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Development of methodology for detection of circulating nucleic acids in cancer patients
Kobrle, Lukáš ; Souček, Pavel (advisor) ; Valihrach, Lukáš (referee)
Cancer diagnosis represents one of the most common causes of death in the world, also linked to an ever-increasing incidence of even some more difficult-to-treat variants. One of the most common types of cancer in the world is colorectal cancer, which ranks second in terms of mortality. For these reasons, various methods have been developed for an early diagnosis of the disease. One of the modern and non-invasive methods is the so-called liquid biopsy, based on the detection of circulating DNA and RNA from the blood of patients. This method is also suitable for choosing a therapy, monitoring its effectiveness, and observing the relapse of the disease. In this work, a methodical workflow was created for the monitoring of circulating nucleic acids for its further use in a cohort of patients with colorectal cancer. Various commercial kits were used for the isolation of circulating nucleic acids from plasma of healthy controls, among which the best ones, in terms of quality and quantity, was chosen. The threshold for hemolysis detection by qPCR was also determined using samples from healthy donors. Everything was then applied to RNA samples extracted from plasma of cancer patients, from whichna library of short RNA molecules was prepared for subsequent sequencing. After processing the results, the...
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Biomarkers of neurodegeneration in serum and cerebrospinal fluid in patients with selected neurological diseases
Nosková, Libuše ; Fialová, Lenka (advisor) ; Tlaskalová - Hogenová, Helena (referee) ; Švarcová, Jana (referee)
Neurofilaments are the key structural component of the cytoskeleton of neurons, where they are essential for many functions. They consist of 3 subunits: light chain (NFL); medium chain (NfM) and heavy chain (NfH). Except neurofilament proteins there is also α-internexin in the central nervous system (CNS) or peripherin in the peripheral NS. Due to various pathophysiological processes, neurofilament proteins are released into the extracellular space, where they can interact with the components of the immune system. While the involvement of the immune system in the pathogenesis of neurodegenerative diseases is obvious, less knowledge about the antibody response to the neurofilament proteins is available. It is eligible to expand our knowledge in this area. Determination of free antibodies against neurofilaments together with their immune complexes with corresponding antigen provides us more detailed insight into the antibody immune response against neurofilaments. We have optimized the ELISA methods to determine free antibodies against light and heavy chain of neurofilaments together with their corresponding immunocomplexes in both serum and cerebrospinal fluid. Implementation of these methods is precondition for analysis of those parameters in serum and cerebrospinal fluid of patients with...
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Vývoj protilátkové imunitní odpovědi proti kryptosporidiovým infekcím
FENCLOVÁ, Jana
This bachelor thesis is focused on the development of humoral immune response against cryptosporidial infections in BALB/c mice. Mice were infected with various Cryptosporidium spp., namely C. parvum, C. proliferans, C. baileyi and C. andersoni. Cryptosporidium-specific IgG antibodies were detected by ELISA. In this work, cross-reactivity of antibodies with different Cryptosporidium species was also monitored.
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Seroprevalence of Cryptosporidium spp. in the human population
KUTSYNA, Valeriy
In 2018 - 2020, 1366 samples of serum from immunocompetent patients, including 860 sera from the Czech Republic and 506 sera from Ukraine, were collected and examined by an indirect ELISA test to detect specific IgG antibodies against Cryptosporidium in human population. Age, sex, and relevant data on previous cryptosporidial infections were obtained from each patient. Oocysts of C. parvum, 105 oocysts per well, were used as a corpuscular antigen. Blood serum samples and secondary antibodies were diluted 1:200 and 1: 20,000, respectively. The age range of patients varied from newborn infant to 89 years old. A total of 639 male and 709 female patients were examined. The resutts showed number of serologically positive patients increased with age. Moreover, females had higher seropositivity than males, regardless of nationality. Additionally, Czechs were more often positive than Ukrainians. The results showed less than 20 % of patients under 15 years of age were seropositive. In patients older than 15 years the seroprevalence ranged from 20 to 42 %, except the age group 41 - 50 years old, where seroprevalence was 50 %.
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The function of telomeres and cell-free DNA in the healthy volunteers and patients with chosen pathological condition
Zinková, Alžběta ; Korabečná, Marie (advisor) ; Vodička, Radek (referee) ; Drábek, Jiří (referee)
More than 70 years have passed since the discovery of cell-free DNA (cfDNA), but the greatest interest in this topic and knowledge has undoubtedly occurred in the last thirty years. It is used mainly in oncology and prenatal diagnostics. While it is routinely used diagnostically in these fields, little is known about its physiological functions in the organism. Our research therefore focuses on understanding this role and in experiments works mainly with samples obtained from healthy individuals. The first study focused on the differences between plasma and serum in healthy individuals. We asked the question whether they differ in cfDNA concentration and telomeric sequences abundance. We found that the serum contains significantly more cfDNA than plasma, on the other hand, plasma is relatively richer in telomeric sequences. In stimulation experiments with THP1 cells, samples cultured with DNase-treated serum (without cfDNA) showed a higher expression of mRNA TNF-α (Tumor necrosis factor α) than samples untreated. The same trend was observed when plasma samples were stimulated. A study involving plasma samples from ten patients with celiac disease and ten healthy controls showed significant differences in mRNA TNF-α expression between experiments in which THP1 cells were stimulated by DNase-treated...
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