Jaroslava Joniova

Optically Trapped Surface-Enhanced Raman Probes Prepared by Silver Photoreduction to 3D Microstructures

3D microstructures partially covered by silver nanoparticles have been developed and tested for surface-enhanced Raman spectroscopy (SERS) in combination with optical tweezers. The microstructures made by two-photon polymerization of SU-8 photoresist were manipulated in a dual beam optical trap. The active area of the structures was covered by a SERS-active silver layer using chemically assisted photoreduction from silver nitrate solutions. Silver layers of different grain size distributions were created by changing the photoreduction parameters and characterized by scanning electron microscopy. The structures were tested by measuring the SERS spectra of emodin and hypericin.

Estimation of PKCδ autophosphorylation in U87 MG glioma cells: combination of experimental, conceptual and numerical approaches

Golgi apparatus (GA) is a center for lipid metabolism and the final target of ceramide pathway, which may result in apoptosis. In this work localization of highly hydrophobic hypericin is followed by time-resolved imaging of NBDC6 (fluorescent ceramide) in U87 MG glioma cells. Decrease of NBDC6 fluorescence lifetimes in cells indicates that hypericin can also follow this pathway. It is known that both, ceramide and hypericin can significantly influence protein kinase C (PKC) activity. Western blotting analysis shows increase of PKCδ autophosphorylation at Ser645 (p(S645)PKCδ) in glioma cells incubated with 500 nM hypericin and confocal-fluorescence microscopy distinguishes p(S645)PKCδ localization between GA related compartments and nucleus. Experimental and numerical methods are combined to study p(S645)PKCδ in U87 MG cell line. Image processing based on conceptual qualitative description is combined with numerical treatment via simple exponential saturation model which describes redistribution of p(S645)PKCδ between nucleus and GA related compartments after hypericin administration. These results suggest, that numerical methods can significantly improve quantification of biomacromolecules (p(S645)PKCδ) directly from the fluorescence images and such obtained outputs are complementary if not equal to typical used methods in biology.

Modulation of the endogenous production of protoporphyrin IX in a yeast - based model organism

The main aim of this study was to assess conditions at which simple yeast-based model organism produces maximal levels of protoporphyrin IX (PpIX) after an exogenous administration of its precursor, 5-aminolevulinic acid (ALA), and the ferrous-ion chelator 2,2’-bipyridyl. We observed that the fluorescing porphyrin, produced after these administrations, was likely to be PpIX since fluorescence spectroscopy of the porphyrins produced endogenously in yeast cells resembles that of PpIX in DMSO and in vivo in the chick’s chorioallantoic membrane model. Also, fluorescence lifetimes of these porphyrins are very similar to that of PpIX in vitro and in vivo. This suggests that PpIX is the main fluorescent compound produced by yeast in our conditions. We found that the conditions at which yeast produces the maximal PpIX were a synchronous administration of 5 μM ALA and 1 mM 2,2’-bipyridyl for yeast incubated in aqueous glucose and 1 mM 2,2’-bipyridyl in the presence of YPD medium. Such a simple model is of high interest to study basic mechanisms involved in the mitochondrial respiration since PpIX, which is produced in this organelle, can be used as an oxygen sensor, or to perform photodynamic therapy and photodiagnosis. Since the absorption and scattering coefficients of this model are much smaller than those of soft tissues over the visible part of the spectrum, a version of this model loaded with appropriated amounts of light absorbing and scattering particles could be designed as a phantom to mimic tumors containing PpIX, a useful tool to optimize certain cancer photodetection set-ups.

The flashlights on a distinct role of protein kinase C δ: Phosphorylation of regulatory and catalytic domain upon oxidative stress in glioma cells

Glioblastoma multiforme are considered to be aggressive high-grade tumors with poor prognosis for patient survival. Photodynamic therapy is one of the adjuvant therapies which has been used for glioblastoma multiforme during last decade. Hypericin, a photosensitizer, can be employed in this treatment. We have studied the effect of hypericin on PKCδ phosphorylation in U87 MG cells before and after light application. Hypericin increased PKCδ phosphorylation at tyrosine 155 in the regulatory domain and serine 645 in the catalytic domain. However, use of the light resulted in apoptosis, decreased phosphorylation of tyrosine 155 and enhanced serine 645. The PKCδ localization and phosphorylation of regulatory and catalytic domains were shown to play a distinct role in the anti-apoptotic response of glioma cells. We hypothesized that PKCδ phosphorylated at the regulatory domain is primarily present in the cytoplasm and in mitochondria before irradiation, and it may participate in Bcl-2 phosphorylation. After hypericin and light application, PKCδ phosphorylated at a regulatory domain which is in the nucleus. In contrast, PKCδ phosphorylated at the catalytic domain may be mostly active in the nucleus before irradiation, but active in the cytoplasm after the irradiation. In summary, light-induced oxidative stress significantly regulates PKCδ pro-survival and pro-apoptotic activity in glioma cells by its phosphorylation at serine 645 and tyrosine 155.

Formation of Large Hypericin Aggregates in Giant Unilamellar Vesicles—Experiments and Modeling

The incorporation of hypericin (Hyp) from aqueous solutions into giant unilamellar vesicle (GUV) membranes has been studied experimentally and by means of kinetic Monte Carlo modeling. The time evolution of Hyp fluorescence originating from Hyp monomers dissolved in the GUV membrane has been recorded by confocal microscopy and while trapping individual GUVs in optical tweezers. It was shown that after reaching a maximum, the fluorescence intensity gradually decreased toward longer times. Formation of oversized Hyp clusters has been observed on the GUV surface at prolonged time. A simplified kinetic Monte Carlo model is presented to follow the aggregation/dissociation processes of Hyp molecules in the membrane. The simulation results reproduced the basic experimental observations: the scaling of the characteristic fluorescence decay time with the vesicle diameter and the buildup of large Hyp clusters in the GUV membrane.

Optimization and characterization of the endogenous production of protoporphyrin IX in a yeast model

Abstract. The availability of reproducible, convenient, and inexpensive model organisms able to generate predictable levels of endogenous porphyrins, including protoporphyrin IX (PpIX), is essential in photomedicine research. Saccharomyces cerevisiae produces endogenous PpIX and was used as a model organism for this study with the aim to maximize endogenous PpIX fluorescence intensity. It was found that PpIX fluorescence was significantly enhanced by administration of 5-aminolevulinic acid (ALA) and 2,2′-bipyridyl. Fluorescence intensity and spectroscopy of PpIX produced endogenously were measured in diluted yeast solutions under various conditions. The optimal protocol was: 5  μM ALA and 1 mM 2,2′-bipyridyl administered synchronously at 32°C. After 3 h, PpIX in yeast demonstrated similar steady-state and time-resolved spectroscopy as that of PpIX in DMSO. Moreover, under hypoxic conditions, the reciprocal lifetime of PpIX delayed fluorescence measured in real time was correlated to the partial pressure of oxygen (pO2) measured concomitantly with a commercially available pO2 probe. These data show that yeast can, in optimal conditions, reproducibly generate PpIX. This is of interest in various fields such as photodiagnosis, photodynamic therapy, and photobiomodulation. Use of this model organism focuses on essential mechanisms, without the complexity of a multicellular organism.