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Nanostructured TiO2 as the surface for the investigation of cell behaviour
Poláková, Kateřina ; Urbánková, Kateřina (referee) ; Fohlerová,, Zdenka (advisor)
This thesis deals with the study of cells on nanostructured surfaces of titanium dioxide, which are produced by the electrochemical method called anodic oxidation. The theoretical part is formed by an overview of manufacturing nanostructured surfaces using anodic oxidation method. It mentions the influence of external factors on the geometric parameters of the structure and description of methods of characterization structures. Furthermore there is processed outline of use for biomedical application and the description of interaction of the cell with surface. The practical part includes description of the production of nanoporous and nano-tubular structures made on thin films of titanium by direct method of anodic oxidation on which was studied the influence of external factors. Described a procedure and production of nanorods structures and nanodots generated using alumina template (AAO) which is subsequently carried out the study of the behavior of cells, which includes tests of adhesion, examination of morphology of cells, assays of proliferation and differentiation. Structures are under investigation of the interaction of cells with the nanostructured layer compared with the smooth surface of the titanium dioxide.
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Fluorescent Nanoprobes Emitting in Near-Infrared Region for Biomedical Applications
Kvaková, Klaudia ; Cígler, Petr (advisor) ; Bogdanowicz, Robert (referee) ; Poláková, Kateřina (referee)
The primary focus of this thesis is to conduct an in-depth investigation into the capabilities of near-infrared photoluminescent (PL) nanoprobes and their application in biomedicine. Specifically, this research explores the potential of fluorescent nanodiamonds (FNDs) and gold nanoclusters (AuNCs). First, the thesis examined the potential of FNDs for in vivo visualization of mice sentinel lymph nodes (SLNs). The study observed that FNDs modified with mannose exhibited greater uptake by macrophages and improved retention within SLNs compared to non- mannosylated FNDs. In addition, FNDs possess a remarkable capacity to accurately measure cellular temperature as was demonstrated by local temperature variation measurements associated to hippocampal neurons firing. Furthermore, the study explored the potential of AuNCs as viable tools for cellular imaging. A simple synthetic approach was developed to create AuNCs with enhanced stability, ligand-based PL enhancement, and prolonged fluorescence lifetime. The thesis also describes the reversible photo- and thermal-induced effects on the PL properties of the synthesized AuNCs. Additionally, the enhancement of AuNCs' PL through their combination with plasmonic nanostructures, specifically gold nanorods, was described. Lastly, a successful visualization of the...
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Metabarcoding and environmental sequencing projects
Kandaurova, Ekaterina ; Hampl, Vladimír (advisor) ; Poláková, Kateřina (referee)
The thesis explores the use of metabarcoding in projects uncovering biological diversity and its applications in various fields of biology. The thesis provides an overview of the principles and workflow of metabarcoding, including the choice of specific molecular markers for various groups of organisms and the bioinformatics aspect of data analysis. Limitations of metabarcoding, such as lack of standardization, primer bias and targeting metabolically inactive organisms are discussed along with potential strategies for overcoming these obstacles. The practical application of metabarcoding is demonstrated on examples of its use in sequencing projects, such as Human Microbiome Project or TARA Oceans expedition. The potential of using metabarcoding for research purposes is discussed from the perspective of paleontology, diet analysis, and conservation biology. Overall, this work aims to assess the potential of metabarcoding as a powerful tool and could serve as an initial guidance for researchers interested in utilizing metabarcoding for their scientific investigations. Keywords: metabarcoding, eDNA, molecular markers, NGS, bioinformatics, biodiversity.
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The diversity of anaerobic ciliates from the subclass Scuticociliatia and their symbionts
Poláková, Kateřina ; Čepička, Ivan (advisor) ; Fiala, Ivan (referee)
Ciliates are the most diversified protists in suboxic and anoxic habitats where they often form symbioses with prokaryotes. Although the diversity of anaerobic ciliates has been overlooked for a long time, anaerobic representatives can be found in most ciliate classes. This study focuses on anaerobic ciliates from the subclass Scuticociliatia, a neglected lineage, which belongs to the species-rich class Oligohymenophorea. One of the main outcomes resulting from this study is the discovery of a novel anaerobic clade of ciliates, from which only one species has been described molecularly to date. We have shown that the clade represents a diversified lineage, likely a new order. Thanks to the sampling of many freshwater and marine anoxic sediments, we have established the largest culture collection of anaerobic scuticociliates in the world. This has enabled us to determine the 18S rRNA gene sequences of 55 cultured anaerobic scuticociliates and to study their morphology both in-vivo and using various silver- impregnation methods. Besides, we applied transmission and scanning electron microscopy techniques to study the ultrastructure of both ciliates and symbionts. To identify the symbionts, we also employed other methods including microbiome sequencing and fluorescence in-situ hybridization. Since all...
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Lineage plasticity of leukemic cells
Slámová, Lucie ; Mejstříková, Ester (advisor) ; Brdička, Radim (referee) ; Machová Poláková, Kateřina (referee)
So far, the lymphoid to myeloid lineage switch during the treatment of B cell precursor acute lymphoblastic leukemia (BCP ALL) was identified only rarely in patients with the MLL gene rearrangement. We discovered a novel BCP ALL subset switching to monocytoid lineage during an early phase of the treatment - swALL ("switching" ALL) with no MLL gene rearrangement. The proportion of swALL cases among BCP ALLs was unexpectedly high (3-4%). All swALLs have expressed the CD2 antigen (LFA-2). The upregulation of C/EBPα gene and hypomethylation of the CEBPA promoter were significant in blasts already at diagnosis, proceeding the lineage switch in the majority of the cases. SwALL patients were characterized by unique subpopulation of the cells coexpressing B lymphoid and monocytoid markers. Changes in the gene expression of M-CSFR, GM- CSFR and other genes accompanied the lineage switch. The lineage switch could be recapitulated in vivo and in vitro. Even if the children patient with swALL respond slowly to initial therapy, the prognosis is comparable to "other" BCP ALLs. Risk-based ALL therapy appears to be the treatment of choice for swALL. Powered by TCPDF (www.tcpdf.org)
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TEL/AML1, BCR/ABL and TEL/ABL Fusion genes in childhood acute lymphoblastic leukemia
Žaliová, Markéta ; Trka, Jan (advisor) ; Machová Poláková, Kateřina (referee) ; Pospíšilová, Dagmar (referee)
Acute lymphoblastic leukemia (ALL) is the most common malignancy in childhood. It represents a group of clinically and biologically heterogenous malignancies that can be subclasified into several subtypes according to the presence of recurrent genetic aberrations. The typical genetic aberrations in childhood ALL are chromosomal translocation, that often result in creation of fusion genes encoding either chimeric kinases or chimeric transcription factors. These recurrent genetic aberations are aquired lesions, they are supposed to be the initial hits (that may arise even prenataly) with a causal role in the process of leukemogenesis, which is, however, in the majority of them not yet fully understood. They further represent specific markes used for the detection of leukemic cells and some of them have also prognostic significance and belong among the factors used for risk group stratification in treatment protocols. Risk group stratification and subsequent risk-adapted therapy together with introduction of new therapeutic approaches (intensive chemotherapeutic regimens involving intrathecal application, hematopoetic stem cell transplantation (HSCT), supportive therapy) account for the significant improvement of the treatment outcomes of childhood ALL in the last decades. In addition to genotype, several...
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Prokaryotic symbionts of free-living anaerobic protists
Poláková, Kateřina ; Čepička, Ivan (advisor) ; Hampl, Vladimír (referee)
Anaerobní prvoci jsou organismy obývající ředí bez přítomnosti kyslíku. Najdeme je anoxických habitatech jako jsou mořské a sladkovodní sedimenty, komunální skládky, nesmíme opomenout zástupce žijící v bachoru přežvýkavců, trávicím traktu švábů a dalších živočichů. Většinou mají anaerobní deriváty mitochondrií, často hydrogenosomy, organely produkující vodík. Metabolismus anaerobních prvoků je ve srovnání s aerobními organismy méně efektivní Časté interakce anaerobních ický my však mohou Symbiózy mezi anaerobními běžné a vznikly u zástupců mnoha anaerobních linií. žít buď endosymbioticky, uvnitř buňky hostitele, nebo ektosymbioticky, na povrchu hostitelské buňky. Dvě ekologicky významné skupiny prokaryot si dokázaly osvojit život symbiotickém vztahu volně žijícími anaerobními prvoky anogenní archebakterie, osídlující hlavně , využívají vodík nické sloučeniny k anu a síran redukující bakterie, žijící hlavně na povrchu buněk, využívají vodík a různé organické sloučeniny k redukci síranu na sulfan. Velmi málo se ví o bližším charakteru těchto vztahů druhové identitě a hostitelské specifitě prokaryotických symbiontů. Další výzkum je nutný pro pochopení fenoménu symbióz v anoxickém světě. Klíčová slova: anaerobní prvoci symbióza anogenní archebakterie síran redukující bakterie
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Study of the regulatory properties of oncogenic microRNAs under normal and pathologically altered conditions in order to detect new tumors.
Dusílková, Nina Borisovna ; Stopka, Tomáš (advisor) ; Machová Poláková, Kateřina (referee) ; Rohoň, Peter (referee)
Oncogenic microRNAs (miRNAs) are small RNA molecules that inhibit post-translational regulatory mechanisms at the epigenetic level. miRNAs are often deregulated in malignancies and due to their stability are detectable in non-cellular fractions of peripheral blood. In our laboratory, we have performed several studies that have investigated and utilized miRNAs as biomarkers for various hematological tumors (e.g., chronic lymphocytic leukemia, Hodgkin`s lymphoma) and solid tumors (e.g., breast cancer). The aim of these studies was to find the association of miRNAs with pathophysiological and clinical aspects of each disease. Here, we confirmed the importance of particular miRNA or its complex during disease monitoring. Combining clinical, molecular biological and statistical analyses, we were able to find miRNA sets that fulfilled not only a diagnostic role but also a prognostic role beyond expectations. The main focus of this thesis is on the investigation of microRNAs in the diagnosis of a hematological malignancy - primary cutaneous T-cell lymphoma (CTCL). Tumor specificity of some miRNAs has been demonstrated. Their aberrant expression in tissue samples of CTCL patients obtained from skin biopsies, correctly distinguished malignant disease from control samples of benign skin lesions. Here, we...
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Identification and Characterization of Genetic Aberrations in Acute Childhood Leukemia
Lukeš, Julius ; Kubričanová Žaliová, Markéta (advisor) ; Machová Poláková, Kateřina (referee) ; Živný, Jan (referee)
Childhood acute leukemias are genetically complex disorders, with recurrent or random aberrations found in most patients. Their proper functional characterization is crucial for understanding the role they play in the process of leukemogenesis. We aimed to identify and characterize the genetic background of two leukemic entities. The transient myeloproliferative disorder (TMD) is a preleukemic condition that occurs in 10% of newborns with Down syndrome. Trisomy 21 together with in-utero gained mutations in the GATA1 gene are essential in TMD and represent an ideal "multi-hit" model to study leukemogenesis. We investigated an alternative pathogenic mechanism enabling TMD development in a confirmed absence of trisomy 21. Novel deletions in the GATA1 and JAK1 genes were described as potential drivers of this TMD. The deletion D65_C228 in GATA1 results in the expression of an aberrant isoform, which is predicted to lose transactivation potential and, more importantly, to partially lose the ability of recognizing physiological DNA binding sites, possibly triggering TMD alone. Our thorough characterization of JAK1 F636del questions its role in TMD development. Analysis of JAK/STAT signaling suggested decrease of kinase activity upon F636 loss. Cells harboring the aberrant JAK1 did not obtain cytokine-...
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