Browsing by Subject "mTOR"

Now showing 1 - 3 of 3
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    Effects of placental mTOR alteration on the pancreatic beta cell mass of embryonic day 17.5 mice
    (2022-05) ORIBAMISE, EUNICE
    The fetal environment during development is a strong determinant for health in adulthood. Pancreatic beta cells development in-utero has an important role in the predisposition to Type 2 Diabetes (T2D). The pancreatic endocrine cells are highly sensitive to nutrient flux during development and very early in life. Clinical studies suggest that signaling of mechanistic target of rapamycin (mTOR) in the placenta regulates fetal birth weight and the offsprings’ metabolic course, partly by modulating maternal to fetal nutrient flux.This study aims at exploring the link between placental mTOR activity and beta cell mass at embryonic day (e) 17.5, a gestational period in which beta cells become more abundant in the pancreatic islets. Pancreatic beta cells are known to be impacted by amino acid (AA) levels during development. We hypothesized that alteration of mTOR in the placenta will affect AA transporters (which are essential for amino acid transport for beta cell growth in-utero), thus affecting the gestational growth and development of beta cells in the offspring. Within beta cells, AAs promote mTOR signaling, which regulates cell growth, proliferation, and protein synthesis. We tested our hypothesis by genetically deleting mTOR or activating mTORC1, via deletion of its negative regulator, Tuberous Sclerosis Complex (TSC2), in placental trophoblast cells using placental-specific Cre recombinase, and by characterizing the effects of placental mTOR on pancreas morphology at e17.5. Interestingly, we revealed that the placental-specific knock out of mTOR led to an increase in protein expression of amino acid transporter SNAT-1 and beta cell mass in e17.5 female offspring. However, the placental-specific knock out of TSC2 showed no effect on total beta cell mass of e17.5 embryos. These results suggest that placental mTOR may regulate AA flux to the growing pancreas, but additional experiments are needed to better understand amino acid placental functions and its effects on pancreatic progenitors and beta cell development.
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    The Impact of Iron Deficiency During Development on Mammalian Target of Rapamycin Signaling, Neuronal Structure, and Learning and Memory Behavior
    (2010-11) Fretham, Stephanie
    Iron deficiency (ID) is the most common micronutrient deficiency, affecting an estimated 2 billion people world wide including 20-30% of pregnant women and their offspring. Many human studies have demonstrated negative effects of early life ID on learning and memory which persist beyond the period of ID despite of prompt iron treatment, observations which are supported by rodent models of early iron deficiency anemia (IDA). In spite of a large, observational literature the mechanisms through which early ID causes acute and persistent brain dysfunction are largely unknown. Mammalian target of rapamycin (mTOR) signaling is an attractive candidate for mediating the effects of early ID because it integrates cellular metabolic status to regulate fundamental aspects of cellular growth and differentiation. The overall goal of the current studies is to understand the role of iron in regulating mTOR signaling during a critical period of development in the hippocampus by using unique genetic mouse models of hippocampal ID to: 1) Determine when iron is required for hippocampal development 2) Determine the role of iron in mTOR signaling 3) Manipulate iron and mTOR to determine effects on hippocampal structure and behavior. The findings from these experiments demonstrate that mTOR signaling is upregulated by neuronal ID during the same period that rapid hippocampal development requires large amounts of iron. Additionally, rescue of behavioral outcomes in adult animals following restoration of mTOR signaling (through either timely iron repletion or pharmacological suppression) provides functional evidence for a connection between mTOR and the persistent effects of early ID.
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    Studies on the regulatory mechanism of the ULK1 complex in the induction of autophagy
    (2012-10) Cao, Jing
    Autophagy, an evolutionarily-conserved cellular process through which organelles and macromolecules are degraded in the lysosome, is induced under nutrient starvation or other unfavorable growth conditions. Unc51-like kinase 1 (ULK1) is a serine/threonine protein kinase that plays a key role in the autophagy induction process, but how ULK1 is regulated by cellular signals for induction of autophagy and how ULK1 regulates the downstream processes in autophagy remain poorly understood. ULK1 interacts with Atg13, focal adhesion kinase family interacting protein of 200 kD (FIP200) and Atg101 to form a large protein complex involved in early steps of the autophagy induction process. To better understand the function of the ULK1 complex, my thesis work has sought to identify binding proteins of the complex. Through a yeast two hybrid screen using a human fetal brain cDNA library with Atg13 as bait, a protein named MCF.2 cell line derived transforming sequence-like 2 (MCF2L2) was identified. Through co-immunoprecipitation and in vitro binding assay, MCF2L2 was determined to directly interact with Atg13 via its N-terminal region independently of ULK1. Knockdown of MCF2L2 inhibited the formation of autophagosome and autophagy flux and led to accumulation of p62/sequestosome-1, a protein degraded through autophagy. Knockdown of MCF2L2 also suppressed the aggregation of WD-repeat protein interacting with phosphoinositides-1, an autophagic isolation membrane marker. MCF2L2 contains a putative Rho-guanine nucleotide exchange factor (GEF) domain in the middle and has a sequence similarity to MCF2L and MCF2, the well-known Rho-GEFs. MCF2L2 overexpression induced a moderate increase in the active forms of Rho GTPases and MCF2L2 colocalized with actin related protein 3, the actin nucleation factor that is regulated by Rho GTPases, implying that MCF2L2 potentially contains GEF activity. MCF2L2 knockdown partially suppressed the distribution of Atg9 from trans-golgi network to the cytoplasm in response to starvation, a process that may depend on actin cytoskeleton. Combined, these results suggest that MCF2L2, as a component of the ULK1 complex, might play an important role in mediating signal transduction between the actin cytoskeleton and autophagy induction.