Inner ear tissue preservation by rapid freezing: improving fixation by high-pressure freezing and hybrid methods

In the preservation of tissues in as ‘close to life’ state as possible, rapid freeze fixation has many benefits over conventional chemical fixation. One technique by which rapid freeze-fixation can be achieved, high pressure freezing (HPF), has been shown to enable ice crystal artefact-free freezing and tissue preservation to greater depths (more than 40μm) than other quick-freezing methods. Despite increasingly becoming routine in electron microscopy, the use of HPF for the fixation of inner ear tissue has been limited. Assessment of the quality of preservation showed routine HPF techniques were suitable for preparation of inner ear tissues in a variety of species. Good preservation throughout the depth of sensory epithelia was achievable. Comparison to chemically fixed tissue indicated that fresh frozen preparations exhibited overall superior structural preservation of cells. However, HPF fixation caused characteristic artefacts in stereocilia that suggested poor quality freezing of the actin bundles. The hybrid technique of pre-fixation and high pressure freezing was shown to produce cellular preservation throughout the tissue, similar to that seen in HPF alone. Pre-fixation HPF produced consistent high quality preservation of stereociliary actin bundles. Optimising the preparation of samples with minimal artefact formation allows analysis of the links between ultrastructure and function in inner ear tissues

Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached in the region of interest. The fluorescence of the region recovers when the unbleached GFP-tagged protein from outside of the region diffuses into the region of interest. The mobility of the protein is then analyzed by measuring the fluorescence recovery rate. This technique could be used to characterize protein mobility and turnover rate

Исследование теплоотдачи отопительного прибора системы теплоснабжения полевого госпиталя, функционирующего в условиях чрезвычайных ситуаций

Criteria dependences for assessment of heat transfer value under conditions natural convection (a horizontal slot) have been obtained in the paper. The dependences are applicable for heaters in heat supply systems.Получены критериальные зависимости для оценки величины теплоотдачи в условиях естественной конвекции (горизонтальная щель). Зависимости применены для отопительных приборов системы теплоснабжения

An actin molecular treadmill and myosins maintain stereocilia functional architecture and self-renewal

We have previously shown that the seemingly static paracrystalline actin core of hair cell stereocilia undergoes continuous turnover. Here, we used the same approach of transfecting hair cells with actin–green fluorescent protein (GFP) and espin-GFP to characterize the turnover process. Actin and espin are incorporated at the paracrystal tip and flow rearwards at the same rate. The flux rates (∼0.002–0.04 actin subunits s−1) were proportional to the stereocilia length so that the entire staircase stereocilia bundle was turned over synchronously. Cytochalasin D caused stereocilia to shorten at rates matching paracrystal turnover. Myosins VI and VIIa were localized alongside the actin paracrystal, whereas myosin XVa was observed at the tips at levels proportional to stereocilia lengths. Electron microscopy analysis of the abnormally short stereocilia in the shaker 2 mice did not show the characteristic tip density. We argue that actin renewal in the paracrystal follows a treadmill mechanism, which, together with the myosins, dynamically shapes the functional architecture of the stereocilia bundle

Two adaptation processes in auditory hair cells together can provide an active amplifier

The hair cells of the vertebrate inner ear convert mechanical stimuli to electrical signals. Two adaptation mechanisms are known to modify the ionic current flowing through the transduction channels of the hair bundles: a rapid process involves calcium ions binding to the channels; and a slower adaptation is associated with the movement of myosin motors. We present a mathematical model of the hair cell which demonstrates that the combination of these two mechanisms can produce `self-tuned critical oscillations', i.e. maintain the hair bundle at the threshold of an oscillatory instability. The characteristic frequency depends on the geometry of the bundle and on the calcium dynamics, but is independent of channel kinetics. Poised on the verge of vibrating, the hair bundle acts as an active amplifier. However, if the hair cell is sufficiently perturbed, other dynamical regimes can occur. These include slow relaxation oscillations which resemble the hair bundle motion observed in some experimental preparations.Comment: 13 pages, 6 figures,REVTeX 4, To appear in Biophysical Journa

The urgency of lifelong learning : a study on the learning of Information and Communication Technologies in populations 50+

O presente artigo visa abordar uma investigação de doutoramento realizada nas universidades seniores do distrito de Castelo Branco no ano letivo 2013/2014, que teve como principal objetivo identificar os fatores socioculturais que influenciam e condicionam a opção pela aprendizagem das TIC e conhecer os impactos desta aprendizagem no Bem-estar (mental e social) ao longo do processo de envelhecimento. Os dados foram recolhidos em 5 universidades seniores deste distrito através de um inquérito por questionário que envolveu 374 cidadãos (50 + anos) e de entrevistas a 5 Diretores, 5 Professores de TIC e 10 Participantes que já frequentaram uma formação em TIC nas respetivas universidades seniores. Os resultados permitiram verificar que a necessidade de comunicação, o combate ao isolamento são os principais fatores socioculturais que influenciaram a aprendizagem das TIC nestes participantes. O exercício da memória e das aptidões intelectuais, a participação e inclusão na sociedade digital e a diminuição da solidão foram os impactos no «Bem-estar mental» e no «Bem-estar social» mais evidenciados— This article aims to address a doctoral research carried out in the senior universities of the district of Castelo Branco in the academic year 2013/2014, whose main objective was to identify the sociocultural factors that influence and condition the option to learn ICT and to know the impacts of this learning on well-being (mental and social) throughout the aging process. Data were collected at 5 senior universities in this district through a questionnaire survey involving 374 citizens (50+ years old) and from interviews to 5 Directors, 5 ICT Teachers and 10 Participants who have already attended ICT training in their respective universities. The results allowed us to verify that the need for communication, the fight against isolation are the main sociocultural factors that influenced the learning of ICT in these participants. The exercise of memory and intellectual skills, participation and inclusion in the digital society and the reduction of loneliness were the most evident impacts on the "mental well-being" and "social well-being"info:eu-repo/semantics/publishedVersio