The mechanism of action and regulation of hepcidin

Iron is vital for bodily function but is potentially toxic and accordingly, tightly controlled in the body. Systemic iron homeostasis is regulated by the liver-produced hormone hepcidin which controls cellular iron efflux through ferroportin, the only known iron exporter. Ferroportin delivers iron into the plasma from duodenal enterocytes which absorb dietary iron, from macrophages in the spleen and the liver which recycle iron from senescent erythrocytes, and from hepatocytes in the liver which store iron and deliver it to plasma when systemic iron requirements increase. Ferroportin is the hepcidin receptor: hepcidin binds to ferroportin, leading to its endocytosis and degradation, thus preventing the entry of iron into plasma. Regulation of hepcidin production is crucial for iron homeostasis. Iron, inflammation, and erythropoiesis are three factors which regulate hepcidin transcription. Iron and inflammation increase hepcidin expression whereas erythropoiesis suppresses it. Dysregulation of hepcidin leads to iron disorders. Hepcidin deficiency causes unrestrained iron absorption resulting in iron overloading disorders, such as hereditary hemochromatosis and β-thalassemia. However, hepcidin excess leads to iron restriction causing anemia, such as iron-refractory iron deficiency anemia.Neither the mechanism of hepcidin action nor the regulation of hepcidin is completely understood, and these are the two areas on which I focused my research. Chapter 1 elucidates the structural basis of hepcidin-Fpn interaction whereas Chapters 2 and 3 examine potential signaling pathways which regulate hepcidin transcription. More specifically, Chapter 1A examines the structural basis of genetic resistance to hepcidin and details an alternative hepcidin mechanism of action. Chapter 1B provides preclinical support for a potential therapy to treat a genetic iron overload disease caused by resistance to hepcidin. Chapter 2 examines the signaling pathway used by erythroferrone, a protein hormone involved in hepcidin regulation by erythropoiesis, and Chapter 3 examines a candidate hepcidin regulator, Nrf2, a transcription factor induced by oxidative stress.In summary, this body of work examines the structural basis of hepcidin action, the mechanism of its regulation by iron and erythropoiesis, and a potential therapeutic application to a genetic iron overload disorder

Erythroferrone and matriptase-2 independently regulate hepcidin expression

Erythroferrone and matriptase-2 independently regulate hepcidin expressio

Quantification of endocytosis using a folate functionalized silica hollow nanoshell platform.

A quantification method to measure endocytosis was designed to assess cellular uptake and specificity of a targeting nanoparticle platform. A simple N -hydroxysuccinimide ester conjugation technique to functionalize 100-nm hollow silica nanoshell particles with fluorescent reporter fluorescein isothiocyanate and folate or polyethylene glycol (PEG) was developed. Functionalized nanoshells were characterized using scanning electron microscopy and transmission electron microscopy and the maximum amount of folate functionalized on nanoshell surfaces was quantified with UV-Vis spectroscopy. The extent of endocytosis by HeLa cervical cancer cells and human foreskin fibroblast (HFF-1) cells was investigated in vitro using fluorescence and confocal microscopy. A simple fluorescence ratio analysis was developed to quantify endocytosis versus surface adhesion. Nanoshells functionalized with folate showed enhanced endocytosis by cancer cells when compared to PEG functionalized nanoshells. Fluorescence ratio analyses showed that 95% of folate functionalized silica nanoshells which adhered to cancer cells were endocytosed, while only 27% of PEG functionalized nanoshells adhered to the cell surface and underwent endocytosis when functionalized with 200 and 900  μg , respectively. Additionally, the endocytosis of folate functionalized nanoshells proved to be cancer cell selective while sparing normal cells. The developed fluorescence ratio analysis is a simple and rapid verification/validation method to quantify cellular uptake between datasets by using an internal control for normalization

Quantification of endocytosis using a folate functionalized silica hollow nanoshell platform.

A quantification method to measure endocytosis was designed to assess cellular uptake and specificity of a targeting nanoparticle platform. A simple N -hydroxysuccinimide ester conjugation technique to functionalize 100-nm hollow silica nanoshell particles with fluorescent reporter fluorescein isothiocyanate and folate or polyethylene glycol (PEG) was developed. Functionalized nanoshells were characterized using scanning electron microscopy and transmission electron microscopy and the maximum amount of folate functionalized on nanoshell surfaces was quantified with UV-Vis spectroscopy. The extent of endocytosis by HeLa cervical cancer cells and human foreskin fibroblast (HFF-1) cells was investigated in vitro using fluorescence and confocal microscopy. A simple fluorescence ratio analysis was developed to quantify endocytosis versus surface adhesion. Nanoshells functionalized with folate showed enhanced endocytosis by cancer cells when compared to PEG functionalized nanoshells. Fluorescence ratio analyses showed that 95% of folate functionalized silica nanoshells which adhered to cancer cells were endocytosed, while only 27% of PEG functionalized nanoshells adhered to the cell surface and underwent endocytosis when functionalized with 200 and 900  μg , respectively. Additionally, the endocytosis of folate functionalized nanoshells proved to be cancer cell selective while sparing normal cells. The developed fluorescence ratio analysis is a simple and rapid verification/validation method to quantify cellular uptake between datasets by using an internal control for normalization

Red-luminescent europium (III) doped silica nanoshells: synthesis, characterization, and their interaction with HeLa cells

A simple method to fabricate Eu3+ doped silica nanoshells particles with 100 and 200 nm diameters is reported. Amino polystyrene beads were used as templates, and an 8 to 10 nm thick silica gel coating was formed by the sol-gel reaction. After removing the template by calcination, porous dehydrated silica gel nanoshells of uniform size were obtained. The Eu3+ doped silica nanoshells exhibited a red emission at 615 nm on UV excitation. The porous structure of the silica shell wall was characterized by transmission electron microscopy measurements, while particle size and zeta potentials of the particles suspended in aqueous solution were characterized by dynamic light scattering. Two-photon microscopy was used to image the nanoshells after assimilation by HeLa cancer cells