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Cancer Immunotherapy exploiting engineered antibody fragments against prostate-specific membrane antigen
Das, Gargi ; Bařinka, Cyril (advisor) ; Vaněk, Ondřej (referee) ; Ormsby, Tereza (referee)
Prostate cancer (PCa) remains a leading cause of male cancer-related mortality, necessitating thus the development of novel therapeutic approaches as conventional treatments have limited efficacy. Prostate-specific membrane antigen (PSMA) is an established biomarker for both imaging and therapy of PCa, as it is highly upregulated in neoplastic PCa tissues and metastatic castration- resistant prostate cancer. Consequently, immunological targeting of PSMA has gained significant attention as a therapeutic platform for the management of the disease. The thesis is focused on engineering of antibody fragments and fusion proteins derived from the high affinity anti-PSMA 5D3 monoclonal antibody that can be used as immune cell engagers to target and eliminate PSMA-positive cells. To this end, we engineered 5D3 single chain variable fragments (scFv) that were subsequently fused to anti-CD3 scFv and CP33 sequences, creating thus immune cell engagers targeting T-cells (BiTE) and monocytes (5D3-CP33), respectively. The engagers were expressed in insect cells, purified to homogeneity and their biophysical and functional characteristics evaluated using size exclusion chromatography, differential scanning fluorimetry, ELISA and flow cytometry. Ensuing cell-based assays revealed that both BiTE and 5D3-CP33 can...
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Dissecting the effects of salicylic acid on redox balance in plant cells using biochemical and fluorescent imaging techniques
Růžičková, Gabriela ; Burketová, Lenka (advisor) ; Janda, Martin (referee)
Topic of bachelor thesis is plant immunity, specifically it focuses on salicylic acid, reactive oxygen species, it also analyses hox salicylic acid effects formation of reactive oxygen species and which proteins are involved in this biosynthesis. Also in this thesis is described basic methods for measuring of reactive oxygen species, what are advantages, disadvantages, how they work and what they can be used for. Salicylic acid is one of phytohormones involved in immune reaction in plant defence, this thesis is describing salicylic acid signalling, associated proteins and overall effect on plant. Reactive oxygen species have wide framework of action in organisms, they can be called double-edged sword, they can help plant, but also, they can harm depending on their concentration and regulation of their forming, the problem of their quenching is also described in this thesis - their formation, antioxidants, effect on plant immunity. Chapter about measuring methods is divide into three parts - fluorescence methods, chemiluminescence methods and spectrophotometric (histochemical) methods. Key words: salicylic acid, reactive oxygen species, plant immunity, plant stress, phytohormones
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Role of oxidative stress in male infertility.
Dolečková, Barbora ; Tlapáková, Tereza (advisor) ; Šanovec, Ondřej (referee)
Oxidative stress is a phenomenon caused by an excess of reactive oxygen species (ROS), or by insufficient activity of antioxidants, that reduces these ROS levels and thereby protect the organism from oxidative damage. ROS have two types of origin: endogenous, which includes leukocytes and immature sperm, and exogenous, which includes factors such as air pollution caused by heavy metals, smoking tobacco products, obesity and others. Low levels of ROS have a positive effect on the physiological functions of the organism, including the process of spermatogenesis, where ROS participates in the course of hyperactivation and capacitation. However, increased levels of ROS trigger a number of cellular pathologies, whether the loss of fluidity of biological membranes due to lipid peroxidation, deformation of enzymatic proteins or DNA fragmentation, which negatively affects individuals' infertility. Due to the significant positive correlation of ROS scavenging by antioxidants with improving sperm parameters of an infertile individual, antioxidant therapy has recently begun to be used as a possible successful component of male idiopathic infertility treatment.
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Mitochondrial dysfunction in brain tumors
Rollerová, Kateřina ; Vaňátko, Ondřej (advisor) ; Zobalová, Renata (referee)
Brain tumors are one of the most serious pathologies of the central nervous system. Brain tumors are aggressive and very hard to treat due to the fragile nature of the nervous system, presence of blood-brain barrier and high recurrence rate. One of the hallmarks of brain tumors is mitochondrial dysfunction. Mitochondria are organelles involved in essential cellular processes, such as energy production, redox and calcium signaling, or the regulation of cell death. Structural and functional abnormalities, mutations in the mitochondrial genome and other mitochondrial dysregulations may cause disruptions in various cellular processes, such as production of reactive oxygen species, migration, proliferation, or regulation of cell death, promoting the development and/or maintenance of brain tumors. The goal of this thesis is to summarize current knowledge about mitochondrial dysfunction in brain tumors. Key words: brain tumors; mitochondria; mitochondrial dysfunction; Warburg effect; apoptosis; reactive oxygen species; isocitrate dehydrogenase
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Semiconductor microstructures for cellular photostimulation
Tvrdoňová, Anna ; Weiter, Martin (referee) ; Glowacki, Eric Daniel (advisor)
Peroxid vodíku (H2O2) je metastabilní reaktivní forma kyslíku, která reguluje mnoho biologických drah. Úloha H2O2 v biologických procesech závisí na jeho lokální koncentraci, v malém množství (nM) může působit jako signální molekula, zatímco vyšší koncentrace mohou mít cytotoxické účinky. Cílem této práce bylo vytvoření fotosenzitivních mikročástic, které by produkovaly fyziologické množství peroxidu vodíku pro regulaci signálních drah v kultivovaných buňkách. Navrhované mikročástice jsou organické fotovoltaické struktury bez substrátu, které produkují H2O2 fotofaradickou redukcí molekulárního kyslíku. Produkce peroxidu probíhá za současné oxidace donoru elektronů přítomného v elektrolytu. Částice mají rozměry v řádu mikrometrů, zatímco jejich tloušťka nepřekračuje 100 nm. Fotofaradické reakce jsou poháněny červeným světlem (660 nm), které může pronikat tkáněmi, což umožňuje potenciální aplikace mikročástic in vivo. Práce se bude zabývat optimalizací výroby mikročástic a charakterizací produkce H2O2.
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Mitochondrial production of reactive oxygen species and its role in physiological regulations
Holzerová, Eliška ; Mráček, Tomáš (advisor) ; Kalous, Martin (referee)
The production of mitochondrial reactive oxygen species and the resulting oxidative stress is an important phenomenon driving long-lasting research and intense discussions. Knowledge of exact mechanisms of reactive oxygen species production and pathways leading to their formation could help us to directly affect their production, a task with potential terapeutic implications. The molecular nature of the production of reactive oxygen species by some enzymes has already been well documented, but others still remain controversial and current theories are obviously far from the truth. Much more interesting is the question of physiological importace of this production. The reactive oxygen species were considered harmful factors clearly distorting the integrity of the organism for a long time. However, recent research suggest that their existence can also be beneficial and effective. Evidently they can serve as a signaling molecules in several metabolic and regulatory pathways occurring in the organism. This bachelor thesis offers insight into the current state of knowledge. It focuses on the most detailed description of the reactive oxygen species production by mitochondrial respiratory chain enzymes. Furthermore, it deals with some signaling cascades, where involvement of mitochondrially generated...
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Molecular mechanism of reactive oxygen species production by flavin dehydrogenases of mitochondrial respiratory chain.
Holzerová, Eliška ; Mráček, Tomáš (advisor) ; Kalous, Martin (referee)
The aim of this thesis is to investigate molecular mechanism of reactive oxygen species production by flavin dehydrogenases mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) and succinate dehydrogenase (SDH). Together, they represent important source of reactive oxygen species in mammalian mitochondria, but the mechanism of electron leak is still poorly understood. Because mechanisms of reactive oxygen species production by other complexes of respiratory chain are better characterized, they can serve as case studies to get insight into mechanisms of reactive oxygen species by flavin dehydrogenases. Relevant knowledge is therefore summarized in the first part of the thesis. To study the production of reactive oxygen species by the isolated flavin dehydrogenases, we used brown adipose tissue mitochondria solubilized by digitonin as a model. Enzyme activity measurements, hydrogen peroxide production studies by Amplex UltraRed fluorescence and luminol luminescence revealed flavin as the most likely source of electron leak in SDH under in vivo conditions, while we propose coenzyme Q binding site as the site of reactive oxygen species production in the case of mGPDH. Distinct mechanism of this production by the two dehydrogenases is also apparent from induction of reactive oxygen species...
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Oxidative damage to cellular components after oxidative stress induction by specific herbicides
Kramná, Barbara ; Wilhelmová, Naďa (advisor) ; Ryšlavá, Helena (referee)
Oxidative stress is caused by overproduction and overaccumulation of ROS (reactive oxygen species). This state is responsible for cellular damage during unfavorable environmental conditions such as drought, low temperatures, salinity. In order to directly study oxidative stress at tobacco plants (Nicotiana tabacum cv. Xanthi) I used specific herbicides, MV (methyl viologen) and 3-AT (3- aminotriazole). There were several markers used for monitoring oxidative damage to cellular components: DNA damage detected by a comet assay, lipid peroxidation, carbonylated proteins and modification of activities of antioxidant enzymes CAT (catalase) and APX (ascorbate peroxidase). Fluorescent microscopy documented changes in a redox state of tobacco cells and a specific signal for peroxisomes was observed after treatment with higher concentrations of MV and 3-AT. Application of both herbicides caused significant DNA damage, while they worked in a different concentrations, MV in µM and 3-AT in mM. Another convincing oxidative stress marker for MV was protein carbonylation. The inhibition of antioxidant enzymes CAT and APX was less significant when compared to the effects of 3-AT. Decreasing membrane stability proved to be an universal oxidative stress marker for both herbicides. On the other hand, lipid...
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Myocardial tolerance to ischemia/reperfusion injury - possible protective mechanisms
Alánová, Petra ; Neckář, Jan (advisor) ; Nováková, Olga (referee) ; Vaněčková, Ivana (referee)
Ischemic heart disease is the leading cause of death and disability worldwide. The effects of ischemic heart disease are usually attributable to the detrimental effects of acute myocardial ischemia/reperfusion (I/R) injury. The aim of the thesis was to contribute to current effort to clarify the basis of mechanisms that could save the heart from I/R injury. The whole thesis is based on four studies; while the first three are published, the fourth one has been under revision. In the first study, we proved the involvement of nitric oxide (NO) in the cardioprotective mechanism of chronic hypoxia (CH). We described that exogenously increased availability of NO as well as inhibition of phosphodiesterase type 5 led to increased myocardial tolerance of normoxic and chronically hypoxic rats. The effects of both interventions were not additive, suggesting that NO is included in cardioprotective signaling of CH. Second study continued in investigating molecular mechanisms underlying cardioprotection induced by CH. We showed that infarct size-limiting effect of adaptation to CH was accompanied by increased myocardial concentration of tumor-necrosis factor alpha (TNF-α) and TNF-α receptor R2. In the third study, we examined the effect of dexrazoxane (DEX), the only clinically approved drug against...
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Leaf structural changes induced by tropospheric ozone.
Češpírová, Zdeňka ; Lhotáková, Zuzana (advisor) ; Hála, Michal (referee)
Tropospheric ozone is an important pollutant formed by the photochemical reactions of ultraviolet radiation and nitrogenous. Recently, its concentration in the air is increasing due to the promoting climate change and antropogenic activities, mainly because of combustion of the fossil fuels. The ozone is highly reactive molecule degrading to the reactive oxygen species having negative effects on the leaf physiological functions and structure. Ozone penetrates the leaf through the stomata, thereafter it reacts with the cells and their singular compartments. The products formed due to ozone degradation are able to cause the disintegration of plasma membranes and other oxidative damage, for example accumulation of plastoglobuli inside the chloroplasts, changes in the shape of chloroplasts, mitochondria or peroxisomes. At the macroscopic level the visible lesions, chloroses and necroses are observed. In addition, the faster senescence of leaves is observed under the effect of ozone. The main aim of present thesis is to review knowledge of the ozone-induced changes in the leaf structure beginning on the cell structure level and ending at the whole plant body level and to explain physiological mechanisms leading to these damages. Key words: ozone, oxidative stress, leaf structure, mesophyll, reactive...
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