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Molecular mechanisms of tamoxifen resistance in breast cancer
Tomková, Veronika ; Truksa, Jaroslav (advisor) ; Brábek, Jan (referee) ; Mráček, Tomáš (referee)
The resistance to tamoxifen, a drug used in the adjuvant therapy for hormone sensitive breast cancer, represents a major clinical obstacle. Although various mechanisms leading to tamoxifen resistance have been described and intensively studied, a significant number of patients still become resistant to the treatment and eventually relapse. Tamoxifen therapy has been shown to enrich tumors with cancer stem cells (CSCs), which are naturally resistant, and have self-renewal ability and the potential to form secondary tumors. Metabolic rewiring, altered iron metabolism and upregulation of ATP-binding cassette (ABC) transporters have been shown to be important in the maintenance of CSC phenotype. Therefore, we investigated these mechanisms as possible contributors to tamoxifen resistance in vitro in two tamoxifen resistant (Tam5R) cell lines that we established. We show that Tam5R cells have dramatically disassembled and less active mitochondrial supercomplexes (SCs) and higher level of mitochondrial superoxide, together with a fragmented mitochondrial network. Such dysfunction of mitochondria results in the AMP-activated protein kinase (AMPK) activation and metabolic rewiring towards glycolysis. Importantly, cells lacking functional mitochondria are significantly more resistant to tamoxifen, supporting...
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Role of sulfhydryl oxidase 1 in cancerogenesis
Beranová, Lea Marie ; Truksa, Jaroslav (advisor) ; Šuťák, Róbert (referee)
Disulfide bridges play a significant role in protein-folding as well as en- zyme activity and thus regulate many intra- and extracellular processes. Sulfhydryl oxidase QSOX1 forms S-S bridges de novo, modulating the activity of its substrates and thus directly or indirectly influences vital cel- lular processes. The first part of this thesis focuses on characterization of the role of QSOX1 in cancerogenesis, using breast cancer cell lines (MCF7, MDA-MB-231) and pancreatic cancer cell line (Panc-1), while the second part emphasizes the regulation of QSOX1 expression by different oxygen concentrations. To study the effect of QSOX1 on proliferation of triple-negative cancer cells MDA-MB-231, two genetically modified cell lines - QSOX1-overexpressing and QSOX1 knockout cell lines - were constructed. While increased QSOX1 protein levels do not have a significant effect, the absence of QSOX1 leads to a decreased cellular growth. Lack of QSOX1 also results in visible change in cellular morphology. QSOX1 knockout cells can be mostly characterized as more round-shaped with less noticeable or completely missing lamellipo- dia. This finding is with agreement with to-date literature suggesting that QSOX1 is important not only for cellular proliferation but also for migration and invasiveness. While authenticating the theory of...
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Mechanisms of resistance and iron metabolism in cancer stem cells
Lettlová, Sandra ; Truksa, Jaroslav (advisor) ; Kovář, Jan (referee) ; Brábek, Jan (referee)
(EN) Analogously to normal stem cells within the tissues, cancer stem cells (CSCs) have been proposed to be responsible for maintenance and growth of tumours. CSCs represent a small fraction of cells within the tumour, which is characterised by self-renewal capacity and ability to give rise to a tumour when grafted into immunocompromised mice. Cells with increased stemness properties are believed to be responsible for tumour resistance, metastases formation and relapse after tumour treatment. The first part of this work concentrates on resistance of the tumours, which is often associated with increased expression of ATP-binding cassete (ABC) transporters pumping chemotherapeutics out of the cells. For the purposes of this study, we utilized an in vitro model of CSCs, based on cultivation of cells as 3D "spheres". Expression profiling demonstrates that our model of CSCs derived from breast and prostate cancer cell lines express higher mRNA level of ABC transporters, particularly ABCA1, ABCA3, ABCA5, ABCA12, ABCA13, ABCB7, ABCB9, ABCB10, ABCC1, ABCC2, ABCC3, ABCC5, ABCC8, ABCC10, ABCC11 and ABCG2 among the cell lines tested. The protein level of ABC transporters tested in breast CSCs showed higher expression of ABCB8, ABCC1, ABCC2, ABCC10 and ABCG2 but downregulation of ABCB10 and ABCF2 proteins....
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Analyzing the role of the p130Cas SH3 domain in p130Cas-mediated signaling
Gemperle, Jakub ; Rösel, Daniel (advisor) ; Vomastek, Tomáš (referee) ; Truksa, Jaroslav (referee)
The adaptor protein p130Cas (CAS, BCAR1) represents a nodal signaling platform for integrin and growth factor receptor signaling, and influences normal development and tissue homeostasis. Its altered expression drives many pathological conditions including tumor growth, metastasis and drug resistance in many cancer types. How p130Cas contributes to many of these pathologies is still poorly understood. Therefore, the overall aim of my PhD work was to provide new insights to p130Cas signaling and its regulation. The SH3 domain is indispensable for p130Cas signaling, but the ligand binding characteristics of the p130Cas SH3 domain, and the structural determinants of its regulation were not well understood. To be able to study various aspects of p130Cas signaling we identified an atypical binding motif in p130Cas SH3 domain by establishing collaborations with Dr Veverka (Structural biology) and Dr Lepšík (Computational biochemistry; Academy of Sciences, CZ) which gave new insight into this binding interface. Through these collaborations I generated chimeras of p130Cas SH3 domain with its ligands for structural NMR analysis and learned how to visualize and analyze structures. Furthermore, my work expanded our knowledge of p130Cas SH3 ligand binding regulation and led to a novel model of Src-p130Cas- FAK...
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Male infertility in context of testicular cancer
Cimlerová, Markéta ; Komrsková, Kateřina (advisor) ; Truksa, Jaroslav (referee)
This bachelor thesis focuses on male infertility in a connection to testicular cancer. Testicular cancer is the most common malignancy among young men in a reproductive age and the worldwide incidence of testicular cancer is on the rise. A lot of attention is also given to an increasing rate of infertility in a context of testicular cancer. For these reasons, the aim of this thesis is to clarify several non-physiological changes, such as hormone levels, spermatogenesis and sperm parameters, which take place in a male body and influence the chance to become a biological father. These pathological changes can be due to the disease itself but also due to the treatment. A cryopreservation of semen is also going to be discussed as the way to preserve male fertility and as an option for couples with the infertility problems due to male factor which rely on help of the assisted reprodiction. Kye words: testicular cancer, cancer treatment, male infertility, sperm parameters, mitochondria, assisted reproduction
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Mitochondria as a target for anti-cancer therapy
Monschizadeh Tehrany, Shahin ; Truksa, Jaroslav (advisor) ; Kalous, Martin (referee)
Cancer is a complex disease that is characteristic by its heterogeneity in forms and symptoms. This diversity is caused by various mutations in oncogenes and tumor suppressor genes, which then makes cancer a very unclear and indistinct target. However numerous neoplastic characteristics are linked to the functions of mitochondria. This then makes mitochondria a center of interest of cancer research. Many cancer cells show the switch to the metabolism of aerobic glycolysis witch is characteristic by the increased glucose uptake, increased activity of biosynthetic pathways and lower oxidative capacity of mitochondria. Another mitochondria-linked modification is the increased production of reactive oxygen species in cancer cells, which are a source of new mutations and enhance cell proliferation. An increased transmembrane potential on the inner mitochondrial membrane is another very common feature that also promotes cell division and directly correlates with cancer malignancy. In this context a group of antitumor drugs called mitocans was discovered, that acts on the altered mitochondria of tumor cells. The activity of mitocans is ranging from the restoration of the function of pro-apoptotic molecules to the enforced cell death caused by oxidative damage done to the mitochondria of cancer cells....
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Iron containing cofactors in anaerobic parasite Giardia intestinalis
Pyrih, Jan ; Tachezy, Jan (advisor) ; Martásek, Pavel (referee) ; Truksa, Jaroslav (referee)
Iron is an essential element in nearly all organisms. It is present mainly as a component of iron sulfur (FeS) clusters or as a heme iron. These cofactors enable proteins to transfer electrons or diatomic gasses, signal sensing and enzyme catalysis. Numerous FeS and heme depending proteins are involved in photosynthesis and respiratory chain pathways, which are well described processes. However, there is still much to learn about more recently discovered pathways such as formation of FeS clusters in various cell compartments and about roles of novel FeS or heme proteins. Particularly, only limited information is available about how FeS clusters are assembled or how heme is used in anaerobic protists, in which cytochrome-dependent respiration and photosynthesis does not occur. We decided to focus on iron cofactors in anaerobic parasite Giardia intestinalis. This organism undergone dramatic reductive evolution that resulted in formation of one of the smallest eukaryotic genome and the most reduced form of mitochondria, the mitosome. We characterized some components of mitochondrial (ISC) and cytoplasmic (CIA) FeS assembly machineries. We have detected ISC components in mitosome by proteomic analysis. Furthermore we investigated the presence and subcellular localization of CIA proteins in Giardia. In...
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Iron metabolism in cancer cells
Beranová, Lea Marie ; Truksa, Jaroslav (advisor) ; Čermák, Vladimír (referee)
1Abstract: Cancer is one of the major causes of death in the present world. As the research of this disease has progressed, the attention of some scientists has been focused on a metabolism of iron and how it can be used to fight these rapidly proliferating invasive cells and stop their spreading. This work should serve as a brief review of iron metabolic processes from the iron absorption from dietary resources and recycled cell iron, to its usage in heme- or Fe/S clusters-proteins and storage in a form of ferritin, while highlighting the points that differ in cancer cells. It also gives a modest overview on the regulatory pathways of iron uptake and use, and mentions iron metabolism disorders such as iron-depletion and overload. Simultaneously it is denoting possible differences that could be targeted in tumor treatment, and, at least but not last, the perspectives and future work that could bring a new methods and approaches to this matter. Keywords: iron metabolism, iron, cancer, hepcidin
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The plasticity of melanoma cell invasiveness
Gandalovičová, Aneta ; Brábek, Jan (advisor) ; Truksa, Jaroslav (referee)
and keywords: During metastasis, cancer cells can invade the extracellular matrix using various strategies. When invading individually, they employ either the amoeboid invasion mode, during which the cell body dynamically deforms by enhanced contractility to squeeze through pores within the matrix, or protease dependent mesenchymal migration that takes advantage of the possibility to digest the surrounding matrix. Cells migrating in one mode can actively switch to the other by mesenchymal-amoeboid (MAT) or amoeboid-mesenchymal transitions (AMT). This enables escape mechanisms and considerably complicates anti-metastatic treatment. It is well known that Rho GTPases are master regulators of cytoskeleton re-arrangements and thus, unsurprisingly, play a major role in both invasion modes and can directly drive the transitions. However, upstream activation of these pathways is still largely unclear. This thesis aimed to optimize 3D conditions suitable for studying plasticity of cell invasion in vitro, establish AMT and MAT in melanoma cells based on manipulation of Rho GTPases and verify novel candidates regulating cell invasion plasticity based on previous RNA sequencing of cells before and after MAT. Last, by synthesis of published data, results from sequencing and new findings presented in this...
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The role of autophagy and selected beta-tubulin isotypes in taxane resistance in breast cancer cells
Kábelová, Adéla ; Jelínek, Michael (advisor) ; Truksa, Jaroslav (referee)
Drug resistance in cancer cells is a frequent cause of breast cancer therapy failure. The aim of this thesis was to elucidate mechanisms of resistance to taxanes, that are used in therapy of various types of cancer, including breast cancer. We particularly assessed the role of autophagy and changes in βII- and βIII isotype gene expression in development of taxane resistance. As model of breast cancer we used human sensitive cell lines SK-BR-3, MCF-7 a T47-D and resistant sublines SK-BR-3-PAC/REZ a MCF-7- PAC/REZ which grow in paclitaxel concentration lethal for sensitive sublines. In cell lines SK-BR-3 and MCF-7, taxane application decreased the level of autophagy, however in cell line T47-D led to its activation. We detected no difference between basal levels of autophagy in sensitive subline SK-BR-3 compared to resistant subline SK-BR-3-PAC/REZ, but we observed increased basal level of autophagy in sensitive subline MCF-7 compared to the resistant subline. Increase or decrease level of autophagy did not affect taxane resistance, except activation of autophagy in resistant subline SK-BR-3-PAC/REZ, that further increased the resistance to paclitaxel. Taxane application in cell line T47-D increased the levels of βII- and βIII-tubuline expression, however we did not find any similar effect in other tested...
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