The growth of vertebrates is primarily regulated by the growth hormone-insulin-like growth factor (GH-IGF) system, but not in isolation. The central question of this dissertation was how do other hormones peripheral to the GH-IGF system interact with the system, including feedbacks by GH and IGF themselves on various tissues in rainbow trout (Oncorhynchus mykiss)? The representative hormones selected were thyroxine, cortisol, and the sex steroids testosterone and estrogen, along with GH and IGF. These hormones were chosen because they are known to affect overall growth and development during specific life events, but exactly what target genes and what mechanisms are involved are only at the early stages of being delineated in fish. Liver and gill tissues were selected as representative tissues to assess the in vitro effects on growth-related genes of the GH-IGF system. A total of more than thirty experiments were conducted, including time- and concentration-response, inhibitory studies, hormone combination studies, and radio-receptor binding assays. Hormones were applied to whole tissue cultures and real-time quantitative-PCR was used to measure hormonal effects on GHR, IGF, and IGFR1 genes. Microsomal preparations were treated with selected hormones and radio-labeled GH or IGF. A gamma counter was used to measure receptor-ligand activity. GH and IGF were found to possess autocrine and/or paracrine actions in self-regulating target growth genes. Thyroxine had no direct effects on targeted growth genes but may interact with other molecules or hormones to elicit its effects on growth and development. Cortisol directly influenced target growth genes in a tissue-specific and isoform-specific manner. Finally, sex steroids differentially regulated the growth genes: estradiol inhibited growth genes while testosterone directly stimulated growth genes. These findings contribute to understanding how hormones peripheral to the GH-IGF system interact with the growth system. / National Science Foundation grant IOS 0920116 to Dr. Mark Sheridan

Progression of metazoans through different developmental programs requires temporal control that is achieved by molecules originating from endocrine tissues that diffuse throughout the whole body of the animal to coordinate program execution by activating cell specific gene expression patterns. These programs then define cascades of successive, distinct developmental stages or the choice between alternative fates for the same stage. A model for this developmental control is found in the nematode C. elegans, where environmental cues signal through insulin and TGF-beta cascades to regulate the daf-12/nuclear hormone receptor (NHR) ligand synthesis that then coordinates organism wide developmental timing and fate choice. For cell intrinsic aspects of C. elegans temporal control of development, microRNAs play an important role but their connection to organism wide endocrine control is unknown. This work shows how the DAF-12/NHR directly activates let-7 family microRNAs during the L3 stage to repress L2 stage activator hbl-1 to prevent L2 stage programs from reoccurring. The interaction of upstream transcription factors with the downstream cis-regulatory elements in promoters of the let-7 family microRNAs are further analyzed in detail and identify potential DAF-12 coregulators that might connect daf-12 endocrine signaling also to later stage developmental control. These observations are the first to integrate microRNAs into establishedendocrine signaling cascades. In addition they reveal specific details about how organism wide upstream, endocrine signaling pathways induce downstream cell intrinsic changes of gene expression and developmental progression. This work postulates a "molecular switch" that actively drives stage transitions, consisting of a NHR that directly activates microRNAs to actively repress mediators of old stages while directly activating translation of protein coding genes mediating the new stage.

No description available.

<p> Obesity is a growing concern as 42.3% of people in the U.S were considered obese in the years 2017- 2018. Little is known about the genetic components that contribute to weight gain. In humans, the hormone glucagon is a major contributor to the body’s energy demand as it helps break down lipids. Therefore, learning more about this pathway could enable a range of therapeutics. In fact, studies have shown that glucagon treatments have helped patients with both weight loss and appetite suppression. In this project, we analyzed candidate genes that modify the glucagon pathway in <em>Drosophila melanogaster.</em> We reduced the expression of the fly version of the glucagon receptor (AKHR) in our model. This induces fat retention in the L3 larvae, which mimics obesity in humans. We then crossed our model to the DGRP and looked for natural variation in fat content using a density assay. The density assay examines the relative fat levels of the larvae by slowly increasing the amount of sucrose in water. This enables us to observe whether we have lean larvae which float later or fat larvae which float early on. We used the variation in floating concentration to identify candidate modifier genes through GWA or genome-wide association study. We crossed our <em>AKHR</em> RNAi model to RNAi for various candidate modifier genes that may enhance or suppress fat retention. We screened these candidates initially with the same density assay used in the original study. This resulted in four candidate genes that significantly impacted the density of the larvae: <em>THADA</em>, <em>AmyD</em>, <em>GluRIIC</em>, and <em>CG9826</em>. We further characterized these candidates using biochemical assays to analyze stored metabolites such as triglycerides, glucose, glycogen, and protein. These have been further analyzed under control, high sugar, and high fat conditions to see if the larvae are resistant to environmental changes. <em>CG9826</em> showed significant increase in stored fats across all environments. <em>THADA</em> RNAi showed an increase in fat in the high fat environment. Overexpression of <em>THADA</em> showed a decrease in fat storage in the high fat environment. Our goal is to advance our understanding of the glucagon signaling pathway, obesity, and lipid metabolism. We are also hopeful to provide candidate genes that can be regarded as future therapeutic targets. </p>

No description available.

<p> The liver X receptors (LXRs) are members of the nuclear hormone receptor (NHR) superfamily of transcription factors and are activated by oxysterols. As such, LXRs act as "cholesterol sensors" and play an integral role in cholesterol homeostasis by modulating the expression of genes involved in lipid transport and metabolism as well as inflammation. </p> <p> This thesis begins by describing the modulation of LXR transactivation by PKC. Specifically, transactivation by LXRα is decreased upon activation of PKC signalling pathways as assessed by LXR reporter gene analysis and endogenous target gene expression. These findings reveal a mode of regulation of LXRα that may be relevant to disease conditions where aberrant PKC signalling is observed. </p> <p> The second and third part of the thesis turns the attention to the role of LXR in human airway smooth muscle (hASM), a crucial effector cell in asthma progression. For the first time, research described here indicates that primary human ASM cells express functional LXRs. Moreover, LXR target genes ABCA 1 and ABCG I were highly induced upon the addition of LXR agonists leading to enhanced cholesterol efflux to apoAI and HDL, a process dependant entirely on ABCA I. Furthermore, activation of LXR inhibited the expression of multiple cytokines in response to inflammatory mediators and inhibited the proliferation and migration of hASM cells, two important processes that contribute to the airway remodelling observed in the asthmatic lung. </p> <p> This body of work suggests that modulation of LXR offers prospects for new therapeutic approaches in the treatment of asthma. Furthermore, it establishes a critical role for ABCA 1 in lipid transport in ASM cells and suggests that dysregulation of cholesterol homeostasis in these cells may be important. These findings have broad implications in the association of hypercholesterolemia and AHR and places LXR at the forefront of novel therapeutic avenues to treat inflammatory lung disease. </p> / Thesis / Doctor of Philosophy (PhD)

No description available.

No description available.

No description available.

No description available.