ABSTRACT Thyroid hormones play an important role in early development and growth, as well as adult phenotypes affecting oxidative metabolism, cardiac output, energy balance, fertility, CNS function, and others. Complete absence of thyroid hormone is incompatible with life. Thyroglobulin (Tg) is the precursor protein used in the biosynthesis of thyroxine (T4) and tri-iodothyronine (T3). Tg is synthesized in the endoplasmic reticulum (ER) of thyrocytes and undergoes intracellular trafficking and secretion to the apical (extracellular) lumen of thyroid follicles, where it is iodinated, triggering formation of T4 and T3. The function of thyrocytes is regulated by pituitary-derived Thyroid Stimulating Hormone (TSH), which stimulates thyrocyte transcription and translation of hormonogenic genes, as well as thyrocyte proliferation resulting in gland growth. Humans with biallelic TG mutation (and murine models) have congenital hypothyroidism; in this disease, the mutant Tg protein misfolds in the ER and fails to undergo anterograde trafficking in the secretory pathway, resulting in insufficient thyroid hormone synthesis with an increase of circulating TSH. Additionally, misfolding of mutant Tg triggers severe ER stress, which is accompanied by thyrocyte cell death (although growth can outpace death, favoring goiter development). Notably, the dead thyrocytes are found in the apical follicle lumen. The finding of dead thyrocytes in the follicle lumen either signifies cell death caused by the severe ER stress followed by extrusion into the apical lumen, or hyperstimulation by TSH triggering cell growth into the follicle interior that exposes thyrocytes to an inhospitable luminal environment, which causes cell death. In my F30 project, I will study homozygous TG-KO mice (created via targeted gene disruption). My preliminary data indicate that the animals have severe hypothyroidism, yet they survive to adulthood despite expressing no Tg mRNA (by qPCR) or Tg protein (by Western blotting). I seek to address two general questions. First, since Tg is by far the most highly expressed thyroid secretory protein of the ER, do the thyrocytes of TG-KO animals also experience thyrocyte cell death, and if so, does this involve ER stress or occur independently of ER stress? The result of this question can help us understand the real driving force behind the thyroid cell death observed in hypothyroid humans synthesizing mutant Tg. Second, as some level of thyroid hormone synthesis is required for survival, how do animals lacking Tg protein make thyroid hormone? Altogether, this project affords me significant educational opportunity, as I will develop the techniques and tools of basic cell biology (ER stress analysis, proliferation and cell death analysis) with a deeper understanding of the growth and function of the thyroid gland (a critical endocrine system), with clear clinical implications.