The embryonic mouse inner ear was used as a model with which to study ototoxicity and tissue interactions. The inner ear anlage can be explanted and cultured in vitro from about the 12th gestational day (gd), and will differentiate parallel with the inner ear developing in vivo until a time corresponding to birth (21st gd). During this period the ovoid sac develops into the labyrinth. In the present thesis work, otic anlagen from gd 12, 13, 13.5, 15 and 16 were used. As a rule the explants were kept in culture until a time point equivalent to the 21st gd. Analyses using freeze-fracture technique and transmission electron microscopy showed that in cultured 13th gd otocysts the development of junctional complexes followed the same principal pattern as in vivo. Tight junctions develop into many strands lying parallel to the apical surface of all epithelial cells. Uncoupling of the hair cells occurs with loss of gap junctions. Some tight junctions had an aberrant appearence, with in part very thick strands and strands running at right angles to the apical surface. All aminoglycosides are potentially ototoxic. In the inner ear, outer hair cells of the organ of Corti and vestibular type I hair cells are affected by these antibiotics. The access route to the hair cells and the sites and mechanisms of action of aminoglycosides are not precisely defined. The uptake of tritiated tobramycin in 16th gd inner ears was studied. An initial rapid uptake of the drug, within 10 min, was followed by a slower accumulation, reaching a steady state after 60 min. Most of the tobramycin was bound reversibly, at least after a short period of incubation (2 h). The irreversibly bound fraction was of the same magnitude as the uptake within 10 min. Uptake took place against a concentration gradient. The otocyst can differentiate even without the statoacoustic ganglion. The interaction of the sensory epithelium with the ganglion was investigated by explanting the statoacoustic ganglion without target tissue. Twenty-five percent of the ganglions survived and had outgrowth of neurites but there was no differentiation into either the cochlear or vestibular type of neuron cells. Exposure of cultured otocysts (13 or 13.5 gd) to l-azetidine-2-carboxylic acid, a 1-proline analog that disrupts formation of collagen, resulted in retarded morphogenesis of the labyrinth and a dose- dependent derangement of the basal lamina. The expression of intermediate filaments (IFs) was analysed using monoclonal antibodies. The same IF pattem was found in cultured inner ears as in vivo. Explants were taken on 13th, 15th or 16th gd. Exposure to gentamicin, ethacrynic acid or cisplatin did not alter the IF composition. Cytokeratins (CKs) 8 and 18 were identified in all inner ear epithelia. In addition CKs 7 and 19 were visualized in the epithelia involved in maintaining endolymph homeostasis. The ganglion cells showed coexpression of CK, vimentin and neurofilaments. The elemental composition of the endolymph compartment of 16th gd inner ears cultured for 5 days was studied using energy-dispersive X-ray microanalysis. Na to K ratios characteristic of endolymph were found. / <p>S. 1-34: sammanfattning, s. 37-88: Härtill 6 uppsatser</p> / digitalisering@umu

Diss. (sammanfattning) Linköping : Univ., 2003. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 7 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Thesis (Ph.D.). / Written for the Dept. of Medicine, Division of Experimental Medicine. Title from title page of PDF (viewed 2008/07/23. Includes bibliographical references.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Vita. Bibliography: 155-170.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 78-98).

Thesis (Ph.D. in Neuroscience) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 145-165). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (Ph.D.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 118 pages. Includes Vita. Includes bibliographical references.

A disintegrin and metalloprotease (ADAM) 17 cleaves and releases membrane-tethered pro-forms of several signalling molecules from the plasma membrane, including the inflammatory cytokine tumour necrosis factor alpha (TNFα) and ligands of the epidermal growth factor receptor (EGFR). Due to the important functions of its substrates, ADAM17 activity has to be tightly controlled, and its misregulation has implications for inflammation and cancer. The multi-pass membrane proteins iRhom1 and iRhom2 are members of the conserved rhomboid-like superfamily and control ADAM17 activity by several mechanisms throughout the secretory pathway. First, iRhoms facilitate trafficking of the catalytically inactive proenzyme form of ADAM17 from the endoplasmic reticulum (ER) to the Golgi apparatus, where the inhibitory pro-domain of ADAM17 is removed. Subsequently, iRhoms exert a different form of control of ADAM17 at the plasma membrane, this time on stimulus-induced ADAM17 activity, its substrate specificity, and its stability. iRhoms ultimately regulate the release of ADAM17 substrates, and are consequently key players in TNF&alpha; and EGFR signalling. However, it remains unclear how iRhom function itself is regulated posttranslationally, and whether iRhoms require co-factors to exert their roles as ADAM17 regulators. The goal of my project was to shed light into these questions by identifying new iRhom interaction partners. I developed a mass spectrometry-based screen to identify new binding partners of human iRhoms using co-immunoprecipitation. The top hit of the screen was the poorly characterised FERM domain-containing protein 8 (FRMD8), which binds to both iRhom1 and iRhom2. FRMD8 was found to play a crucial role in the iRhom/ADAM17 pathway because FRMD8 knockdown and knockout in HEK293T cells significantly reduced the levels of mature ADAM17 and the release of ADAM17 substrates. The closely related metalloprotease ADAM10 was not affected by the loss of FRMD8, implying that FRMD8 is not a general regulator of ADAM metalloproteases. Interaction studies revealed that FRMD8 binds to the cytosolic N-terminus of iRhom2 throughout the entire secretory pathway. FRMD8 loss does not affect the ER-to-Golgi trafficking of iRhom2 but plays a role in stabilising iRhom2 at the plasma membrane by preventing the lysosomal degradation of both iRhom2 and mature ADAM17. Using human induced pluripotent stem cell (hiPSC)-derived macrophages, I showed that FRMD8 regulates mature ADAM17 levels and the ADAM17-dependent release of TNF&alpha; in human macrophages. Studies in FRMD8 knockout (KO) mice confirmed the reduced mature ADAM17 levels in all mouse tissues tested, further supporting the conclusion that FRMD8 is a novel regulator of the iRhom/ADAM17 pathway with physiological relevance in mammals. Finally, I showed that the interaction of FRMD8 and iRhom, which are both conserved from Drosophila to human, is also conserved. Furthermore, loss of the FRMD8 orthologue in flies, Bili, leads to motility defects and shows similarity to the loss of iRhom in flies. These results suggest that FRMD8 is a novel regulator of iRhom function in mammals and Drosophila.