Recent attention has focused on the use of renewable resources as an alternative to petroleum-based polymers. Fudge et al. (2010) demonstrated that hagfish slime threads, which are composed of “keratin-like” intermediate filament (IF) proteins undergo an α→β transition when strained and when exposed to glutaraldehyde, mechanical properties further improved. Negishi et al. (2012) showed that fibres produced from solubilized hagfish slime threads did not possess comparable mechanical characteristics to native slime threads and were unable to assemble into 10 nm filaments. In this study, fibres were produced from solubilized recombinant vimentin protein and assembled vimentin filaments. Solubilized protein fibres did not display mechanical properties as impressive as fibres made from filaments assembled in the presence magnesium and glutaraldehyde. Additionally, X-ray diffraction analysis of fibres cross-linked with magnesium showed an α→β transition when draw-processed. These data show that fibres produced using IFs can potentially be used for production of sustainable protein polymers.

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Prior studies have demonstrated the effect of diazoxide in protecting against apoptosis via mitochondrial KATP channel opening in vitro. The current investigations are designed to determine if sildenafil, a phosphodiesterase-5 inhibitor and known mitochondrial KATP channel opener, would protect against chronic doxorubicin cardiomyopathy both in vivo and in vitro.Male ICR mice were randomized to 1 of 4 treatments: saline, sildenafil (0.7 mg/kg IP), doxorubicin (5 mg/kg IP), and sildenafil (0.7 mg/kg IP)+doxorubicin. Apoptosis was determined using the terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling and in situ oligo ligation methods. Desmin distribution was determined via immunofluorescence. Bcl-2 was analyzed by Western blot. Left ventricular function was measured in Langendorff mode. Electrocardiographical analysis measured changes indicative of doxorubicin cardiotoxicity (ST-prolongation). In vitro studies using adult ventricular cardiomyocytes were exposed to doxorubicin (1 μM), sildenafil (1 μM) with or without NG-nitro-L-arginine methyl ester (L-NAME; 100 μM), or 5-hydroxydecanoate (5-HD; 100 μM) 1 hour before doxorubicin and incubated for 18 hours. Doxorubicin-treated mice demonstrated increased apoptosis and desmin disruption, which was attenuated in the sildenafil+doxorubicin group. Bcl-2 decreased in the doxorubicin group but was maintained at basal levels in the sildenafil+doxorubicin group. Left ventricular developed pressure and rate pressure product were significantly depressed in the doxorubicin group but attenuated in the sildenafil+doxorubicin group. ST-interval significantly increased in the doxorubicin group over 8 weeks. In the sildenafil+doxorubicin group, ST-interval remained unchanged from baseline. Doxorubicin significantly increased apoptosis, caspase-3 activation, and disruption of mitochondrial membrane potential in vitro,. In contrast, sildenafil significantly protected against doxorubicin cardiotoxicity; however, protection was abolished by both L-NAME and 5-HD. Cell viability studies using spectrophotometer and flow cytometric techniques demonstrated that sildenafil did not affect the antitumor efficacy of doxorubicin in PC-3 cells in vitro. In fact, flow cytometry data indicate that sildenafil, when combined with doxorubicin, was synergistic in the antineoplastic action of doxorubicin. Prophylactic treatment with sildenafil prevented apoptosis and left ventricular dysfunction in a chronic model of doxorubicin-induced cardiomyopathy. Moreover, these studies provide relevant clinical data on the safety and efficacy of sildenafil, leading the way for clinical trials in humans receiving doxorubicin chemotherapy.

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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

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Pendant la morphogenèse, la réparation des tissus et le cancer, les cellules peuvent migrer en manière mésenchymateuse et collective. Le cytosquelette est essentiel pour la migration, mais alors que l'actine et les microtubules ont été largement étudiés, le rôle des filaments intermédiaires (FIs) est encore largement inconnu. La déplétion des FI diminue souvant la vitesse de migration et les FI sont fréquemment surexprimé dans les tumeurs invasives. Pour ces propriétés, nous supposons que les FIs peuvent jouer un rôle clé dans la mécanique cellulaire pendant la migration.Pour étudier le rôle des FI dans la migration collective, nous avons utilisé des astrocytes, les principales cellules gliales du système nerveux central. Les astrocytes migrent collectivement pendant le développement et l'astrogliose en réponse à des signaux pathologiques ou traumatiques. Les astrocytes expriment trois principales FI cytoplasmiques: nestine, GFAP (protéine acide fibrillaire fibreuse) et vimentine, qui forment un réseau dense. Les FI sont surexprimé pendant l'astrogliose et dans les glioblastomes, des tumeurs cérébrales hautement invasives et létales. On ignore si la surexpression des FI est responsable de l'invasion du glioblastome.Au cours de la migration collective dans un test de blessure, les FI contrôlent le positionnement du noyau, la polarité et la migration. On montre que les FI régulent la migration collective dirigée de manière dépendante de la rigidité. Ils agissent avec la protéine connecteur cytoplasmique plectine pour contrôler les point focaux et les jonctions adhérentes. Les FI contrôlent la dynamique et l'organisation de l'actine et régulent la distribution des tractions cellulaires et des contraintes dans la monocouche migrante. Ces résultats démontrent le rôle crucial des FI dans les propriétés mécaniques des cellules migrantes. / During morphogenesis, tissue repair and cancer, cells can migrate in a mesenchymal and collective manner. The cytoskeleton is essential for migration, but whereas actin and microtubules have been extensively studied, the role of intermediate filaments (IFs) is still largely unknown. IF depletion generally decreases migration speed and IF proteins are frequently found upregulated in invasive tumours. Because of IF properties, we hypothesise that they may be key players in cell mechanics during migration. To study the role of IFs in collective migration we used astrocytes, the main glial cells of the central nervous system. Astrocytes migrate collectively during development and astrogliosis in response to pathological or traumatic signals. Astrocytes express three main cytoplasmic IFs: nestin, GFAP (Glial Fibrillary Acidic Protein) and vimentin, which form a dense network. IF proteins are upregulated during astrogliosis and glioblastomas, highly invasive and lethal brain tumours. Whether upregulation of IFs is responsible for glioblastoma invasion is still unknown. During wound-induced collective migration, IFs control nuclear positioning, polarisation and migration. We found that IFs regulate collective directed migration in a stiffness-dependent manner. They act in concert with the cytolinker protein plectin to control focal adhesions and adherens junctions. IFs control actin dynamics and organisation and regulate the distribution of cell tractions and stresses across the migrating cell monolayer. These results demonstrate the crucial role of IFs in the mechanical properties of migrating cells.

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