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Proteomic analysis of posttranslation modifications in breast cancer cell line profiles
Predná, Nikola ; Laštovičková,, Markéta (referee) ; Strouhalová,, Dana (advisor)
Estrogenové a progesteronové receptory, stejně jako HER2 protein, jsou v současnosti klinicky nejužitečnějšími metabolickými markery u karcinomu prsu. Tyto markery umožňují určit typ nádoru a nejlepší možnosti jeho léčby. Jeden z nejagresivnějších typů tohoto onemocnění, triple-negative breast cancer (TNBC), však tyto klinicky stanovené biomarkery postrádá. To znamená, že hormonální terapie nebo cílené léky nepřicházejí v úvahu, takže je na výběr méně možností léčby. Aby bylo možné vyvinout nové léky na míru, je zásadní pochopení molekulárního základu onemocnění. V poslední době se mnoho studií zaměřuje na hledání biomarkerů na úrovni proteinů pomocí proteomiky. Proteiny, zejména jejich post-translační modifikace (PTM), jsou jádrem mnoha buněčných událostí a jejich odhalení může pomoci při pochopení mechanismů rakoviny prsu. Pro objevení molekulárních rysů TNBC, je cílem této studie porovnat proteomická data neléčených rakovinných buněčných linií s buňkami, které podstoupily retinoidní terapii. Důraz bude kladen na PTM, zejména glykosylaci a fosforylaci, Vimentinu a CD44, které byly navrženy jako potenciální biomarkery TNBC v předchozích studiích. Proteinová separace bude provedena pomocí 1D a 2D gelové elektroforézy nebo pomocí SEC-HPLC. Vzorky budou také podrobeny enzymatickému štěpení před identifikací pomocí MALDI-TOF hmotnostní spektrometrie. V případě fosfoproteinového selektivního záchytu bude obohacení provedeno afinitní chromatografií s použitím hrotů pro obohacení fosfopeptidu TiO2 (TopTip). Glykosylované proteiny budou obohaceny pomocí WGA lektinové afinitní chromatografie. Proteiny s významnými rozdíly v PTM mezi ošetřenými a neošetřenými buňkami budou blíže hodnoceny pomocí proteinových databází (MASCOT, STRING a další). Data získaná ze studie budou případně použita k navržení potenciálních biomarkerů pro TNBC.
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Proteomic approach for the study of cancer cell line profiles.
Predná, Nikola ; Langová, Denisa (referee) ; Strouhalová,, Dana (advisor)
Triple-negativní karcinom prsu (TNBC), velice agresivní podtyp rakoviny prsu, je známý svou nepříznivou prognózou a omezenými možnostmi léčby. V tuto chvíli je chemoterapie považována za hlavní způsob léčby. Za účelem vyvinutí nových účinných léčiv je snaha pochopit molekulární základ této nemoci. V důsledku toho bylo již několik potenciálně aktivních látek pro tento konkrétní typ rakoviny prsu podrobeno výzkumu. V poslední době se mnoho studií zabývající touto záležitostí provádí za použití proteomiky jakožto prostředku ke studiu proteomů rakovinných buněk. Rakovinové buňky obsahují klíčové rozdíly v proteinech, které regulují mechanismy buňky. Mapování těchto mechanismů může nakonec umožnit diagnostikovat stav organismu. Tato práce se zaměřuje na proteomické studium buněk TNBC a porovnává neošetřené buňky s buňkami, které byly podrobeny léčbě retinoidy. Separace proteinů a peptidů byla úspěšně provedena elektroforézou na 1D a 2D gelu. Kromě toho byly vzorky podrobeny enzymatickému štěpení vybraných proteinů, které byly poté identifikovány pomocí hmotnostní spektrometrie MALDI-TOF (MS). Proteiny, které se podílejí na procesu epiteliálně-mezenchymálního přechodu (EMT), byly poté kvantifikovány a porovnány mezi vzorky.
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Influence of autism on facial morphology
Cenková, Klára ; Velemínská, Jana (advisor) ; Harnádková, Katarína (referee)
Autism (ASD) is a severe pervasive neurodevelopmental disorder with an as yet unclear etiology but increasing incidence. Early diagnosis and early initiation of therapy are crucial to the success of its treatment and to improving the quality of life of individuals with PAS. The identification of biomarkers in the form of specific facial features of individuals with autism can contribute significantly to facilitating diagnosis. These facial dysmorphologies have their basis in the prenatal period as a consequence of the common and interacting embryogenesis of the face and brain and reflect the neurodevelopmental basis of autism. Using special anthropometric methods, specific facial phenotypes can be determined, which can also be used to classify individuals with PAS into groups according to the severity of the disorder. Key words: Autism, facial dysmorphology, ASD, biomarkers, anthropometry
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Carotid Artery Stenosis
Svoboda, Norbert ; Beneš, Vladimír (advisor) ; Peisker, Tomáš (referee) ; Herzig, Roman (referee)
The basis of the doctoral thesis consists of three studies focused on the diagnosis of carotid stenosis. The first study examines the histological structure of carotid plaques and their relationship to clinical manifestations. The study included 269 patients with 280 analyzed plaques. Multiple logistic regression demonstrated a significant association between symptomatic stenosis and foam cells (OR=1.9, p=0.03) as well as plaque thrombosis (OR=3.5, p=0.02). A significant correlation was also found between symptomatic plaque and unstable plaque according to the comprehensive AHA plaque classification (OR=1.8, p=0.03).The second study addresses the accuracy of carotid stenosis diagnosis using digital subtraction angiography (DSA). Carotid stenosis was measured on DSA and histologically processed plaques obtained during carotid endarterectomy in 644 patients. The study did not establish a correlation between the findings on the histological plaque and DSA (correlation coefficient of 0.22 for ECST and 0.20 for NASCET). The last study focuses on the analysis of the biomarker of oxidative stress (malondialdehyde, MDA) as a predictor of unstable carotid plaques. The study involved 35 patients. Patients with asymptomatic carotid stenosis and those who experienced amaurosis fugax had significantly lower...
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Mitophagy biomarkers in the continuum of Alzheimer's disease
Katonová, Alžbeta ; Veverová, Kateřina (advisor) ; Bohačiaková, Dáša (referee)
The findings of recent years have shown that impaired mitophagy is involved in the pathophysiology of Alzheimer's disease (AD) and other neurodegenerative diseases. Studies on brain biopsies of AD patients, cellular and animal models of AD show that age-dependent decline in mitophagy is a significant contributor to AD pathology, and that the levels of mitophagy proteins are altered. However, whether these changes are reflected in the biofluids of individuals with AD, and whether mitophagy proteins could be potential biomarkers of AD, is unknown.The aim of the diploma thesis was to compare the level of mitophagy markers in blood serum and cerebrospinal fluid (CSF) of patients in various stages of AD with cognitively healthy controls (CU) and determine its relationship to the degree of cognitive impairment and standard Alzheimer's biomarkers (amyloid beta (Ab42), total tau (T-tau) and tau phosphorylated at threonine 181 (P-tau181)). We have shown that mitophagy is impaired in individuals with AD, manifested by increased levels of PINK1 and BNIP3L (activators of mitophagy) and decreased levels of TFEB (master regulator of lysosomal biogenesis) compared to CU. Moreover, these changes were associated with more advanced AD pathology, manifested by increased AD biomarker positivity and cognitive...
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Involvement of the neuroimmune system in Alzheimer's disease
Chaloupková, Barbora ; Hejnová, Lucie (advisor) ; Vašek, Daniel (referee)
Alzheimer's disease afflicts more and more people with increasing life expectancy. The causes of this disease are still not fully understood and explained. An effective treatment is still lacking. One of the reasons is a lack of effective biomarkers of the disease in its early stages before the onset of cognitive deficits. Current research focuses on the neuroimmune system. Emerging evidence shows that changes in its function play a significant role in Alzheimer's disease. This bachelor's thesis describes the interaction of components of the neuroimmune system in the preclinical stages and progression of Alzheimer's disease, their use as biomarkers in the diagnosis of preclinical and clinical stages of AD, and subsequently their potential use in the therapeutic treatment of AD. Key words: Alzheimer's disease, neuroimmune system, neuroinflammation, therapeutic treatment, biomarkers
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Detecting biomarkers of extremophiles in Martian analogues
Němečková, Kateřina ; Jehlička, Jan (advisor) ; Hauer, Tomáš (referee) ; Vandenabeele, Peter (referee)
The main focus of this thesis is on the examination of endolithic microorganisms within gypsum from Sicily. This region is of particular interest because gypsum outcrops are common and accessible there, but the environment is not considered to be extreme in terms of solar radiation or other factors. The research aims to detect endolithic biomarkers and analyse their distribution in their natural environment, to identify endoliths with a focus on phototrophs, and to assign the detected biomarkers to their producers. One of the key tools used in this study was a portable Raman spectrometer with a 532 nm laser, used for the detection of carotenoids under field conditions. Further, the Raman fingerprints of scytonemin and gloeocapsin can be used for the taxonomic identification of some cyanobacteria. With the use of a combination of different excitation wavelengths (445nm, 532 nm, 780 nm), it was possible to detect good quality spectra of various pigments in situ, conditions also in isolated cells and colonies. The results of the molecular analysis also suggest that gypsum habitats in non-extreme regions have a similar microbial composition as those found in extreme regions; i.e., Cyanobacteria being the most abundant group of these microorganisms. This has previously been described as the "hypothesis...
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Prognostic markers of disease course in early stages of multiple sclerosis
Petržalka, Marko ; Marusič, Petr (advisor) ; Taláb, Radomír (referee) ; Lízrová, Jana (referee)
Prognostic markers of disease course in early stages of multiple sclerosis ABSTRACT This thesis analyzes the biomarkers of unfavorable prognosis assessed at early stages of multiple sclerosis. Because specific biomarkers often providerather complex information,the theoretical part provides a summary of biomarkers used not only in prognosis assessment, but also in diagnostics and therapy monitoring. In the empirical part, we have investigatedthe prognostic value of intrathecal synthesis of class M immunoglobulins, serum and cerebrospinal fluid concentrations of interleukin 2, interleukin 6, interleukin 10, chitinase 3-like 2 protein and neurofilament heavy chains, assessed from samples acquired during diagnostic procedures. 58 patients early after the first manifestation of multiple sclerosis were included. All patients initiated therapy with glatiramer acetate, teriflunomide, or interferon beta. To assess the evolution of the disease, we followed the patients clinically and with MRI for two years. Interleukin 2 to interleukin 6 ratio of cerebrospinal fluidvalues ˂0.48 (p=0.0028), interleukin 2 in cerebrospinal fluid ≥1.23 (p=0.0260), chitinase 3-like 2 in cerebrospinal fluid ≥7900 (p=0.0330), age at clinical onset ˂22 years (p=0.0312) and baseline Expanded Disability Status Scale ≥1.5 (p=0.0481), proved to...
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The biological significance of changes in the vascular wall and atherosclerotic plaque for the development of atherosclerotic disease of the coronary arteries
Horváth, Martin ; Hájek, Petr (advisor) ; Poledne, Rudolf (referee) ; Kovárník, Tomáš (referee)
Acute complications of atherosclerosis are typically caused by arterial occlusion due to acute thrombosis forming over a ruptured vulnerable atherosclerotic plaque (VP). Early identification of such VP prior to their rupture could help prevent these events. At the same time, methods for assessing cardiovascular risk are focused on measures at the population level and are not sensitive and specific enough to assess the risk of individual patients. We therefore focused on the possibilities of in vivo detection of VP using biomarkers and invasive imaging methods. First, we focused on the detection of microRNAs (miRs), which have been investigated in recent years as promising biomarkers for a number of diseases, including atherosclerosis. We determined miR levels using quantitative polymerase chain reaction in peripheral venous blood samples from patients in very early phase of ST-elevation myocardial infarction (STEMI), which served as the closest clinical model of acute VP rupture. The results led to the identification of two miRs (miR-331 and miR-151-3p) that were deregulated in STEMI and could be biomarkers of VP. In the next part of the research, we dealt with the possibilities of invasive imaging of VP using intravascular ultrasound and near- infrared spectroscopy (IVUS and NIRS). We confirmed...
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Study of Genotoxicity of Organic Compounds Using DNA Biosensors
Augustín, Michal ; Vyskočil, Vlastimil (advisor) ; Trnková, Libuše (referee) ; Labuda, Ján (referee)
Proposed dissertation thesis presents novel hybrid electrochemical DNA biosensors based on distinct forms of pyrolytic graphite ("edge-plane" and "basal-plane") and their potential in terms of applicability in the field of DNA electrochemistry. Testing of the applicability of the presented bioanalytical tools was preceded by thorough optimization process involving selection of the optimal values for the most crucial operational parameters of the electrochemical DNA biosensors fabricated by means of electrostatic adsorption - deposition potential (Edep), deposition time (tdep), optimal DNA concentration for electrostatic adsorption (cg(DNA)), and possible involvement of convection during the adsorption procedure. Initially, applicability of presented bioanalytical tools was investigated in relation to monitoring DNA damage by utilization of triplet of external DNA damaging sources - UV light irradiation, one- electron oxidants, and hydroxy radicals. Outcome of the damaging processes was monitored directly (via monitoring changes in the redox signals of DNA) or indirectly (via monitoring changes in the redox signals of redox indicator) by the use of voltammetric methods - square- wave voltammetry (SWV), cyclic voltammetry (CV), linear-sweep voltammetry (LSV), and electrochemical impedance...
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