and presents a new system for in vitro study of stress granules. Together, the findings shed new light on the composition, function, structure and regulation of P bodies and stress granules in yeast.

Viruses infecting bacteria (phages) are the most abundant and ubiquitous entities on Earth and likely critical to any ecosystem, as they influence nutrient cycling, mortality and evolution. Ultimately, their impact depends on whether phage—host interactions lead to intracellular phage coexistence (temperate phage) or cell death (lytic phage). Temperate phages in the lysogenic cycle replicate their genome (either integrated into the host chromosome or extrachromosomally), until induced to become lytic, when they create and release progeny via cell lysis. While knowledge on lytic versus lysogenic outcomes is vast, it largely derives from few model systems that underrepresent natural diversity. Further, less is known about the efficiency of phage—host interactions and the regulation of optimal versus sub-optimal lytic infections, which are predicted as relevant under environmental (nutrients, temperature) and host (availability, density) conditions that are common in the ocean. In this dissertation I characterize the phage—host interactions in a new marine model system, phage ϕ38:1 and its Cellulophaga baltica bacterial host, member of the ubiquitous Bacteroidetes phylum. First, I show ϕ38:1’s ability to infect numerous, genetically similar strains of the C. baltica species, two of which display contrasting infection outcomes–lytic versus sub-optimally lytic or lysogenic on the original versus alternative hosts, respectively. Second, I collaboratively apply new gene marker-based approaches (phageFISH and geneELISA) to study ϕ38:1’s infection at the single-cell level and show that it is sub-optimal on the alternative host, rather than lysogenic. Third, I collaboratively develop whole-genome transcriptome datasets for ϕ38:1 infecting both, the optimal and sub-optimal hosts, to characterize the cellular response to infection and hypothesize potential transcriptional and post-transcriptional regulation of the sub-optimal infection. Together, these findings advance our knowledge of naturally-occurring phage—host interactions with a focus on nearly-unstudied sub-optimal infections.

Sex differences exist in the progression of heart disease, as premenopausal women are protected from developing severe hypertension, aortic stenosis, myocardial infarction and hypertrophic cardiomyopathies. The susceptibility and progression of cardiovascular disease increases in post-menopausal women. This is at least partially underlined by a pronounced decrease in circulating estrogen levels. Estradiol (E2), the most abundant estrogen in premenopausal women, is known to be cardioprotective. Recently, AMP-activated protein kinase (AMPK) has emerged as a prominent player in the development of cardiac hypertrophy and heart failure. AMPK is central to the energetic metabolism of the cell and is activated in response to energy deprivation. E2 has been shown to activate AMPK, by yet an unknown mechanism. The first part of this dissertation focuses on describing the molecular mechanism behind this AMPK activation. We found that E2 activates AMPK through a non- genomic pathway and involves direct interaction of classical estrogen receptors (ERα and ERβ) with the α-catalytic subunit of AMPK. These receptors also associate with the upstream kinase LKB1, which is required for E2-dependent activation of AMPK. Furthermore, the two estrogen receptors play opposite roles, where ERα increases AMPK activation, and ERβ acts as a repressor, inhibiting AMPK phosphorylation. To translate our findings to heart disease, the next step was to determine the effect of ovarian failure, underlined by E2 loss, on AMPK signaling during the progression of cardiac hypertrophy. We hypothesized that ovarian failure decreases cardiac AMPK signaling, translating in worsening of hypertrophy. We found that the status of cardiac AMPK signaling depends on the nature of the hypertrophic stimulus and the timing of ovarian failure in relation to the onset of hypertrophy. Furthermore, we did not detect any differences in the development of cardiac hypertrophy between wild type mice and mice in ovarian failure, which most likely occur down the line. In summary we described a novel mechanism of AMPK activation by the hormone E2. We also explored the effect of estrogen loss on cardiac AMPK activity, and found that it is dependent on factors such as the pathological state of the heart and timing of the intervention. These findings add to our understanding of the molecular mechanisms behind sex differences in energy handling and in the future could be translated into better therapeutics for the treatment of cardiac pathologies.

A cell's ability to sense and respond to mechanical signals highlights the significance of physical forces in biology; however, to date most biomedical research has focused on genetics and biochemical signaling. We sought to further understand the physical mechanisms that guide the cellular migrations that occur in a number of biological processes, such as tissue development and regeneration, bacterial infections and cancer metastasis. We investigated the migration of single cells and determined whether the biomechanics of these cells could be used to elucidate multi-cellular mechanisms. We first studied Borrelia burgdorferi (Bb), the bacterium that causes Lyme disease. We created a mathematical model based on the mechanical interactions between the flagella and cell body that explained the rotation and undulation of the cell body that occurs as the bacterium swims. This model further predicts how the swimming dynamics could be affected by alterations in flagellar or cell wall stiffnesses. Fitting the model to experimental data allowed us to calculate the flagellar torque and drag for Bb, and showed that Treponema pallidum (Tp), the syphilis pathogen, is biomechanically similar to Bb. Next, we used experimentally-determined parameters of Bb's motility to develop a population-level model that accounts for the morphology and spreading of the "bulls-eye" rash that is typically the first indicator of Lyme disease. This work supported clinical findings on the efficacy of antibiotic treatment regimes. Finally, we investigated the dynamics of epithelial monolayers. We found that intracellular contractile stress is the primary driving force behind collective dynamics in epithelial layers, a result previously predicted from a biophysical model. Taken together, these findings identify the relevance of physics in cellular migration and a role of mechanical signaling in biomedical science.

A fundamental problem in developmental and evolutionary biology is understanding the developmental genetic basis of morphological diversity. The current paradigm holds that a genetic and developmental program, or developmental genetic "toolkit", conserved across hundreds of millions of years patterns development in all metazoans. However, outside of a few well-characterized signal transduction pathways and developmental processes, overly broad strokes have been used to paint this "toolkit" metaphor as a hypothesis. Arthropoda, one of the largest groups of metazoans, represent the most morphologically diverse groups of metazoans, making them of particular interest for studies of morphological diversity and its evolution. Arthropoda is also home to one of the most well-understood model systems for developmental and genetic studies, the fruit fly Drosophila melanogaster. However, Drosophila is highly derived among arthropods with respect to the molecular genetic mechanisms that function during its development. As it is expected that all arthropods have access to the same development "toolkit", some changes are expected based on the observable differences in morphology, making arthropods extremely powerful tools for comparative genomic and molecular genetic studies. In this dissertation I characterize how modifications to the developmental "toolkit" contribute to the evolution of morphological diversity using emerging model arthropod systems. First, as part of a collaboration, I show that several genes expected to be conserved in all arthropods, belonging to the Hox family of transcription factors, have been lost from the genome of a phylogenetically basal arthropod, the two-spotted spider mite Tetranychus urticae. Second, I perform a genomic survey and find an overall reduction in the conservation of Drosophila orthologs from several major signal transduction pathways in the Tetranychus genome in comparison with findings from previous insect surveys. Third, I show that arthropod Hox genes, expected to be found in a tightly linked genomic cluster in most arthropod genomes, are not as tightly clustered as previously thought. Fourth, I show that changes in the genomic arrangement of Tetranychus Hox genes correspond with shifts in their expression and morphological change. Finally, I show the terminal Hox gene Abdominal-B is required for proper axial elongation and segment formation (both segment identity and number) during embryogenesis and metamorphosis in the red-flour beetle Tribolium castaneum. Taken together, these findings advance our knowledge of the evolution of morphological change, with a primary focus on Hox genes and their contribution to axial patterning during development.