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Acute kidney injury (AKI) is a common life-threaten disease without specific treatment. Tissue protective receptor, erythropoietin receptor/β common receptor (EPOR/βcR), mediates the migration of injury to adaptive repair in ischaemia/reperfusion (IR)-induced AKI. This project further studied the dynamic expression, role and mechanism of EPOR/βcR, its association with complement properdin, in murine IR kidneys and tubular epithelial cells (TECs). The potential of EPOR/βcR guided cell/organ target caspase-3 (executing apoptosis and inflammation) siRNA delivery was also explored. A time course model of renal IR from 6 h to 1 w uncovered the gradual increase of EPOR/βcR in kidneys, localized mainly in TECs, providing an evidence of EPOR/βcR highly expressed in damaged TECs at the early stage of injury. At 72 h post IR, EPOR/βcR expression was increased greater in properdin deficient (PKO) mouse kidneys with aggravated damage, in which properdin might act as a pattern recognition molecule facilitating phagocytosis and clearing damaged cells. HBSP, a ligand of EPOR/βcR, preserved renal function andstructure in wild type and PKO mice, and further reduced interstitial apoptosis in PKO IR kidneys. PKO further elevated EPOR/βcR enhanced the renoprotection of HBSP by complementing deficient phagocytosis, reducing apoptosis and inflammation and promoting repair. Isolated TECs from wild type and PKO mice provided additional evidence that properdin released from TECs, but not serum, affected apoptosis and phagocytosis. In the 2-w renal IR model, HBSP and caspase-3 siRNA administered at the onset of injury synergistically decreased injury mediators and preserved kidney structure. In addition, fluorescein iridium labeled HBSP was reveled on TECs, and the apical surface of tubules predominately in IR kidneys. EPOR/βcR, therefore, associated with properdin, phagocytosis, apoptosis and inflammation, limits injury and promotes repair. Cell/organ target siRNA delivery might be achieved by siRNA conjugated HBSP guiding by highly expressed EPOR/βcR in injured cells of the organ.

Inhibin hormones play a significant role in reproductive health, regulation of bone metabolism, and adrenal gland growth. Inhibins act to suppress follicular stimulating hormone through a negative feedback loop across the hypothalamic-pituitary-gonadal axis. Decline in inhibin levels during menopause correlates with anomalous reproductive hormone levels and osteoporosis. Inhibins are grouped with the larger Transforming Growth Factor Beta (TGF-β) superfamily which is responsible for regulating cell growth/differentiation and adult tissue homeostasis. TGF-β ligands form disulfide-linked dimers that potentiate intracellular gene transcription through the assembly of a ligand-receptor complex on the cell surface. Inhibins were the first identified naturally forming heterodimers in the family. Inhibins share a gene with activins, other well-established TGF-β ligands. Inhibins are distinct beyond the heterodimeric makeup in that inhibins form non-signaling complexes with activin receptors acting as antagonists of activin signaling. Expression and purification of inhibins for study is a challenging process since inhibins and activins are co-expressed due to their common gene. It is unclear how inhibins can potently and specifically target activin signaling without affecting other TGF-β ligands. The purpose of the following thesis work is to gain a better understanding of the inhibin mechanism of antagonism including insights into TGF-β ligand dimerization and signaling modulation. These studies use activin as a model for comparison when characterizing inhibin engagement with shared activin-binding partners including receptors or other antagonists such as follistatin. Collectively, these findings have contributed to the previously lacking specifics of inhibin function and the understanding of the growing significance of heterodimers in the TGF-β superfamily.

Actin is a highly conserved protein that assembles into many three-dimensional structures, each with a unique subset of actin-binding proteins that produces a unique three-dimensional architecture capable of performing a specific function. These actin networks are central to cellular processes such as motility, adhesion, endocytosis, and division. Yet how actin filaments can recruit the correct subset of actin-binding proteins while avoiding the incorrect ones remains unclear. Recent literature demonstrated that in non-muscle cells, a different tropomyosin isoform(s) is present at every single actin network. Tropomyosins are coiled-coil proteins that regulate access to actin filament binding sites and now widely believed to determine which actin structure will be built. Despite this, we know very little about the assembly and function of tropomyosins on actin filaments. This is partly due to the fact that mammalian cells express over 20 different tropomyosin isoforms. In the Mullins lab, we have chosen Drosophila melanogaster S2 cells as the model system for the study of tropomyosins because S2 cells are easy to grow, highly susceptible to gene inhibition using RNAi, have well-characterized actin networks, and most importantly, only express three tropomyosin isoforms. In this thesis, we investigate how these Drosophila tropomyosins assemble on actin filaments. In Chapter 1, we designed a microfluidic system that allows us to manipulate microscopy experiments at a level never before achieved in the Mullins lab. In Chapter 2, we characterize binding and dynamic properties of the classic Drosophila tropomyosin Tm1A. In Chapter 3, we discovered how to get Drosophila tropomyosins Tm1J and Tm2A to bind actin filaments and then characterize these tropomyosins.

Bivalent drug design is an efficient strategy for increasing potency and selectivity of many drugs. We devised a strategy to prepare agonist-benzodiazepine heterodimers that could simultaneously bind to agonist and BZD sites of the GABAAR. We synthesized a benzodiazepine-MPEG model compound that relied on physiological GABA to elicit flux. We established that a tether at the N1 position of the BZD would not prevent binding to the receptor. However, coupling of GABA amides with long chain PEG tethers studied by another group member resulted in complete loss of agonist activity. We therefore ceased research in this particular area. 1,4-Benzodiazepin-2,5-diones display a wide range of pharmacological activities. Compounds containing the tricyclic proline-derived subtype have received attention as potent anxiolytic agents and as starting materials for anthramycin-inspired anticancer agents. More recently enantiopure (S)-proline-derived 1,4-benzodiazepin-2,5-diones have been recognized as selective α5 GABAA receptor ligands. Despite the impressive diversity of 1,4-benzodiazepine-2,5-diones prepared to date, enantiopure examples possessing a quaternary stereogenic center have been largely unexplored. "Memory of chirality" (MOC) is an emerging strategy for asymmetric synthesis. This technique enables the memory of a sole chiral center in the substrate to be retained in a process that destroys that center. We have used this technique to prepare a library of quaternary proline-derived, thioproline-derived and hydroxyproline-derived 1,4-benzodiazepin-2,5-diones, in high ee. We have developed an efficient synthetic method for preparing oxaproline-derived 1,4-benzodiazepin-2,5-diones in high yields, and by applying the MOC strategy we have prepared quaternary derivatives in acceptable %ee. We envision oxaproline-derived 1,4-benzodiazepin-2,5-diones may exhibit similar or more potent pharmacological properties than proline-derived 1,4-benzodiazepin-2,5-diones. Using density functional theory (DFT) methods, we modeled the formation of an enantiopure, dynamically chiral enolate intermediate and the slow racemization of the enolate on the alkylation reaction time scale.