Uterine disorders are common and can be debilitating, but they are poorly understood. Endometriosis is the most common cause of secondary dysmenorrhea, yet existing treatments have substantial side effects and are not able to fully resolve pain for most patients. Many studies have shown that endometrial tissue from individuals with endometriosis has differential gene expression and DNA methylation patterns compared to endometrial tissue from controls. Data sharing efforts have made large amounts of genetic and epigenetic data available to researchers. However, it is technically challenging to compare datasets from different platforms and study designs, and these resources are underutilized. In this dissertation, I explore methods of combining DNA methylation and gene expression data to gain additional insights into endometrial disorders. First, I demonstrate that DNA methylation age can be used to understand ectopic endometriosis lesions. Then, I correlate DNA methylation age changes caused by hormonal treatment with differential gene expression in endometriosis. Given a candidate pathway, I then use datasets of infertility to determine whether these expression changes could provide insight into clinical phenotypes. Finally, I explore whether clinical phenotypes of menstrual pain resistant to non-steroidal anti-inflammatory drugs (NSAIDs) can predict altered signaling of related pathways in menstrual-derived tissues. This work explores methods of utilizing the vast amounts of genetic and epigenetic data that have already been generated, while taking into consideration both the unique dynamic properties of uterine tissue and the aspects of uterine dysfunction that are most disruptive to quality of life.

Hematopoiesis is a highly complex developmental process that is remarkably conserved at the molecular level across higher order eukaryotes. An organ termed the lymph gland resides within the Drosophila larva which is the site of the majority of hematopoietic processes in the organism. The lymph gland contains a population of differentiating blood cells in addition to a pool of quiescent hematopoietic progenitors that are maintained by a small niche. While there has been a number of signaling pathways and mechanisms characterized in the regulation of lymph gland homeostasis, questions still remain unanswered in regards to the nature and requirement of progenitor cells and the mechanisms that promote differentiation in the lymph gland.

In this dissertation, we use genetic approaches to characterize the roles of the Hippo Pathway effectors Yorkie and Scalloped in regulating the maintenance and differentiation of hematopoietic progenitors through modulation of conserved signaling pathways. We show that differentiation of the crystal cell lineage of hemocytes is reliant on Yorkie and Scalloped dependent regulation of the Notch ligand Serrate in lineage specifying cells. Furthermore, Scalloped regulates organ size and differentiation of progenitors through modulation of the secreted factor PVF2 and its receptor PVR, respectively. Finally, we utilized an immune challenge model to interrogate the function of Yorkie and Scalloped in the cell fate decisions that are incurred during the cellular immune response induced by wasp parasitizaion. Using this immune challenge model, we demonstrated that wasp parasitization induces unique changes to Yorkie and Scalloped expression in the lymph gland. We also report that Serrate down-regulation in lineage specifying cells is critical for the cellular immune response, leading to a loss in crystal cell differentiation. Finally, we demonstrate a unique mechanism where-in Scalloped expression is enforced in the Posterior Signaling Center (PSC) of the lymph gland upon immune challenge by wasp parasitization as Scalloped function is required for maintenance of the PSC and differentiation of lamellocytes in the cellular immune response.

Measurement of head circumference is a common practice in the care of a developing child as it is a high yield, low cost clinical test to monitor brain development. Macrocephaly, or head circumference greater than two standard deviations above the mean (>98th %ile), can be caused by abnormal brain growth, impaired cerebral spinal fluid drainage, or bone growth. While head circumference is a normally distributed trait with normal variations in the population, extreme variants in head circumference are often associated with underlying disease pathogenesis and, in many cases, accompanying behavioral phenotypes. Understanding the causes of reproducible macrocephaly allows us to better grasp the governing principles of brain growth and furthermore the associated behavioral abnormalities. The clinical workup for a child presenting with macrocephaly is important as it not only dictates testing, but also can be indicative of a more insidious disease process with comorbidities. With the wide spread use of massively parallel sequencing technologies the genetic etiologies of numerous de novo, sporadic, and rare genetic syndromes have been recently discovered. We have used this powerful detection tool to identify mutations in a cohort of cases with Macrocephaly. We present the phenotypic information for cases with identified mutations in the genes PTEN, Ptch1, SUFU, mTOR, and DICER1. Furthermore we have modeled these mutations in human cell lines and show that their mechanism of action converges on activation of mTOR signaling. Lastly we demonstrate that infection of with Toxoplasmosis Gondi mirrors this mTOR activation and explains why congenital infections manifest with macrocephaly. We have dissected the exact molecular mechanism resulting from each mutation and proposed novel precision medical approaches to treat these and future cases. The work presented in this dissertation demonstrates the power and utility of a precision medical approach to patients presenting with rare disease and abnormal phenotypes.

The tight regulation of hematopoietic progenitors is required to continuously supply blood cells during the lifespan of an organism and to make cells rapidly available in response to stress conditions, such as injury or infection. In Drosophila, a large pool of multipotent blood progenitors are maintained in the larval hematopoietic organ, the lymph gland (LG), by a complex network of signaling pathways that are mediated by niche-, progenitor-, or differentiated hemocyte-derived signals. Despite extensive studies in the LG during recent years, several important questions remain unanswered. In particular, the nature of the signals required to regulate the stereotypical proliferation and differentiation of LG blood progenitors during larval development remain unidentified.

In this dissertation we identify the Target of Rapamycin (TOR) and Fibroblast Growth Factor Receptor (FGFR) signaling pathways as critical regulators of growth and differentiation of the Drosophila LG. In particular, we demonstrate that the tumor suppressors TSC and PTEN control blood progenitor proliferation through a common TOR- and 4EBP-dependent pathway. Tsc or Pten deficiency in progenitors increases TOR signaling and causes LG overgrowth by increasing the number of actively dividing cells during a critical window of growth. These phenotypes are associated with increased reactive oxygen species (ROS) levels in the LG, and scavenging ROS in progenitors is sufficient to rescue overgrowth. Differences between Tsc and Pten function become apparent at later stages. While loss of Tsc specifically expands the number of intermediate progenitors and limits terminal differentiation, absence of PTEN induces the myeloproliferative expansion of terminally differentiated blood lineages. This increased malignancy is associated with non-autonomous TOR activation within peripheral differentiating hemocytes, culminating in their premature release into circulation.

Since reduced TOR signaling in progenitors also induces increased differentiation, our findings demonstrate that although TOR signaling is sufficient, it is not required for inducing differentiation in the LG during development. In contrast, we identified the Drosophila FGFR, Heartless (Htl), and its two ligands, Pyramus and Thisbe, to be both required and sufficient for inducing blood progenitor differentiation in the LG. This Htl-mediated differentiation response of progenitors is dependent on two transcriptional regulators, the ETS protein, Pointed, and the Friend-of-GATA protein, U-Shaped, as well as TOR signaling, which is required specifically downstream of Thisbe-mediated Htl activation. Finally, we identify the Drosophila heparan sulfate proteoglycan (HSPG), Trol, as a critical negative regulator of FGF signaling in the LG, and suggest that sequestration of this potent differentiation signal by the extracellular matrix is a unique mechanism employed in blood progenitor maintenance in the Drosophila LG.