Browsing by Subject "oxidative stress"
Now showing items 1-6 of 6
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(2020)Reactive oxygen species (ROS) are one of the prominent groups of signal compounds that are produced in stress conditions such as excess light. Nuclear protein RADICAL-INDUCED CELL DEAT (RCD1) is sensitive to ROS and controls the expression of organelle components, e.g. mitochondrial alternative oxidases (AOX), thus balancing the redox-status of a plant cell. Plants have fast responses to fluctuating light conditions that happen even before gene expression: i.e. readjusting the capability to receive light energy between the two photosystems by state transitions and increasing the capacity to remove excess energy by non-photochemical quenching (NPQ). Various small auxiliary proteins function in these fast acclimation events. However, many of them are identified on gene level only. The goal of this master’s thesis is to describe the role of a hypothetical protein, PPD8 in Arabidopsis thaliana. We evaluate how PPD8 is associated with RCD1 and a chloroplast thiol-regulator enzyme NTRC. We created double (rcd1 ppd8) and triple mutant plant lines (rcd1 ppd8 ntrc) by crossing single knockout lines ppd8, rcd1 and ntrc. Photosynthetic performance, NPQ and sensitivity to ROS were observed in each line by using two different chlorophyll fluorescence measurement methods: pulse-amplitude-modulation (PAM) and novel OJIP imaging fluorometry. The leaves were exposed to methyl viologen (MV), which accelerates the chloroplastic ROS production in light, and also to hypoxic conditions in order to study how the effect of MV is altered in low concentrations of oxygen. Additionally, we examined the amount of photosynthetic proteins and stoichiometry of photosystems in ppd8, rcd1 and rcd1 ppd8 by immunological methods. Finally, PPD8 gene with attached hemagglutinin encoding tags was generated by cloning and reintroduced back to the ppd8 knockout lines. Plants lacking RCD1 are very tolerant against MV and ROS, but when rcd1 was crossed with ppd8 the resistance was suppressed. Both rcd1 ppd8 and ppd8 exhibited elevated chlorophyll fluorescence and NPQ values. The removal of PPD8 gene had an impact on the abundance and the stoichiometry of photosynthetic proteins reducing the plants’ performance. When RCD1, PPD8 and NTRC were simultaneously absent the plants had major defects: their NPQ and fluorescence values were drastically increased. Furthermore, several results hinted towards possible issues in the function of ATP synthase in ppd8 background plants. It is also known that NTRC regulates ATP synthase: taken together, the results suggest that PPD8 is necessary for a fully operative ATP synthase and photosynthetic machinery. By reintroducing PPD8 to knockout line ppd8, the phenotype could be reverted back to wild type -like, thus confirming the significance of the PPD8 gene product in plant.
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(2023)Both microplastics (MPs, size 0.1 to 5 mm) and pharmaceuticals and personal care products (PPCPs) are ubiquitous in aquatic environments globally. Polystyrene microplastic (PS-MP) and diclofenac (DCF) are among the most widespread and commonly occurring MPs and PPCPs in the environment. Exposure to these chemicals in single has been reported to cause oxidative stress, immobilization, changes in growth and reproduction and even mortality in Daphnia magna. Moreover, MPs have the ability to act as a vector for PPCPs, and several laboratory studies have reported that PS-MP is able to absorb DCF. However, the combined toxicity of MPs and PPCPs remains largely unknown, especially as is the case for the combined toxicity of PS-MP and DCF in D. magna. In this study, D. magna was exposed to PS-MP and DCF, both in single and combined, at a concentration of 0.5 mg/L each. The aim was to assess the combined toxicity of the two, and determine whether they had a synergistic, additive, or antagonistic effect in D. magna. Whole organism endpoints monitored included mortality, immobilization, and moulting. Physiological endpoints monitored focused on oxidative stress and antioxidant defence, including reactive oxygen species (ROS), superoxidase dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST). The results indicated that both PS-MP and DCF in single were able to significantly alter D. magna antioxidant and biotransformation enzyme activities by inhibition of SOD and GST, while there were no observed impacts on the monitored whole organism endpoints or ROS and CAT. However, similar significant inhibition of SOD and GST was not observed in the combined exposure, which indicates that the combined toxicity of PS-MP and DCF in D. magna was most likely antagonistic. These results highlight that the combined toxicity of MPs and PPCPs is a complex topic that still needs further investigation to fully understand the complex interactions between MPs and PPCPs.
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MANF-Knockout Human Embryonic Stem Cells React to Oxidative Stress Differently than Wild-Type Cells (2023)Introduction: Oxidative stress occurs in cells when reactive oxygen species are generated as a by-product of oxygen metabolism and start to accumulate excessively. While extensive oxidative stress is highly detrimental to the cells, trophic factors help them survive. Trophic factor MANF has interested especially Parkinson’s disease researchers, but recent findings suggest that MANF plays a role in many diseases, also ones with an early childhood-onset. For this reason, it is important to investigate MANF function in different cell types. We have studied how MANF-knockout human embryonic stem cells react to oxidative stress compared to wild-type human embryonic stem cells, by exposing the cells to hydrogen peroxide and ethanol. Results: MANF-knockout human embryonic stem cells were more sensitive to oxidative stress than wild-type cells, but the variation between measurements was remarkable and the differences were statistically insignificant. We found that a transcription factor of our interest localized in the cell nuclei of MANF-knockout cells upon oxidative stress exposure. Such a nuclear translocation did not occur in wild-type cells. Moreover, we found that high concentrations (>2%) of ethanol reduced the viability of cells in only four hours. Discussion: Our findings suggest that MANF-knockout human embryonic stem cells react to oxidative stress differently than wild-type cells. Additional studies are necessary to clarify whether MANF-knockout human embryonic stem cells are indeed more sensitive to oxidative stress than wild-type cells. In the future, it would be interesting to inspect whether MANF protects human embryonic stem cells when the cells are exposed to physiologically relevant ethanol concentrations for longer periods of time.
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(2020)Microplastics are widely studied subject and have raised concern towards water security worldwide but the vector effect of microplastic has not yet fully understood. In this study the ability of microplastic to attach hydrophobic organic compounds is tested with a nonsteroidal anti-inflammatory drug diclofenac. The ability to attach hydrophobic organic compounds has been proved by microplastics but not with diclofenac. Diclofenac is also causing water security threats nearby wastewater treatment plants because it is biologically active and can cause stress to the aquatic organisms even in small quantities. The aim of this study is to see if microplastic has vector effect for the diclofenac. If microplastic retains diclofenac on its surface area it would decrease the stress factor effect of diclofenac towards the investigated macroalgae Aegagropila linnaei. The possible change of oxidative stress levels in A. linnaei is measured by peroxidase enzyme activity. The aim is to see if the enzyme activity raises or decreases when A. linnaei is exposed to microplastic with and without diclofenac. If the peroxidase enzyme activity decreases in macroalgae while exposing A. linnaei to both microplastic and diclofenac it would strengthen the vector effect hypothesis. As a result, the peroxidase enzyme activity seems to have a decreasing trend when the diclofenac concentrations increase. Diclofenac affected to peroxidase enzyme activity but microplastic does not show any signs of binding of diclofenac in this study, and therefore microplastic cannot act as a vector for diclofenac.
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(2015)Parkinson's disease is a progressive neurodegenerative movement disorder which is characterized by the death of dopaminergic neurons in the substantia nigra. In addition, other neuropathological features of the disease are intracytoplasmic protein inclusions as well as oxidative and ER stress. Currently there is no cure for Parkinson's disease so there is a need for novel therapies which could stop the disease progression. Because neurotrophic factors can promote the survival of neurons they might be beneficial for the treatment of Parkinson's disease. Cerebral dopamine neurotrophic factor (CDNF) has proven to be neuroprotective and neurorestorative in rodent models of Parkinson's disease. However, the development of new therapies requires relevant disease models. The defects of the current models of Parkinson's disease increases the need for better and more descriptive disease models. The literature review of this thesis presents an overview of ER stress and oxidative stress. Their role in Parkinson's disease 6-OHDA, MPTP, α-synuclein and rotenone models is also reviewed. The experimental part consists of three studies. The aim of the first study was to establish a preformed α-synuclein fibril mouse model of Parkinson's disease. The development of the lesion was studied by testing the motoric skills with balance beam, rotarod, wire hanger and cylinder test. In addition, TH and α-synuclein immunostainings from striatum and substantia nigra sections was performed. In the second study the effect of CDNF on mice behaviour and TH- and α-synuclein-immunoreactivity in the α-synuclein fibril mouse model was examined. The same motoric behaviour tests as in the first study were used. The purpose of the third experimental part was to investigate the effect of CDNF and GDNF on ER stress proteins in 6-OHDA rat model of Parkinson's disease. The levels of ER stress markers GRP78 and phosphorylated eIF2α were analyzed by Western Blot. The results of the studies were promising. In further studies the effect of α-synuclein fibrils on mouse behaviour and TH- and α-synuclein-immunoreactivity could be studied for longer time. The effect of CDNF on α-synuclein aggregation could also be studied further. The expression levels of other ER stress markers could be investigated so it would clarify the effect of CDNF and GDNF on ER stress.
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(2015)The honey bee is ecologically important and nowadays, it is intensively studied species because of its decline worldwide. Vitellogenin (vg) is a pleiotropic gene that affects especially yolk formation but also e.g. longevity, immunity and labour division in honey bee. Recently, three vitellogenin-like genes have been found, but their functions are not yet discovered. They are named vitellogenin-like-A, vitellogenin-like-B and vitellogenin-like-C. Objectives of this MSc thesis were: i) Are vg and vg-like-genes expressed when the bee is exposed to oxidative stress, ii) Is Vg protein removed from the bee’s hemolymph after shielding her, and iii) Are vg-like-genes expressed abundantly in overwintering bees, like vg is. Oxidative stress was induced by injecting Paraquat. Sham injection was done with physiological saline. Control group had no injections. We found that inflammation by injection (both sham and Paraquat) had a surprisingly strong upregulating effect on vg and vg-like-A expression. Vg-like-B was only significantly upregulated by Paraquat, and vg-like-C had very low expression level with no treatment effect. When measuring Vg protein from fat body and hemolymph samples, no differences were found between treatments. Winter bees expressed high amounts of vg and vg-like-A. Taken together, it looks like vg-like-A works the same shielding way as vg, and vg-like-B slightly too, but vg-like-C might have specialized in other functions.
Now showing items 1-6 of 6