Proteins are the major target for reactive oxygen species as a result of their abundance in biological systems. Stress conditions induce the upsurge in the level of reactive oxygen species causing protein oxidation recognized by the formation of protein radicals, fragments, aggregates, and subsequent degradation. In the current study, we used the immuno-spin trapping technique for the detection of protein radicals in vivo and in vitro. We performed western blotting for the characterization of proteins involved in the formation of protein radicals, fragments, and aggregates. We observed that light stress triggered the upsurge in the level of hydroxyl radical and singlet oxygen. Hydroxyl radical and singlet oxygen induced protein oxidation, which is recognized by the formation of reactive carbon-centered (alkyl) and oxygen-centered (peroxyl and alkoxyl) protein radicals. Generation and kinetic behavior of reactive oxygen species and molecular oxygen in spinach leaves have been studied under abiotic stress. We used electrochemical biosensor techniques for the real-time monitoring of superoxide anion radical and hydrogen peroxide formation and two-dimensional imaging of light-induced oxygen evolution. Immuno-spin trapping technique for the detection and characterization of protein radical formation is imperative for better understating of the mechanism of oxidative modification in PSII proteins under high light stress and electrochemical biosensor techniques can offer precise information on the qualitative determination of superoxide anion radical, hydrogen peroxide and molecular oxygen.
Anotace v angličtině
Proteins are the major target for reactive oxygen species as a result of their abundance in biological systems. Stress conditions induce the upsurge in the level of reactive oxygen species causing protein oxidation recognized by the formation of protein radicals, fragments, aggregates, and subsequent degradation. In the current study, we used the immuno-spin trapping technique for the detection of protein radicals in vivo and in vitro. We performed western blotting for the characterization of proteins involved in the formation of protein radicals, fragments, and aggregates. We observed that light stress triggered the upsurge in the level of hydroxyl radical and singlet oxygen. Hydroxyl radical and singlet oxygen induced protein oxidation, which is recognized by the formation of reactive carbon-centered (alkyl) and oxygen-centered (peroxyl and alkoxyl) protein radicals. Generation and kinetic behavior of reactive oxygen species and molecular oxygen in spinach leaves have been studied under abiotic stress. We used electrochemical biosensor techniques for the real-time monitoring of superoxide anion radical and hydrogen peroxide formation and two-dimensional imaging of light-induced oxygen evolution. Immuno-spin trapping technique for the detection and characterization of protein radical formation is imperative for better understating of the mechanism of oxidative modification in PSII proteins under high light stress and electrochemical biosensor techniques can offer precise information on the qualitative determination of superoxide anion radical, hydrogen peroxide and molecular oxygen.
Klíčová slova
biosensor, electrochemical techniques, light, oxidative stress, photosynthesis, photosystem II, protein oxidation, reactive oxygen species
Klíčová slova v angličtině
biosensor, electrochemical techniques, light, oxidative stress, photosynthesis, photosystem II, protein oxidation, reactive oxygen species
Rozsah průvodní práce
55
Jazyk
AN
Anotace
Proteins are the major target for reactive oxygen species as a result of their abundance in biological systems. Stress conditions induce the upsurge in the level of reactive oxygen species causing protein oxidation recognized by the formation of protein radicals, fragments, aggregates, and subsequent degradation. In the current study, we used the immuno-spin trapping technique for the detection of protein radicals in vivo and in vitro. We performed western blotting for the characterization of proteins involved in the formation of protein radicals, fragments, and aggregates. We observed that light stress triggered the upsurge in the level of hydroxyl radical and singlet oxygen. Hydroxyl radical and singlet oxygen induced protein oxidation, which is recognized by the formation of reactive carbon-centered (alkyl) and oxygen-centered (peroxyl and alkoxyl) protein radicals. Generation and kinetic behavior of reactive oxygen species and molecular oxygen in spinach leaves have been studied under abiotic stress. We used electrochemical biosensor techniques for the real-time monitoring of superoxide anion radical and hydrogen peroxide formation and two-dimensional imaging of light-induced oxygen evolution. Immuno-spin trapping technique for the detection and characterization of protein radical formation is imperative for better understating of the mechanism of oxidative modification in PSII proteins under high light stress and electrochemical biosensor techniques can offer precise information on the qualitative determination of superoxide anion radical, hydrogen peroxide and molecular oxygen.
Anotace v angličtině
Proteins are the major target for reactive oxygen species as a result of their abundance in biological systems. Stress conditions induce the upsurge in the level of reactive oxygen species causing protein oxidation recognized by the formation of protein radicals, fragments, aggregates, and subsequent degradation. In the current study, we used the immuno-spin trapping technique for the detection of protein radicals in vivo and in vitro. We performed western blotting for the characterization of proteins involved in the formation of protein radicals, fragments, and aggregates. We observed that light stress triggered the upsurge in the level of hydroxyl radical and singlet oxygen. Hydroxyl radical and singlet oxygen induced protein oxidation, which is recognized by the formation of reactive carbon-centered (alkyl) and oxygen-centered (peroxyl and alkoxyl) protein radicals. Generation and kinetic behavior of reactive oxygen species and molecular oxygen in spinach leaves have been studied under abiotic stress. We used electrochemical biosensor techniques for the real-time monitoring of superoxide anion radical and hydrogen peroxide formation and two-dimensional imaging of light-induced oxygen evolution. Immuno-spin trapping technique for the detection and characterization of protein radical formation is imperative for better understating of the mechanism of oxidative modification in PSII proteins under high light stress and electrochemical biosensor techniques can offer precise information on the qualitative determination of superoxide anion radical, hydrogen peroxide and molecular oxygen.
Klíčová slova
biosensor, electrochemical techniques, light, oxidative stress, photosynthesis, photosystem II, protein oxidation, reactive oxygen species
Klíčová slova v angličtině
biosensor, electrochemical techniques, light, oxidative stress, photosynthesis, photosystem II, protein oxidation, reactive oxygen species
Zásady pro vypracování
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Zásady pro vypracování
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Seznam doporučené literatury
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Seznam doporučené literatury
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Přílohy volně vložené
Publications
I. Kumar, A., Prasad, A., Sedlárová, M., Pospíšil, P. (2019). Organic radical imaging in plants: focus on protein radicals. Free Radical Biology and Medicine, 130, 568-575.
II. Kumar, A., Prasad, A., Sedlářová, M., Pospíšil, P. (2018). Data on detection of singlet oxygen, hydroxyl radical and organic radical in Arabidopsis thaliana. Data in Brief, 21, 2246-2252.
III. Kumar, A., Prasad, A., Sedlářová, M., Pospíšil, P. (2019). Characterization of Protein Radicals in Arabidopsis. Frontiers in Physiology, 10, 958.
IV. Prasad, A., Kumar, A., Suzuki, M., Kikuchi, H., Sugai, T., Kobayashi, M., Pospíšil, P., Tada, M., & Kasai, S. (2015). Detection of hydrogen peroxide in Photosystem II (PSII) using catalytic amperometric biosensor. Frontiers in Plant Science, 6, 862.
V. Prasad, A., Kumar, A., Matsuoka, R., Takahashi, A., Fujii, R., Sugiura, Y., Kikuchi, H., Aoyagi, S., Aikawa, T., Kondo, T., Yuasa, M., Pospíšil, P., Kasai, S. (2017). Real-time monitoring of superoxide anion radical generation in response to wounding: electrochemical study. PeerJ, 5, e3050.
VI. Kasai, S., Sugiura, Y., Prasad, A., Inoue, K., Sato, T., Honmo, T., Kumar, A., Pospíšil, P., Ino, K., Hashi, Y., Furubayashi, Y., Matsudaira, M., Suda, A., Kunikata, R., Matsue, T. (2019). Real-time imaging of photosynthetic oxygen evolution from spinach using LSI-based biosensor. Scientific Reports, 9, 12234.
Přílohy vázané v práci
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Převzato z knihovny
Ano
Plný text práce
Přílohy
Posudek(y) oponenta
Hodnocení vedoucího
Záznam průběhu obhajoby
Student nejdříve přednesl výsledky své disertace. Následně školitel doc. P. Pospíšil, Ph.D. přečetl hodnocení práce studneta. Poté přečetl svůj posudek recenzent prof. Komanda, včetně dotazů. Student na dotazy odpovídal a recenzent byl s odpovědi spokojený. Následně přečetl svůj posudek 2. recenzent, doc. Špunda, včetně otázek, na které student postupně odpovídal a se kterými byl recenzent spokojený. Následovaly dotazy členů komise. Doc. Pavlovič se ptal, jestli tvorba ROS je součástí získané odolnosti a jestli daný detekční systém může být pro tento případ užitý. Doktorand odpověděl, že tvorba ROS je součást získané odolnosti a že k tomu lze systém užít. Doc. Špundová se ptala, zdali užili k osvětlení pouze bílé světlo a doktorand odpověděl, že ano. Prof. Ilík se ptal, proč si vybral LHCb3 complex při studiu oxidace pomocí ROS.
Poté probíhalo hlasování a sdělení výsledku doktorandovi.