Navegando por Palavras-chave "Fluorescence Microscopy"

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    Análise molecular do tráfego intracelular do canal para prótons sensível à voltagem por microscopia de fluorescência
    (Universidade Federal de São Paulo (UNIFESP), 2021) Silva, Luisa Ribeiro [UNIFESP]; Miranda Filho, Manoel de Arcisio [UNIFESP]; Universidade Federal de São Paulo
    Voltage-gated proton channel presents a physiological role of great importance to phagocytosis and sub sequential death of microorganisms in phagocytic cells, such as neutrophils and microglia. Recently various studies described the contribution of this channel in a higher malignity of tumors and its proliferation. Additionally, HV1 channels have been related to augmented tissue damage in cerebral stroke. Even though these pathological processes have been associated with a higher superficial expression of HV1 channels, little is known about how their abundance in the plasma membrane is regulated. Thus, in this work we have studied the molecular mechanisms of HV1 channel’s intracellular trafficking, and the relevance of cytoskeleton to its transport and function. Through advanced microscopy techniques, we showed that when overexpressed in a heterologous expression system, CHO-K1 cells, the channels are present diffuse in the plasma membrane and aggregated in vesicles in the region close to the plasma membrane. The movement of these vesicles containing HV1 was characterized for the first time in literature, as well as their fusion to the plasma membrane through kiss-and-run and/or kiss-and-linger mechanisms. Furthermore, high-resolution microscopy analysis of HV1 intracellular localization through co-transfection with 8 different intracellular organelles markers fused with EGFP resulted in a co-localization with caveolae, secretory pathway, late endosome, and recycling endosome. However, only the association with the recycling endosome was evident after dynamic co-localization analysis. The functional relationship between cytoskeleton and HV1 channels was analyzed using disruptive agents of microtubules and actin. Actin depolymerization resulted in a reduction of the density of activation and deactivation currents and diminished time to half peak of activation currents. The same effects were observed with microtubule depolymerization along with a higher activation voltage. On the other hand, cytoskeleton disruption did not arise alterations in the superficial expression and mobility of the channel, except for a lower diffusion coefficient with the loss of microtubules. Overall, these results suggests that HV1 channels are associated with recycling endosomes and are functionally modulated by both components of the cytoskeleton.
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    Caracterização biofísica de vesículas catiônicas de dodab contendo C24:1 beta-glicosilceramida (βGlcCer)
    (Universidade Federal de São Paulo (UNIFESP), 2019-05-30) Martins, Leticia de Souza [UNIFESP]; Rozenfeld, Julio Henrique Kravcuks [UNIFESP]; http://lattes.cnpq.br/4232139220639334; http://lattes.cnpq.br/8580993475540515; Universidade Federal de São Paulo (UNIFESP)
    Objective: To characterize the structure of DODAB vesicles containing βGlcCer using differential scanning calorimetry (DSC), electron paramagnetic resonance (EPR) spectroscopy and fluorescence microscopy. Methods: Pure vesicles of DODAB, βGlcCer, and also mixed vesicles of DODAB plus βGlcCer were prepared from lipid films resulting from the evaporation of chloroform: methanol solutions. EPR, DSC and fluorescence microscopy experiments were performed using 5 mol%, 9 mol% and 16 mol% of βGlcCer in relation to the total lipid concentration. Results: It was shown that βGlcCer destabilized the gel phase of DODAB vesicles by decreasing the gel phase packing. Thus, βGlcCer induced a decrease in the phase transition temperature and cooperativity of DODAB vesicles, as seen in DSC thermograms. EPR spectra also showed that βGlcCer induced an increase in the order and / or rigidity of the DODAB fluid phase. Despite their different structures, a similar effect of loosening of the gel phase and stiffening of the fluid phase was observed when low cholesterol fractions were incorporated into the DODAB vesicles. Conclusion: βGlcCer is completely miscible with DODAB at all fractions tested, since no domains were observed by fluorescence microscopy or ESR spectra. Structural characterization of DODAB vesicles containing βGlcCer may be important for the development of novel immunotherapeutic tools such as vaccine adjuvants.